Label-Free Investigations on the G Protein Dependent Signaling Pathways of Histamine Receptors

G protein activation represents an early key event in the complex GPCR signal transduction process and is usually studied by label-dependent methods targeting specific molecular events. However, the constrained environment of such “invasive” techniques could interfere with biological processes. Although histamine receptors (HRs) represent (evolving) drug targets, their signal transduction is not fully understood. To address this issue, we established a non-invasive dynamic mass redistribution (DMR) assay for the human H1–4Rs expressed in HEK cells, showing excellent signal-to-background ratios above 100 for histamine (HIS) and higher than 24 for inverse agonists with pEC50 values consistent with literature. Taking advantage of the integrative nature of the DMR assay, the involvement of endogenous Gαq/11, Gαs, Gα12/13 and Gβγ proteins was explored, pursuing a two-pronged approach, namely that of classical pharmacology (G protein modulators) and that of molecular biology (Gα knock-out HEK cells). We showed that signal transduction of hH1–4Rs occurred mainly, but not exclusively, via their canonical Gα proteins. For example, in addition to Gαi/o, the Gαq/11 protein was proven to contribute to the DMR response of hH3,4Rs. Moreover, the Gα12/13 was identified to be involved in the hH2R mediated signaling pathway. These results are considered as a basis for future investigations on the (patho)physiological role and the pharmacological potential of H1–4Rs.


Introduction
G protein-coupled receptors (GPCRs), also termed seven-transmembrane-domain receptors (7TMs), are integral membrane proteins that transduce a broad variety of extracellular stimuli, ranging from photons and various small molecules to polypeptides, into the cell. As the largest superfamily of proteins in the human genome, GPCRs are involved in many (patho)physiological processes and represent important drug targets in the treatment of numerous diseases [1,2]. Canonical GPCR signal transduction occurs by binding of an agonist to a receptor, stabilizing an active receptor conformation and allowing the receptor to activate heterotrimeric G proteins, composed of Gα, Gβ and Gγ subunits. Upon GPCR activation, the G proteins dissociate from the receptor and split up into Gα and Gβγ subunits. Subsequently, both can modulate specific downstream effectors. The Gα proteins are divided into four major classes (Gα q/11 , Gα s , Gα i/o and Gα 12/13 ), based on sequence similarity and their functional properties [3] and are predominantly associated with certain events in the signaling cascade, such as increase in intracellular Ca 2+ and IP 3 (Gα q/11 ), in-or decrease in cAMP level (Gα s , Gα i/o , respectively) or activation of Rho GTPase (Gα 12/13 ) [3,4]. By contrast, the effects of the Gβγ subunit are more diffuse [5,6]. Historically, GPCR signaling was assumed to occur via activation of a single class of Gα proteins, and therefore the receptors were typically classified accordingly [7]. The label-free DMR technology detects changes in the refractive index caused by mass redistribution inside a cell, triggered by receptor stimulation, relative to a baseline. Alteration of the refractive index is measured with a biosensor, integrated in each well of a microplate (adapted from Schröder et al. [38]). (B) Schematic summary of the signal transduction of H1-4Rs according to IUPHAR [56] and Panula et al. [57] (adapted from Panula et al. [57]). Canonical Gα protein signaling is indicated by solid lines. Involvement of secondary Gα proteins is indicated by dashed lines. AC, adenylyl cyclase; CAM, calcium-modulated protein; CTX, cholera toxin; DAG, diacylglycerol; IP3, inositol-1,4,5-trisphosphate; PI3Kγ, phosphoinositide 3-kinase-γ; PIP2, phosphatidylinositol-4,5-bisphosphate; PKA, protein kinase A; PKB, protein kinase B; PKC, protein kinase C; PLC-β, phospholipase C-β; PTX, pertussis toxin. The label-free DMR technology detects changes in the refractive index caused by mass redistribution inside a cell, triggered by receptor stimulation, relative to a baseline. Alteration of the refractive index is measured with a biosensor, integrated in each well of a microplate (adapted from Schröder et al. [38]). (B) Schematic summary of the signal transduction of H 1-4 Rs according to IUPHAR [56] and Panula et al. [57] (adapted from Panula et al. [57]). Canonical Gα protein signaling is indicated by solid lines. Involvement of secondary Gα proteins is indicated by dashed lines. AC, adenylyl cyclase; CAM, calcium-modulated protein; CTX, cholera toxin; DAG, diacylglycerol; IP 3 , inositol-1,4,5-trisphosphate; PI3Kγ, phosphoinositide 3-kinase-γ; PIP 2 , phosphatidylinositol-4,5-bisphosphate; PKA, protein kinase A; PKB, protein kinase B; PKC, protein kinase C; PLC-β, phospholipase C-β; PTX, pertussis toxin.
Histamine receptors (HRs) represent important drug targets in the treatment of disorders, such as allergy and reflux diseases [58]. They transmit their signals predominantly via three classes of G proteins: H 1 via Gα q/11 , H 2 via Gα s and H 3 + H 4 via Gα i/o ( Figure 1B). However, for the H 1 and H 2 receptors, evidence is emerging for promiscuous activation of Gα proteins [59][60][61]. By contrast, less information is available on the involvement of non-canonical G protein subunits in the signal transduction processes of the H 3,4 Rs. The first aim of the study was to establish a DMR assay for the entire histamine receptor family to compare the signaling patterns of the H 1-4 Rs in the same experimental setup. For this purpose, the four human receptor subtypes (hH 1-4 Rs) were stably expressed in HEK cells. HEK cells were chosen as they constitutively express the four relevant Gα classes (Gα s , Gα q/11 , Gα 12/13 and Gα i/o ) at comparable levels [62]. The contribution of G proteins to the integrated DMR response of hH 1-4 Rs was investigated by pursuing two different approaches. Firstly, in a classical pharmacological approach the G protein signaling pathways in HEK hH 1-4 R cells were silenced using G protein modulators (PTX, CTX, FR and gallein). Secondly, in a molecular biological approach CRISPR/Cas 9 modified Gα knock-out HEK cells (∆Gα x HEK) lacking either the Gα s/l (∆Gα s/l HEK) [63], the Gα q/11 (∆Gα q/11 HEK) [44] or the Gα 12/13 (∆Gα 12/13 HEK) [64] gene were stably transfected with hH 1-4 Rs. Moreover, cells lacking six Gα proteins (∆Gα s/l, q/11, 12/13 = ∆Gα six HEK) [41], stably expressing the hH 1-4 Rs were used. The results for both approaches were compared and discussed with respect to the impact of G protein inactivation on the hH 1-4 R mediated DMR response.

Characterization of HEK hH 1-4 R Cells
To investigate the effect of endogenously expressed G proteins on the DMR response, HEK hH 1-4 R cells were generated. For this purpose, the human histamine H 1 , H 2 , H 3 or H 4 receptor (hH 1-4 Rs) was inserted into a pIRESneo3 vector encoding the signaling peptide (SP) of the murine 5-HT 3A receptor and a FLAG tag to give the pIRESneo3-SP-FLAG-hH 1-4 R constructs. Both parental and ∆Gα x HEK cells were stably transfected with these constructs to give HEK hH 1-4 R and ∆Gα x HEK hH 1-4 R cells. For HEK hH 1-4 R cells, single clones of the stable transfectants were picked, selected, and screened by DMR for the highest signal elicited by 100 µM histamine (data not shown; (structure is presented in supplementary Figure S1). The expression of the hH 1-4 Rs in HEK cells was confirmed by radioligand saturation binding using live cells (Supplementary Figure S2). For the characterization of the ∆Gα x HEK hH 1-4 R cells see Section 2.4.1 and supplementary Figure S3. The expression levels of hH 1-4 Rs in HEK hH 1-4 R cells were calculated using B max and the specific activity (a s ) of the corresponding radioligand and the cell number (Table 1). Despite identical receptor cloning and transfection procedures of the hH 1-4 Rs, the expression level of hH 3 R and hH 4 R was lower compared to the hH 1 R and hH 2 R. The pK d values determined for the respective radioligands at HEK hH 1-4 R cells were in good agreement with literature data (Table 1).
To further characterize the HEK hH 1-4 R cells, radioligand competition binding experiments were performed with histamine (HIS) and one receptor-specific, inverse agonist (diphenhydramine (DPH) at the hH 1 R, famotidine (FAM) at the hH 2 R, pitolisant (PIT) at the hH 3 R and thioperamide (THIO) at the hH 4 R) using live cells (structures are presented in supplementary Figure S1). The displacement curves are shown in supplementary Figure S4 and the pK i values are summarized in Table 2. As expected, HIS had a markedly higher affinity to hH 3,4 Rs compared to hH 1,2 Rs (Table 2). In the literature, pK i values were determined either with cell membranes/homogenates or with live cells. In general, the pK i values determined for HIS and the inverse agonists using HEK hH 2-4 R cells were in the same range as reported in the literature with live cells (Supplementary Table S1). To the best of our knowledge, pK i values for HIS at the hH 1 R in live cells have not yet been reported. Compared to reference values from membranes/homogenates ranging between 4.3-5.9 [65][66][67], the affinity of HIS for the hH 1 R was lower in live cells (pK i = 3.37 ± 0.29).  (Figure 2A), where both the signal maximum and the time course varied depending on the HR subtype. Of note, no DMR response was detected in non-transfected HEK wildtype (wt) cells, neither for HIS nor for inverse agonists (Supplementary Figure S6A), demonstrating that the ligand induced DMR responses observed in HEK hH 1-4 R cells were HR mediated.
The highest amplitude and fastest increase in the DMR response was observed in HEK hH 1 R cells (1000 pm after 15 min; 10 µM HIS).
This kinetic profile of HIS induced DMR in HEK hH 1 R cells shows similarity to that observed in HeLa cells, which express the hH 1 R endogenously [71]. In HeLa cells, the positive DMR showed a peak response of approx. 300 pm within 3-5 min upon HIS addition, which decreased slightly and remained stable thereafter [71]. In A431 cells, which also express the hH 1 R endogenously, the positive DMR signal increased to a maximum value of approx. 500 pm within approx. 5 min after addition of HIS [72]. Afterwards, the DMR signal decreased steadily to the level of the baseline [72]. A similar kinetic profile was observed previously in our group using genetically engineered HEK293T-CRE-Luc-H 1 R-hMSR1 cells where the hH 1 R was co-expressed with the human macrophage scavenger receptor 1 (hMSR1), introduced to enhance the adhesion of HEK cells [20]. In HEK293T-CRE-Luc-H 1 R-hMSR1 cells, the positive DMR peaked at approx. 600 pm within 10 min after HIS addition and gradually decreased afterwards back to baseline [20,73]. These disparate kinetic profiles observed after stimulation of the hH 1 R with HIS were not surprising, as many characteristics of the different cell models used, e.g., receptor expression, expression patterns of (G) proteins, and/or cell adhesion, can affect the kinetic profile of the DMR response [73,74]. When comparing the kinetics of HIS in HEK hH 1 R cells with DMR traces of purinergic P2Y or muscarinic M3 receptors (both also Gα q coupled and heterologously expressed in HEK cells) [44], no similarities were found.
This kinetic profile of HIS induced DMR in HEK hH1R cells shows similarity to that observed in HeLa cells, which express the hH1R endogenously [71]. In HeLa cells, the positive DMR showed a peak response of approx. 300 pm within 3-5 min upon HIS addition, which decreased slightly and remained stable thereafter [71]. In A431 cells, which also express the hH1R endogenously, the positive DMR signal increased to a maximum value of approx. 500 pm within approx. 5 min after addition of HIS [72]. Afterwards, the DMR signal decreased steadily to the level of the baseline [72]. A similar kinetic profile was observed previously in our group using genetically engineered HEK293T-CRE-Luc-H1R-hMSR1 cells where the hH1R was co-expressed with the human macrophage scavenger receptor 1 (hMSR1), introduced to enhance the adhesion of HEK cells [20]. In HEK293T-CRE-Luc-H1R-hMSR1 cells, the positive DMR peaked at approx. 600 pm within 10 min after HIS addition and gradually decreased afterwards back to baseline [20,73]. These disparate kinetic profiles observed after stimulation of the hH1R with HIS were not surpris- The histamine induced DMR responses were reversible in HEK hH 1-4 R cells. The HEK hH 1-4 R cells were pre incubated with histamine at concentrations corresponding to the respective pEC 80 value (hH 1 R = 316 nM, hH 2 R = 794 nM, hH 3 R = 1995 nM, hH 4 R = 501 nM, indicated by the filled arrow ) and the DMR response was recorded for 60 min (hH 1-3 R) or for 40 min (hH 4 R). Subsequently, a receptor specific antagonist was added (hH 1 R 10 µM MEP, hH 2 R 10 µM DE257, hH 3 R 10 µM THIO, hH 4 R 10 µM JNJ, empty arrow ) and the DMR was recorded for additional 60 min. (C) Constitutive activity was detected in HEK hH 1-4 R cells. Inverse agonism was observed at the hH 1 R for DPH, at the hH 2 R for FAM, at the hH 3 R for PIT, at the hH 4 R for THIO. Traces shown in (A-C) were corrected for the buffer and represent mean ± SEM of the technical triplicate. Traces shown in (B) were additionally normalized to the value recorded after 60 min (100%).
Compared to HEK hH 1 R cells, the DMR response recorded for HEK hH 2 R and HEK hH 3 R cells were markedly different, showing no sharp maxima upon stimulation with HIS at a concentration of 10 or 100 µM within 60 min. Instead, the DMR signal increased slower, but steadily, reaching a highest amplitude ranging between 500-600 pm after 60 min. A unique feature of the hH 2 R mediated DMR response was a slight signal dip (Zoom-in in supplementary Figure S5) immediately after HIS addition, a phenomenon that was not observed within this study for any other HR subtype under the same experimental conditions. A signal dip was also observed for the Gα s coupled GPCRs [29,37], e.g., EP2/4, which was stably expressed in HEK cells [37]. Ye Fang [29] explained such a signal dip by the fact that downstream signaling components involved in the signal transduction process are already compartmentalized and located at or near the cell membrane. Therefore, the recruitment of intracellular signal transduction components to activated receptors is less pronounced and other cellular signaling events are more salient leading to an initial decrease in local mass density [29]. However, one should be careful to interpret this as a reliable feature of Gα s coupling.
Although the hH 2 R is reported as Gα s coupled [57] and the hH 3 R is described as a Gα i/o coupled receptor [57], the DMR traces recorded upon stimulation with HIS were similar in both signal amplitude and time course, except for the signal dip in the case of the hH 2 R (Figure 2A). This was surprising as we had expected that different G protein coupling would be associated with distinct DMR signaling profiles. Moreover, it was interesting that the signal amplitudes were similar because the expression level of the hH 3 R was approximately 20-fold lower compared to that of hH 2 R (Table 1), conflicting with the assumption that the signal amplitude is positively correlated with the level of receptor expression. Instead, it can be speculated that the receptor-specific signal transduction pathway plays a role.
Even though both the hH 3,4 Rs are structurally related and considered as Gα i/o coupled receptors ( Figure 1B), the recorded DMR traces of HEK hH 3 R and hH 4 R cells differed in both time course and signal amplitude ( Figure 2A). Among the four human HR subtypes analyzed in this study, the lowest DMR response was recorded in HEK hH 4 R cells. The signal reached its maximum of approx. 300-400 pm within 10-20 min at the highest HIS concentration of 10 µM, and then declined continuously. Various Gα i/o coupled receptors expressed in HEK cells (DP2 [41], CRTH2 [44]) or in CHO cells (NOP [75]) showed comparable kinetic profiles in DMR assays.

Reversibility of HIS Induced DMR
To demonstrate the reversibility of the DMR responses, HEK hH 1-4 R cells were first treated with histamine at concentrations corresponding to the respective pEC 80 value (hH 1 R = 316 nM, hH 2 R = 794 nM, hH 3 R = 1995 nM, hH 4 R = 501 nM; indicated by the filled arrow in Figure 2B) and the DMR was recorded for 60 min with HEK hH 1,2,3 R cells, or for 40 min with HEK hH 4 R cells. In the second step, a receptor-specific antagonist was added (10 µM mepyramine (MEP) for hH 1 R, 10 µM DE257 for hH 2 R, 100 µM thioperamide (THIO) for hH 3 R and 10 µM JNJ7777120 (JNJ) for hH 4 R; indicated by the empty arrow in Figure 2B; structures of antagonists are presented in supplementary Figure S1). As a control, HEK hH 1-4 R cells were also stimulated with HIS, but in the second step, instead of an antagonist, HIS was added at a concentration corresponding to the pEC 80 . This was to ensure that the observed effect was induced by the antagonist and not by the addition procedure disturbing the system. For all four HR subtypes, the HIS-induced signal was suppressed by addition of a receptor subtype-specific antagonist, and no decrease in the signal was observed in the controls, showing reversibility of the DMR signal.

Constitutive Activity
Previously, all four HR subtypes have been reported as constitutively active in heterologous expression systems in canonical assays [23,[76][77][78][79][80][81]. Constitutive (basal) activity describes the ability of GPCRs to produce a biological response in the absence of agonist binding by spontaneously adopting an active conformation [82]. Usually, the measurement of constitutive activity occurs by comparing the basal activity of a system comprising active-state receptors (e.g., transfected cells or high receptor expression) and without receptors (e.g., not transfected cell, low receptor expression) [83]. The basal activity should increase with increase in receptor expression. To assess the constitutive activity of HRs in the DMR assay we compared the DMR traces of the buffer controls (assay buffer w/o ligand) recorded for not transfected HEK cells with that recorded for HEK hH 1-4 R cells (Supplementary Figure S7). After an equilibration period of about 40 min, higher basal activity was measured in HEK hH 1,3,4 Rs compared to not transfected HEK wt cells. We interpret this as an indication that the receptors in this system are constitutively active. However, no difference in the basal activity was observed between HEK hH 2 R cells and HEK wt cells (Supplementary Figure S7). This may imply that the hH 2 R is either not constitutively active in this system, or that this activity is too weak to be detected in this system. To explore measurement of inverse agonism by DMR, HEK hH 1-4 R cells were stimulated with a receptor specific inverse agonist (hH 1 R: DPH, hH 2 R: FAM, hH 3 R: PIT, hH 4 R: THIO) at increasing concentrations. Constitutive activity, manifesting as negatively deflected DMR traces, was observed at all four receptor subtypes, differing in intensity depending on the HR-ligand combination. ( Figure 2C). The weakest inverse activity was measured for the hH 2 R when stimulated with FAM. This implies that the hH 2 R is constitutively active, but much lower compared to hH 1,3,4 R. We previously anticipated this to be the case in view of supplementary Figure S7. We can rule out off-target effects for any HR-ligand combination, as none of the ligands elicited a DMR response in not transfected HEK wt cells (Supplementary Figure S6A).

Assay Quality
For data analysis, the area under curve (AUC) was calculated for the DMR traces, which is a commonly applied concept for the assessment of dynamic pharmacological processes [38,73]. Compared to single point measurements, the integration over time provides a more accurate estimate of the overall response to a drug [84]. To assess assay quality, signal-to-background (S/B) ratios were estimated based on AUC over the entire measurement period of 60 min (AUC 60 ) for both HIS and a respective inverse agonist using HEK hH 1-4 R cells. We are aware that the calculation of the S/B ratio using AUC appears problematic as the DMR signal does not represent an absolute measure, but rather a shift of the wavelength relative to the baseline. To alleviate this problem, we considered the stable baseline as the zero point and used the modulus of AUC for the estimation of the S/B ratio. This approximation is possible because the EnSpire software records and uses the last measuring point (repeat) in the baseline run as the calibration offset, which is subtracted from all repeats of the baseline and the final record (last repeat in the baseline was set to zero) [85].
For HIS, high S/B ratios were determined at the hH 1 R, hH 2 R, hH 3 R and hH 4 R, amounting to 308, 277, 218 and 123, respectively ( Figure 3). Compared to HIS, the S/B ratios for the standard inverse agonists were markedly lower (S/B ratios: DPH (hH 1 R) = 53, FAM (hH 2 R) = 33, PIT (hH 3 R) = 25 and THIO (hH 4 R) = 30) as shown in Figure 3. In comparison, S/B ratios for HIS in [ 35 S]GTPγS or miniG assays ranged from 2 to 30 [80]. Among other factors, high S/B ratios are beneficial for signal deconvolution studies, assessing efficacies and potencies of ligands, and investigating constitutive activities of receptors.

Conversion of the DMR Responses to Concentration-Response-Curves (CRCs)
The optical traces (representations in Figure 2A, C) were converted to CRCs by calculating the AUC 60 and plotting these values against the logarithmic concentrations of a compound ( Figure 4A). The determined pEC 50 and E max values are summarized in Table 2. However, when calculating the S/B ratios, a slight dependency on the time interval used for the AUC calculations was observed (described in SM Text S1, Impact of the time interval used for calculations of AUC on S/B ratios, supplementary Figure S8 and supplementary  Table S3). Moreover, a time-dependent potency of agonists was observed at the muscarinic M 3 [86] and the neurotensin NTS 1 [53] receptor in DMR assays. Therefore, we investigated whether the time interval used to calculate the AUC had an impact on the pEC 50 and E max values of HIS and the receptor specific inverse agonists. and the respective inverse agonist (10 µM DPH in HEK hH1R cells, 10 µM FAM in HEK hH2R cells, 10 µM PIT in HEK hH3R cells, 100 µM THIO in HEK hH4R cells), and divided by the respective AUC60 value determined for the buffer. For HIS, the positive AUC (above baseline) was used, whereas for the inverse agonists the negative AUC (below baseline) was calculated. The S/B ratios are presented as mean ± SEM from at least three independent experiments, each performed in triplicate.

Conversion of the DMR Responses to Concentration-Response-Curves (CRCs)
The optical traces (representations in Figure 2A, C) were converted to CRCs by calculating the AUC60 and plotting these values against the logarithmic concentrations of a compound ( Figure 4A). The determined pEC50 and Emax values are summarized in Table  2. However, when calculating the S/B ratios, a slight dependency on the time interval used for the AUC calculations was observed (described in SM Text S1, Impact of the time interval used for calculations of AUC on S/B ratios, supplementary Figure S8 and supplementary Table S3). Moreover, a time-dependent potency of agonists was observed at the muscarinic M3 [86] and the neurotensin NTS1 [53] receptor in DMR assays. Therefore, we investigated whether the time interval used to calculate the AUC had an impact on the pEC50 and Emax values of HIS and the receptor specific inverse agonists.   60 ) was calculated for the highest concentrations of HIS (10 µM in HEK hH 1,2,4 R cells and 100 µM in HEK hH 3 R cells) and the respective inverse agonist (10 µM DPH in HEK hH 1 R cells, 10 µM FAM in HEK hH 2 R cells, 10 µM PIT in HEK hH 3 R cells, 100 µM THIO in HEK hH 4 R cells), and divided by the respective AUC 60 value determined for the buffer. For HIS, the positive AUC (above baseline) was used, whereas for the inverse agonists the negative AUC (below baseline) was calculated. The S/B ratios are presented as mean ± SEM from at least three independent experiments, each performed in triplicate. Figure 3. Signal-to-background (S/B) ratios estimated for HIS and inverse agonists using HEK hH1-4R cells. For the estimation of S/B values, the modulus of area under curve (AUC60) was calculated for the highest concentrations of HIS (10 µM in HEK hH1,2,4R cells and 100 µM in HEK hH3R cells) and the respective inverse agonist (10 µM DPH in HEK hH1R cells, 10 µM FAM in HEK hH2R cells, 10 µM PIT in HEK hH3R cells, 100 µM THIO in HEK hH4R cells), and divided by the respective AUC60 value determined for the buffer. For HIS, the positive AUC (above baseline) was used, whereas for the inverse agonists the negative AUC (below baseline) was calculated. The S/B ratios are presented as mean ± SEM from at least three independent experiments, each performed in triplicate.

Conversion of the DMR Responses to Concentration-Response-Curves (CRCs)
The optical traces (representations in Figure 2A, C) were converted to CRCs by calculating the AUC60 and plotting these values against the logarithmic concentrations of a compound ( Figure 4A). The determined pEC50 and Emax values are summarized in Table  2. However, when calculating the S/B ratios, a slight dependency on the time interval used for the AUC calculations was observed (described in SM Text S1, Impact of the time interval used for calculations of AUC on S/B ratios, supplementary Figure S8 and supplementary Table S3). Moreover, a time-dependent potency of agonists was observed at the muscarinic M3 [86] and the neurotensin NTS1 [53] receptor in DMR assays. Therefore, we investigated whether the time interval used to calculate the AUC had an impact on the pEC50 and Emax values of HIS and the receptor specific inverse agonists.   60 . The E max values determined for the inverse agonists wee normalized to the highest histamine concentration applied for the respective receptor subtype. (B) pEC 50 values for HIS resulting from CRCs constructed by using AUC 20 , AUC 40 or AUC 60 at the hH 1-4 Rs. (C) pEC 50 values calculated for inverse agonists (DPH at the hH 1 R, FAM at the hH 2 R, PIT at the hH 3 R and THIO at the hH 4 R) resulting from CRCs constructed by using AUC 20 , AUC 40 or AUC 60 at the hH 1-4 Rs. (D) E max values determined for DPH at the hH 1 R, FAM at the hH 2 R, PIT at the hH 3 R and THIO at the hH 4 R using the AUC 20 , AUC 40 or AUC 60 . E max values were normalized to the highest HIS concentration applied for the corresponding HR subtype. (A-D) All values are means ± SEM of at least three independent experiments, each performed in triplicate. Statistical difference relative to AUC 60 was analyzed by one-way ANOVA followed by Dunnett's multiple comparison test calculated as * p ≤ 0.05, ** p ≤ 0.01.
For this purpose, additional CRCs were constructed using AUC calculations after 20 or 40 min (AUC 20,40 ) and compared to those from AUC 60 (Supplementary Figure S9  and supplementary Table S4). For HEK hH 1 R cells, the time interval had no impact on the mean pEC 50 values for HIS ( Figure 4B). By contrast, in HEK hH 2 R and hH 4 R cells a significant increase in pEC 50 values from AUC 20 to AUC 60 was observed for HIS (hH 2 R: from 6.30 ± 0.05 to 6.57 ± 0.05; hH 4 R: from 6.99 ± 0.05 to 7.15 ± 0.05, respectively), whereas in HEK hH 3 R a gradual decrease in mean pEC 50 values was observed from AUC 20 to AUC 60 (from 6.66 ± 0.07 to 6.49 ± 0.06), which, however, was statistically not significant. For inverse agonists, the calculation of AUC after 20, 40 and 60 min had no significant impact on the mean pEC 50 values ( Figure 4C). Signal transduction of GPCRs involves a complex network of different spatially and temporally resolved events, each of which show individual kinetics and/or amplitudes [53,86,87]. As all this information is bundled in the DMR response, it was not surprising that the temporal component could have an impact on the pEC 50 and E max value depending on the specific signaling cascade triggered by the receptor ligand interaction. The E max values gradually decreased from AUC 20 to AUC 60 for all four HR-inverse agonist combinations ( Figure 4D; exact values in supplementary Table S4) but particularly for THIO at hH 4 R, where the mean E max value showed a significant decrease from AUC 20 to AUC 60 (E max (AUC 20 ) = −20.1 ± 5.0 to E max (AUC 60 ) = −45.0 ± 5.7). The slow kinetics of the DMR response recorded for the inverse agonists can be considered as an explanation here (Figure 2A (HIS) versus Figure 2C (inverse agonists). In view of these results, the inclusion of the entire kinetic information (AUC 60 ) appears preferable and was considered as the standard method to calculate pEC 50 and E max values in the following experiments.

Functional Characterization of (Inverse) Agonists: Label-Free DMR versus Label-Dependent Techniques
There was a discrepancy between competition binding and DMR functional data determined for HIS using HEK hH 1,2 R cells ( Table 2). The pK i values for HIS in live cells were approximately 4 (hH 1 R) or 2 (hH 2 R) orders of magnitude lower compared to the pEC 50 values in the DMR assay. Moreover, a discrepancy between affinity and potency was observed for pitolisant (PIT) at the hH 3 R, where the pK i value was about 2 orders of magnitude larger compared to the pEC 50 value in the DMR assay. As binding data reflect the strength of the receptor-ligand interaction, whereas functional responses are amplified translations of the receptor-ligand interaction, differences in this range are not uncommon and have been reported for example, for dopamine receptors [88]. We have previously stimulated HEK293T-CRE-Luc-hH 1 R-hMSR1 cells with HIS at increasing concentrations in the DMR assay [20]. The CRCs from AUC 40 revealed a pEC 50 value of 7.49 [20], which agrees with the result reported here (pEC 50 = 7.38 ± 0.05).  50 and E max values were determined by converting the DMR traces to CRCs using the positive AUC 60 for HIS or the negative AUC 60 for the inverse agonists (DPH at the hH 1 R, FAM at the hH 2 R, PIT at the hH 3 R and THIO at the hH 4 R). These values were subsequently normalized to AUC 60 for the buffer (0%) and the respective highest histamine concentration (100%). The negative sign of E max values for inverse agonists implies a negative deflection of the originate DMR traces ( Figure 2C). All values represent means ± SEM of at least three independent experiments, each performed in triplicate. n. d. means not determined.
To the best of our knowledge, we are the first to report functional DMR data for the hH 2-4 Rs, so no reference data was available. In order to compare the results, a miniG recruitment assay, recently implemented by Hoering et al. [80] for the entire HR family, was used. As the miniG recruitment assay was also performed with live HEK cells in real time, and the AUC used for data analysis, these results were particularly well suited as a reference. As a canonical alternative, a luciferase reporter gene assay was used. This assay was also performed with HEK cells but represents an endpoint measurement, in contrast to the kinetic measurements of DMR and miniG recruitment assays. Although the three assays measure different processes in the signal transduction cascade of HRs, in general, the pEC 50 values were in good agreement, not differing more than one order of magnitude ( Table 2). Exceptions are HIS at the hH 1 R (DMR vs. miniG) and HIS at the hH 3 R (DMR vs. luciferase). By contrast, higher discrepancies were observed regarding the efficacy of the inverse agonists. In general, inverse agonists were less efficacious in the miniG recruitment assay than in the DMR assay. However, as only one miniG protein-HR interaction was monitored rather than the holistic cellular response as in the DMR assay, this discrepancy is not surprising. A better agreement of E max values was observed between the DMR and the luciferase reporter gene assay for THIO at the hH 4 R.

Impact of Individual Gα Protein Modulators on the DMR Response
As outlined above, depending on the HR subtype, different intensities and time courses of the DMR responses were observed when HEK hH 1-4 R cells were stimulated with HIS at increasing concentrations ( Figure 2A). We investigated whether the receptorspecific DMR response was exclusively the result of an activation of the primary Gα protein dependent signaling pathway described in the literature ( Figure 1B), or whether additional G proteins were involved in the HIS-induced DMR response. The contribution of endogenously expressed Gα proteins was analyzed using G protein pathway modulators FR900359 (FR), pertussis toxin (PTX), and cholera toxin (CTX; mechanisms for all three outlined in Figure 5A). CRCs were recorded for HIS in HEK hH 1-4 R cells in the absence and presence of CTX, PTX (both at concentrations of 1.00, 10.0 and 100 ng/mL) and FR (at concentrations of 0.01, 0.10 and 1.00 µM). In every experiment, HEK hH 1-4 R cells stimulated with HIS without (w/o) modulators served as 100% control. DMR traces recorded at the highest histamine concentration in the absence and presence of the respective modulator were compared ( Figure 5B) and, as before, AUC 60 CRCs were constructed (Supplementary Figure S10). The corresponding E max and pEC 50 values are summarized in supplementary Figure S11.
• hH 1 R Because the Gα q/11 pathway is considered canonical for the hH 1 R [56,57], a strong decline of the DMR response was expected upon incubating the HEK hH 1 R cells with the Gα q/11 modulator FR. When HEK hH 1 R cells were treated with 1.00 µM FR, the time course of the DMR signal for HIS was noticeably altered, but not with 0.01 µM or 0.1 µM FR (5B green traces). In the former case, no maximum was observed and the DMR response was slower. However, even the highest FR concentration of 1.00 µM was not sufficient to eradicate the HIS DMR response ( Figure 5B, green traces), although the E max value was reduced to 41 ± 9.5% ( Figure 6A). Likewise, Lieb et al. were also not able to completely suppress the HIS induced DMR in HEK293T-CRE-Luc-hH 1 R-hMSR1 cells in the presence of 1.00 or 10.0 µM FR [20]. For comparison, a concentration of 1.00 µM FR was enough to completely disrupt the DMR response of the muscarinic M 3 R, which solely couples to Gα q/11 [44]. Thus, we conclude that the failure to completely suppress the DMR signal was not due to insufficient FR concentration, but rather that the residual signal in HEK hH 1 R cells comes from additional (G) protein interactions. The significantly reduced pEC 50 in the presence of 1.00 µM FR (6.81 ± 0.15) could be caused by inactivation of the Gα q/11 protein abolishing the Gα q/11 positive modulation, an effect seen when the G protein stabilizes the active conformation of the receptor [24,90].  [18,41,[44][45][46][47]. PTX selectively and irreversibly silences Gαi/o at a concentration of 100 ng/mL by ADP−ribosylation at the Gα−subunit [20,33,37,[40][41][42]. CTX locks the Gαs protein in its GTP bound state by irreversible ADP-ribosylation leading to a permanent activation of the Gαs protein, which is in turn uncoupled and no longer available for the GPCR [31][32][33]37,43] at a concentration of 100 ng/mL [31,32,37]. As this approach only masks the Gαs protein coupled pathway the results should be interpreted with caution. Gallein (gal) is reported to reversibly bind to the Gβγ subunit (Kd = 422 nM) [92], preventing an interaction with effector proteins [92][93][94]. (B) Representative time courses of the HIS induced DMR response in HEK hH1−4R cells pre−treated with G protein modulator at the indicated concentrations overnight (PTX and CTX) or 30 min (FR and gallein) before measurement of stimulation with HIS (hH1,2,4R at 10 µM HIS, hH3R at 100 µM HIS). All traces were buffer-corrected and normalized to the maximum DMR response (wavelength shift in pm) of the untreated control (w/o). Data are presented as mean ± SEM of a technical triplicate.
• hH2R Pretreatment of HEK hH2R cells with increasing CTX concentrations led to a gradual decrease in the signal amplitude relative to the untreated control, but, in contrast to HEK  [20,33,37,[40][41][42]. CTX locks the Gα s protein in its GTP bound state by irreversible ADP-ribosylation leading to a permanent activation of the Gα s protein, which is in turn uncoupled and no longer available for the GPCR [31][32][33]37,43] at a concentration of 100 ng/mL [31,32,37]. As this approach only masks the Gα s protein coupled pathway the results should be interpreted with caution. Gallein (gal) is reported to reversibly bind to the Gβγ subunit (K d = 422 nM) [92], preventing an interaction with effector proteins [92][93][94]. (B) Representative time courses of the HIS induced DMR response in HEK hH 1-4 R cells pre − treated with G protein modulator at the indicated concentrations overnight (PTX and CTX) or 30 min (FR and gallein) before measurement of stimulation with HIS (hH 1,2,4 R at 10 µM HIS, hH 3 R at 100 µM HIS). All traces were buffer-corrected and normalized to the maximum DMR response (wavelength shift in pm) of the untreated control (w/o). Data are presented as mean ± SEM of a technical triplicate. Surprisingly, masking of the Gα s signaling pathway with CTX had a greater effect on the DMR response of the hH 1 R ( Figure 5B, orange traces) than FR. Even the lowest concentration of 1.00 ng/mL CTX enormously altered both the maximum amplitude, and the time course of the HIS induced DMR response. In this case, the DMR response was slowed down and showed no signal maximum as observed for untreated HEK hH 1 R cells. An increase in CTX concentration to 100 ng/mL further reduced the signal amplitude and led to a deceleration of the DMR signal. Unexpectedly, among the investigated modulators, 100 ng/mL CTX had the strongest effect on E max at the hH 1 R lowering the value to 23 ± 4.9% ( Figure 6A), suggesting that the Gα s protein is involved in the hH 1 R mediated DMR signal. Indeed, it has been shown that the hH 1 R can functionally interact with the Gα s protein in HEK cells overexpressing both the receptor and the Gα s protein [60,91]. The inhibition of Gα s pathway led to a significant increase in the pEC 50 value (7.87 ± 0.19; Figure 6B). It is possible that the uncoupling of Gα s may have enhanced Gα q protein interaction with the hH 1 R, or Gα s may even act as a negative modulator at hH 1 R. Further investigations are necessary to determine the mechanism involved. hH1R cells, did not alter the shape of the DMR time course ( Figure 5B, orange traces). At 100 ng/mL CTX, 62 ± 7.7% of the hH2R signal was retained; a significant effect, but not as pronounced as with the other three HR subtypes ( Figure 6A; hH1R 23 ± 4.9%, hH3R 54 ± 7.6% and hH4R 35 ± 7.9% signal retention). This was unexpected, as the hH2R is commonly considered as a Gαs-coupled receptor [56,95]. Furthermore, 100 ng/mL CTX have been shown to almost completely abolish the agonist induced DMR response of the Gαs-sensitive β2 adrenoreceptor (β2R) expressed by different cell types endogenously or heterologously [31,32,37]. Moreover, the pEC50 value of HIS remained unaffected by the treatment with CTX ( Figure 6B). We expected that uncoupling of the Gαs protein with CTX would negatively affect the pEC50 value of HIS, as was the case with hH1R after the Gαq/11 protein was inactivated by FR. These data suggest that additional signaling pathways contribute to the DMR response in HEK hH2R cells. Scatter plot of the pEC50 values in absence (grey) and presence of G protein modulators at the concentration stated above. The pEC50 were determined by plotting the AUC60 against the respective HIS concentration. (A, B) Data presented are means ± SEM of at least three independent experiments, each performed in triplicate. Statistical difference relative to the control was analyzed by one-way ANOVA followed by Dunnett's multiple comparison test. Significance levels are indicated by asterisks (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).
Apart from Gαs, it is known that the Gαq/11 protein can play a considerable role in H2R signal transduction, dependent on the cellular background [56]. This was not confirmed in the DMR assay as the Gαq/11 modulator FR was almost completely ineffective, even at a concentration of 1.00 µM ( Figure 5B green traces). Although a stepwise decline of the DMR response was observed with increasing PTX concentrations to investigate the involvement of Gαi/o in the HIS induced DMR ( Figure 5B blue traces), the effect was less The inhibition of Gα i/o signaling pathway with PTX reached its maximum effect at a concentration of 10.0 ng/mL at the hH 1 R ( Figure 5B, blue traces). Except for decreasing the signal amplitude to maximum 50 ± 9.3% of E max relative to control cells ( Figure 6A), PTX had no effect on the time course of the DMR signal, suggesting Gα i/o protein involvement in hH 1 R signal transduction. This is in good accordance with the literature [20,59,61]. For example, Lieb et al. showed that the hH 1 R also signals via Gα i/o in the DMR assay using HEK293T-CRE-Luc-hH 1 R-hMSR1, as the DMR signal was completely abolished by 100 ng/mL PTX [20].
• hH 2 R Pretreatment of HEK hH 2 R cells with increasing CTX concentrations led to a gradual decrease in the signal amplitude relative to the untreated control, but, in contrast to HEK hH 1 R cells, did not alter the shape of the DMR time course ( Figure 5B, orange traces). At 100 ng/mL CTX, 62 ± 7.7% of the hH 2 R signal was retained; a significant effect, but not as pronounced as with the other three HR subtypes ( Figure 6A; hH 1 R 23 ± 4.9%, hH 3 R 54 ± 7.6% and hH 4 R 35 ± 7.9% signal retention). This was unexpected, as the hH 2 R is commonly considered as a Gα s -coupled receptor [56,95]. Furthermore, 100 ng/mL CTX have been shown to almost completely abolish the agonist induced DMR response of the Gα s -sensitive β 2 adrenoreceptor (β 2 R) expressed by different cell types endogenously or heterologously [31,32,37]. Moreover, the pEC 50 value of HIS remained unaffected by the treatment with CTX ( Figure 6B). We expected that uncoupling of the Gα s protein with CTX would negatively affect the pEC 50 value of HIS, as was the case with hH 1 R after the Gα q/11 protein was inactivated by FR. These data suggest that additional signaling pathways contribute to the DMR response in HEK hH 2 R cells.
Apart from Gα s , it is known that the Gα q/11 protein can play a considerable role in H 2 R signal transduction, dependent on the cellular background [56]. This was not confirmed in the DMR assay as the Gα q/11 modulator FR was almost completely ineffective, even at a concentration of 1.00 µM ( Figure 5B green traces). Although a stepwise decline of the DMR response was observed with increasing PTX concentrations to investigate the involvement of Gα i/o in the HIS induced DMR ( Figure 5B blue traces), the effect was less pronounced than with CTX (E max = 77 ± 4.2% at 100 ng/mL PTX versus E max = 62 ± 7.7%; Figure 6A). Strikingly, in contrast to the other three HR subtypes, the individual modulators FR, CTX and PTX, had little effect on the HIS induced DMR response in HEK hH 2 R cells. Two explanations can be considered. Firstly, silencing of one pathway may have caused the hH 2 R to switch to other pathways, indicating promiscuous signal transduction of the receptor. Secondly, these results may also indicate the involvement of other effectors, e.g., Gα z or Gα 12/13 [59], in the hH 2 R mediated DMR response.
• hH 3 R As expected, inhibition of the Gα i/o signaling pathway with PTX in HEK hH 3 R cells had a dramatic impact on the DMR response to 100 µM HIS, for both the E max and pEC 50 values. Even 1.00 ng/mL of PTX was sufficient to decelerate the hH 3 R DMR response ( Figure 5A, blue traces) and to reduce the E max to 63 ± 14% (Figure 6A), roughly a 4× more reduction than for hH 1,2 Rs. However, we failed to completely suppress the signal, as at 100 ng/mL PTX 32 ± 7.2% of E max remained. By contrast, Shi et al. described that the HIS response was disrupted by 100 ng/mL of PTX in a CRE-driven luciferase activity assay using HEK cells stably expressing the hH 3 R [42]. Moreover, for other Gα i/o coupled receptors, e.g., the muscarinic M 2 [48], or prostaglandin CRTH 2 [37], PTX at a concentration of 100 ng/mL was sufficient to completely disrupt the DMR signal in CHO or HEK cells. Thus, we expect that 100 ng/mL PTX was sufficient to inactivate Gα i/o mediated signaling and conclude that other (G) proteins were involved in the hH 3 R mediated DMR response. The pEC 50 values declined with increasing PTX concentrations from 6.49 ± 0.06 (control) to 5.75 ± 0.17 and 5.91 ± 0.15 (10.0 and 100 ng/mL of PTX, respectively; Figure 6B). As described above, a similar phenomenon was observed for the hH 1 R when its canonical Gα q/11 signaling pathway was blocked with 1 µM FR. We believe the same hypothesis to be true here, namely that the Gα i/o protein stabilizes an active conformation of the hH 3 R, resulting in decreased pEC 50 values when blocked. Consistent with literature [57], this suggests that the Gα i/o protein plays a major role in hH 3 R mediated signal transduction.
However, as it was not possible to completely abrogate the DMR response with PTX, other G protein (in)dependent signaling pathways might be involved as well.
The Gα q/11 modulator FR at increasing concentrations had no effect on the time course of the HIS induced DMR response, but did decrease the signal amplitude ( Figure 5B, green traces). A decrease in E max to about 95 ± 5.3% was observed in the presence of 0.01 µM FR, whereas 0.10 µM FR significantly reduced the E max value to 60 ± 6.4%. A ten-fold increase in FR concentration to 1.00 µM decreased the E max by only additional 3% compared with 0.10 µM FR, indicating that at the latter concentration of FR the Gα q/11 dependent DMR was almost completely inhibited in HEK hH 3 R cells ( Figure 6A). Strikingly, the pEC 50 value was significantly increased to 7.20 ± 0.05 after treatment with 1.00 µM FR referring to the pEC 50 of 6.49 ± 0.06 in control cells ( Figure 6B). We did not expect this impact of Gα q/11 inhibition because hitherto the hH 3 R has been described as a Gα i/o selective receptor and to date, no evidence has been provided that the hH 3 R is capable of activating a Gα protein other than Gα i/o [91].
Masking the Gα s signaling with CTX did not affect the time course of the HIS-induced hH 3 R mediated DMR response elicited by HIS, but again the signal amplitude was affected ( Figure 5B). The E max values decreased to 77 ± 9.0% or 76 ± 6.3% after treatment with 1.00 or 10.0 ng/mL of CTX, respectively and was significantly reduced to 54 ± 7.61% in the presence of 100 ng/mL CTX compared to control cells ( Figure 6A). In comparison, the E max value at the hH 1 R was already reduced to 43 ± 9.5% at a concentration of 1 ng/mL of CTX. Therefore, we reason that Gα s is not as involved in signal transduction at the hH 3 R as at the hH 1 R. This assumption was further supported by the fact that the pEC 50 value was not significantly affected by the treatment with CTX ( Figure 6B).

• hH 4 R
Pre-incubation of HEK hH 4 R cells with PTX at increasing concentrations to block the Gα i/o protein had no influence on the time course but did affect the signal amplitude of the HIS induced DMR response ( Figure 5B). Similar to the hH 3 R response, even at 1 ng/mL PTX the E max was lowered to 51 ± 6.8% ( Figure 6A). However, we failed to completely displace the HIS induced DMR response at the hH 4 R by PTX, even at a concentration of 100 ng/mL ( Figure 6A; E max = 38 ± 2.9%). Elsewhere, in a luciferase reporter gene assay with HEK293-EBNA cells transfected with the hH 4 R (referred to as GPRv53), 100 ng/mL PTX completely abolished the HIS induced response [96]. However, unlike the hH 3 R response, an increase in PTX concentration had no effect on pEC 50 values in HEK hH 4 R cells ( Figure 6B).
The contribution of the Gα s protein was analyzed by pre-treating the cells with CTX at increasing concentrations. Figure 5B (hH 4 R orange traces) shows that the maximum responses declined stepwise, whereas time courses of the HIS induced DMR remained unaltered ( Figure 5B). Only at the highest CTX concentration of 100 ng/mL, did the signal decrease substantially (E max = 35 ± 7.9%; Figure 6A). Analogous to the hH 3 R, we assume that Gα s is of smaller importance in the signal transduction of the hH 4 R compared to the hH 1 R, where the E max value was reduced to 43 ± 9.5% with 1 ng/mL CTX. Moreover, the pEC 50 value for HIS in HEK hH 4 R cells was not affected in the presence of CTX ( Figure 6B). The Gα q/11 modulator FR at increasing concentrations led to a stepwise decrease in the hH 4 R mediated DMR response. FR at a concentration of 0.10 µM was sufficient to decrease the DMR signal to 71 ± 18% relative to the untreated control, and a further decline was observed in the presence of 1.00 µM FR (E max = 47 ± 4.5%; Figure 6A). Different to the hH 3 R, the pEC 50 value was unaltered by the blockage of the Gα q/11 protein with FR ( Figure 6B).

Impact of the Gβγ Protein Modulator Gallein on the DMR Response upon Stimulation with Histamine
In addition to Gα, the Gβγ dimer is also able to interact with effectors in the signal transduction process. A contribution of Gβγ to the DMR response was assessed by means of the small modulatory molecule gallein [92][93][94]. Pretreatment of HEK hH 1-3 R cells with 20 µM gallein prior to stimulation with HIS led only to a marginal reduction of the E max value to approximately 85% compared to control cells ( Figure 5B, red traces and Figure 6A). In the case of the hH 4 R, the same gallein concentration significantly reduced the E max value to 71 ± 9.3%. The pEC 50 value for HIS remained unaltered by the treatment with gallein ( Figure 6B). The modulatory effect of gallein on E max values was markedly weaker at hH 1-4 Rs than for individual Gα modulators (1.00 µM FR, 100 ng/mL CTX and 100 ng/mL PTX). This may indicate that the endogenous Gβγ subunit plays a minor role in hH 1-4 Rs signal transduction in the DMR assay. Previous investigations using the cAMP-sensitive luciferase reporter gene assay with hH 1,2 Rs stably expressed in HEK293T cells also showed gallein as ineffective at reducing signal response (hH 1 R [20] or hH 2 R [97]). Unfortunately, to the best of our knowledge, comparable investigations with gallein concerning hH 3,4 Rs expressed in HEK cells were not available. Lavenus et al. [45] came to a similar conclusion when investigating the effect of 20 µM gallein on the Angiotensin II-induced response in HEK293-AT 1 R cells using the label-free surface plasmon resonance (SPR) technique. Further experiments are therefore necessary to clarify the involvement of Gβγ dimer in the signal transduction mediated by the hH 1-4 Rs.

Impact of Gα Protein Modulator Combinations on the Histamine Induced DMR Response
None of the four HR subtypes displayed completely suppressed DMR signals with single G protein modulators ( Figure 6A). These results prompted us to investigate whether a complete inhibition of the DMR signal in HEK hH 1-4 R cells is achievable by combining the Gα protein modulators PTX, CTX and FR. At this point it should be noted that PTX and CTX were used at a concentration of 10.0 ng/mL instead of 100 ng/mL to avoid off target effects, which was usually sufficient to achieve the maximum effect (Supplementary Figure S11). HEK hH 1-4 R cells were treated with indicated modulators prior to stimulation with 10 µM HIS (Figure 7).
In HEK hH 1 R cells, each of the modulator combinations changed the time course of the HIS induced DMR response ( Figure 7A), consistent with results from experiments with individual modulators ( Figure 5B). Substantial depression of E max to 14 ± 8.4% was seen in HEK hH 1 R cells after pretreatment with a combination of PTX and CTX ( Figure 7B), corroborating with our previous results for the individual contributions of Gα s/i/o . A stronger reduction of the signal was observed when combining either PTX or CTX with FR, where the signal was reduced almost to the basal level (E max (PTX + FR) = 3.4 ± 3.5%, E max (CTX + FR) = 5.7 ± 0.8%; Figure 7B). The DMR signal was completely removed with a combination of the three modulators (PTX, CTX and FR). Therefore, we hypothesize that the HIS induced DMR response observed in HEK hH 1 R cells were exclusively transmitted via the three main classes of G proteins, namely Gα q/11 , Gα s and Gα i/o proteins.
In accordance with the observations on individually applied Gα protein modulators ( Figure 6), none of the modulator combinations altered the time course of the DMR response in HEK hH 2 R cells ( Figure 7A). Surprisingly, the HIS induced DMR response in HEK hH 2 R cells was not even reduced by half upon treatment with a triple modulator combination (E max (CTX, PTX, FR) = 55 ± 2.2%). In supplementary Figure S11, we showed that for both PTX and CTX, increasing the concentration from 10 ng/mL to 100 ng/mL no longer significantly reduced the DMR signal at the hH 2 R and FR had no effect on the E max value at hH 2 R. Thus, we can exclude that PTX and CTX at a concentration of 10 ng/mL might not have been sufficient to completely inhibit the respective signaling pathways. Beyond this, at the hH 1,3,4 Rs, the same modulator combination caused a more pronounced decrease in the E max value (Figure 7). Both arguments suggest that the weak impact of the triple modulator combination on the E max was a hH 2 R-specific phenomenon. We conclude that in HEK hH 2 R cells the Gα q/11 , Gα s and Gα i/o are not mainly responsible for the HIS induced DMR response, opposed to the hH 1, 3,4 Rs. Referring to the aforementioned hypotheses constructed from the individually applied modulators, it appears that the hH 2 R is not only promiscuous with these Gα proteins, but there is also growing evidence for a possible interaction of hH 2 R with the Gα 12/13 and/or Gα z , which are endogenously expressed in HEK cells [62]. avoid off target effects, which was usually sufficient to achieve the maximum effect (Supplementary Figure S11). HEK hH1-4R cells were treated with indicated modulators prior to stimulation with 10 µM HIS (Figure 7).  Figure 5B). Substantial depression of Emax to 14 ± 8.4% was seen in HEK hH1R cells after pretreatment with a combination of PTX and CTX ( Figure  7B), corroborating with our previous results for the individual contributions of Gαs/i/o. A stronger reduction of the signal was observed when combining either PTX or CTX with FR, where the signal was reduced almost to the basal level (Emax(PTX + FR) = 3.4 ± 3.5%, Emax(CTX + FR) = 5.7 ± 0.8%; Figure 7B). The DMR signal was completely removed with a combination of the three modulators (PTX, CTX and FR). Therefore, we hypothesize that the HIS induced DMR response observed in HEK hH1R cells were exclusively transmitted via the three main classes of G proteins, namely Gαq/11, Gαs and Gαi/o proteins.
In accordance with the observations on individually applied Gα protein modulators ( Figure 6), none of the modulator combinations altered the time course of the DMR response in HEK hH2R cells ( Figure 7A). Surprisingly, the HIS induced DMR response in HEK hH2R cells was not even reduced by half upon treatment with a triple modulator combination (Emax (CTX, PTX, FR) = 55 ± 2.2%). In supplementary Figure S11, we showed that for both PTX and CTX, increasing the concentration from 10 ng/mL to 100 ng/mL no longer significantly reduced the DMR signal at the hH2R and FR had no effect on the Emax value at hH2R. Thus, we can exclude that PTX and CTX at a concentration of 10 ng/mL might not have been sufficient to completely inhibit the respective signaling pathways. Beyond this, at the hH1,3,4Rs, the same modulator combination caused a more pronounced decrease in the Emax value (Figure 7). Both arguments suggest that the weak impact of the triple modulator combination on the Emax was a hH2R-specific phenomenon. We conclude that in HEK hH2R cells the Gαq/11, Gαs and Gαi/o are not mainly responsible for the HIS induced DMR response, opposed to the hH1,3,4Rs. Referring to the aforementioned hypotheses constructed from the individually applied modulators, it appears that the hH2R is not In experiments with individually applied Gα modulators, a marked deceleration of the DMR response was observed in HEK hH 3 R cells in the presence of 10 ng/mL PTX ( Figure 5B). As expected, such a retardation of the DMR signal was observed when HEK hH 3 R cells were pre-treated with modulator combinations comprising 10 ng/mL of PTX ( Figure 7A). Unexpectedly, a combination of 1 µM FR + 10 ng/mL of CTX decelerated the DMR response. Moreover, the same combination (FR + CTX) markedly reduced the E max to 40 ± 12% ( Figure 7B). Both the impact on the time courses and the reduced E max value in the presence of FR + CTX suggest that Gα q/11 and Gα s are involved in the hH 3 R mediated DMR response. However, in comparison, a stronger decrease in E max value was observed when combining 1 µM FR with 10 ng/mL of PTX to jointly inhibit Gα q/11 and Gα i/o signaling pathways. This modulator combination reduced the E max to 12 ± 8.3%, reaching a plateau that was found to be non-suppressible by the triple modulator combination of FR + CTX + PTX (E max = 12 ± 6.4%; Figure 7B) suggesting that Gα i/o and Gα q/11 played a more pronounced role in the hH 3 R mediated DMR response to HIS than Gα s . Again, as with the hH 2 R, the HIS induced DMR response was not completely ablated by the triple modulator combination. Inter alia, one possible explanation for this might be an involvement of additional G proteins such as Gα 12/13 and/or Gα z . However, it should be noted that, unlike for the hH 1,2 Rs, the concentration of CTX in the triple modulator combination (FR + CTX + PTX) is a factor to be considered. In experiments with CTX alone (Supplementary Figure S11), 10 ng/mL CTX were not sufficient to achieve the maximum effect. Precisely, in the presence of 10 ng/mL an E max value of 76 ± 6.3% was obtained, whereas 100 ng/mL CTX reduced the E max value to 54 ± 7.6%. Although this difference was not determined to be significant (one-way ANOVA analysis followed by Tukey's multiple comparison test; p = 0.1980), we still find it worth mentioning.
Likewise, we examined the influence of modulator combinations on the DMR signal in HEK hH 4 R cells. We would like to note that the differences in E max values between the different modulator combinations were subtly nuanced rather than clear, just as with the individual modulators in Section 2.3.1. None of the Gα modulators effected the time courses of the HIS induced DMR responses when applied individually ( Figure 5B). However, in combination, FR + PTX and FR + CTX + PTX altered the time course of the DMR response to HIS ( Figure 7A). In both cases the DMR signal showed no peak and did not decline continuously, as observed in control experiments without modulators (Figure 2A). Instead, the DMR response increased steadily over time upon stimulation with HIS ( Figure 7A). We took this as a hint that Gα q/11 and Gα i/o have more impact on the signal transduction of hH 4 R in HEK cells than Gα s ; nevertheless, the involvement of the latter should not be neglected. This opinion was enforced when E max values were considered ( Figure 7B); the treatment of HEK hH 4 R cells with CTX + PTX decreased the E max to 44 ± 7.7%, whereas addition of FR (FR + CTX + PTX) reduced the E max to a final value of 14 ± 8.0%, relative to control cells. It is also remarkable that a jointly inhibition of Gα q/11 and Gα i/o signaling pathways with FR + PTX decreased the maximum response by almost the same level (E max = 19 ± 7.7%) as the triple combination FR + CTX + PTX. Unexpectedly, the E max value in presence of 10 ng/mL CTX + 10 ng/mL PTX was higher (E max = 44 ± 7.7%) than that upon treatment with 10 ng/mL of PTX alone (E max = 32 ± 1.9%). This might be due to the mechanism of action of CTX, as CTX does not directly inhibit the Gα s protein, but rather masks the Gα s dependent signaling pathway by permanent Gα s protein activation. Similar to the hH 3 R, the inhibition of the three signaling pathways was not sufficient to completely remove the hH 4 R mediated response, as 14 ± 8.0% of E max remained after treatment with FR + CTX + PTX ( Figure 7B). Again, as with the hH 3 R, this demonstrates that signal transduction of the hH 4 R overexpressed in HEK cells occurred mainly through activation of Gα i/o , Gα s and Gα q/11 proteins, but the DMR signal might also arise from either Gβγ, Gα 12/13 and/or Gα Z proteins.
Of note, the two structurally related receptor subtypes hH 3 R and hH 4 R have similar coupling specificities to Gα proteins, and so it is unsurprising that so that inhibition of the corresponding Gα signaling pathways led to comparable reduction in E max values.

Expression of hH 1-4 Rs in ∆Gα s HEK Cells
In addition to the concept of classical pharmacology, namely the employment of specific G protein modulators as molecular tools to elucidate cellular processes, we explored a molecular biology approach to better understand the contribution of individual Gα isoforms to the hH 1-4 R mediated DMR response using CRISPR/Cas9 modified HEK cells devoid of distinct Gα proteins (∆Gα x HEK cells). For the generation of ∆Gα x HEK hH 1-4 R cells the ∆Gα s/l HEK [63], ∆Gα q/11 HEK [44], ∆Gα 12/13 HEK [64] and ∆Gα six HEK [41] cells were stably transfected with the pIRESneo3-SP-FLAG-hH 1-4 R constructs and used as polyclonal cell lines. We confirmed the expression of the hH 1-4 Rs in ∆Gα x HEK hH 1-4 R cells by radioligand saturation binding using live cells (Supplementary Figure S3). The expression levels of hH 1-4 Rs in ∆Gα x HEK hH 1-4 R cells were calculated as mentioned for HEK hH 1-4 R cells (Table 1). When comparing the expression levels of respective HR subtypes in HEK hH 1-4 R cells with those in ∆Gα x HEK hH 1-4 R cells (e.g., expression of the hH 1 R in HEK hH 1 R versus in ∆Gα x HEK hH 1 R cells), we noted that the expression levels of hH [2][3][4] Rs were in the same range. In the case of the hH 1 R, the expression level was determined to be 10-fold lower in ∆Gα s/l, q/11, 12/13 HEK hH 1 R cells compared to HEK hH 1 R cells. On the one hand, this difference may be due to the single clone selection procedure by which only the highest response HEK hH 1 R cells were obtained, thereby having the highest receptor expression level. The fact that only the binding capacity but not the affinity of the radioligand [ 3 H]MEP was affected would argue in favor of this. However, this is contradicted by the fact that no difference in expression level was observed between the single clone HEK hH 2-4 R and polyclonal ∆Gα x HEK hH 2-4 R cells. On the other hand, we cannot exclude that knock-out of Gα proteins might have impaired either the expression of the hH 1 R or the detection of the binding capacity of the radioligand [ 3 H]MEP to the hH 1 R in ∆Gα x HEK hH 1 R cells. This suspicion arose when we failed to detect the expression of the hH 1 R in ∆Gα six hH 1 R cells by radioligand saturation binding (Supplementary Figure S3), although a concentration-dependent signal was detected in the DMR assay when these cells were stimulated with HIS (Section 2.4.2 and supplementary Figure S12). By contrast, no DMR signal was observed in ∆Gα six HEK cells devoid of the hH 1 R (Supplementary Figure S6), suggesting that the hH 1 R was expressed in ∆Gα six HEK hH 1 R cells. For MEP, which has been reclassified as an inverse agonist [98], multiple binding sites differing in affinity and binding capacity for the H 1 R have been reported [99,100]. Moreover, the intrinsic negative efficacy of MEP is thought to be due to the stabilization of a G-protein-coupled state of the H 1 R that is not capable of eliciting a response [100]. Considering this, we argue in favor of the latter hypothesis, namely that the absence of Gα proteins may have affected the binding of [ 3 H]MEP to the hH 1 R. However, as this research project is focused on the results in the DMR assay, we have not pursued this issue. The pK d values determined with both HEK hH 1-4 R and ∆Gα x HEK hH 1-4 R cells were in very good agreement with literature data (Table 1), except for ∆Gα 12/13 hH 2 R and ∆Gα six hH 2 R. In both cases, the pK d value increased significantly to 7.98 + 0.05 (∆Gα 12/13 hH 2 R, p < 0.0001) and 7.86 + 0.06 (∆Gα six hH 2 R, p = 0.0004) relative to the value determined using HEK hH 2 R cells (pK d = 7.19 + 0.06). Apparently, the absence of Gα 12/13 facilitates the binding of the radioligand [ 3 H]DE-257 to the hH 2 R.
The impact of Gα protein knock-out on the affinity of HIS to the hH 1-4 Rs was analyzed by radioligand competition binding with HIS as a competitor using live ∆Gα x HEK hH 1-4 R cells (Figure 8 and supplementary Table S2). Of note, such experiments were not performed with ∆Gα six HEK hH 1 R, as the expression of hH 1 R was not detectable in saturation binding experiments. Unfortunately, in ∆Gα s/l, q/11 HEK hH 1 R cells a pK i value could not be determined for HIS due to an ambiguous curve fit of the data. Although not statistically significant (p = 0.0545, t-test two-tailed), the pK i value for HIS in ∆Gα 12/13 HEK hH 1 R cells (pK i = 2.23 ± 0.37) was approx. one order of magnitude lower than at HEK hH 1 R cells (pK i = 3.37 ± 0.29). While the absence of Gα s/l or Gα q/11 proteins in ∆Gα s/l, q/11 HEK hH 2 R cells had no impact on the pK i of HIS (pK i = 3.68 ± 0.09 and 4.23 ± 0.11, respectively), the value decreased approx. two-fold at ∆Gα six HEK hH 2 R cells (pK i = 1.82 ± 0.28) compared to HEK hH 2 R cells (pK i = 4.32 ± 0.38). The listed discrepancy of the pK i values of HIS at the hH 1,2 Rs were not surprising, as saturation binding experiments (Table 1) demonstrate that the absence of Gα proteins can positively or negatively impact ligand binding at the hH 1,2 Rs. The pK i values determined for HIS using ∆Gα x HEK hH 3,4 R cells were in good agreement with literature data and the results determined with HEK hH 3,4 R cells (Supplementary Table S2).

Stimulation of ∆Gα x HEK hH 1-4 R Cells in the DMR Assay with HIS
A schematic illustration of the ∆Gα x HEK hH 1-4 R cells with regard to G protein knock-out is given in Figure 9A. The ∆Gα x HEK hH 1-4 R cells were stimulated with HIS at increasing concentrations and the DMR response was recorded for 60 min. Throughout, the DMR traces showed a positive deflection and were concentration dependent ( Figure 9B; AUC 60 CRCs in supplementary Figure S12). By contrast, stimulation of ∆Gα q/11 HEK, ∆Gα 12/13 HEK and ∆Gα six HEK cells devoid of hH 1-4 Rs with HIS did not provoke a DMR signal. However, with ∆Gα s HEK cells, devoid of a HR subtype, a slight increase in the DMR signal was observed, but only at high HIS concentrations (1.00 and 10.0 µM). Therefore, we considered this DMR increase as negligible due to its low intensity (Supplementary Figure S6). To evaluate the effect of Gα protein knock-out on the DMR response, the AUC 60 at the respective HIS concentration (hH 1,2,4 Rs 10 µM HIS and hH 3 R 100 µM HIS) using ∆Gα x HEK hH 1-4 R cells was compared to the mean AUC 60 of HEK hH 1-4 R cells, in which all G proteins were present (100% control, Figure 10). This approximation was reasonable, because mostly the expression of the different receptor subtypes was comparable (Table 1). value decreased approx. two-fold at ΔGαsix HEK hH2R cells (pKi = 1.82 ± 0.28) compared to HEK hH2R cells (pKi = 4.32 ± 0.38). The listed discrepancy of the pKi values of HIS at the hH1,2Rs were not surprising, as saturation binding experiments (Table 1) demonstrate that the absence of Gα proteins can positively or negatively impact ligand binding at the hH1,2Rs. The pKi values determined for HIS using ΔGαx HEK hH3,4R cells were in good agreement with literature data and the results determined with HEK hH3,4R cells (Supplementary Table S2).  HIS (hH3,4Rs), each at a final concentration of 10 µM. The non−specific binding was subtracted from the total binding to receive the specific binding. Specific binding was normalized to the buffer value (100%) and the corrected non−specific binding value (0%). Each point represents mean ± SEM of at least three independent experiments, each performed in triplicate.

Stimulation of ΔGαx HEK hH1-4R Cells in the DMR Assay with HIS
A schematic illustration of the ΔGαx HEK hH1-4R cells with regard to G protein knock-out is given in Figure 9A. The ΔGαx HEK hH1-4R cells were stimulated with HIS at increasing concentrations and the DMR response was recorded for 60 min. Throughout, the DMR traces showed a positive deflection and were concentration dependent ( Figure  9B; AUC60 CRCs in supplementary Figure S12). By contrast, stimulation of ΔGαq/11 HEK, ΔGα12/13 HEK and ΔGαsix HEK cells devoid of hH1-4Rs with HIS did not provoke a DMR signal. However, with ΔGαs HEK cells, devoid of a HR subtype, a slight increase in the DMR signal was observed, but only at high HIS concentrations (1.00 and 10.0 µM). Therefore, we considered this DMR increase as negligible due to its low intensity (Supplementary Figure S6). To evaluate the effect of Gα protein knock-out on the DMR response, the AUC60 at the respective HIS concentration (hH1,2,4Rs 10 µM HIS and hH3R 100 µM HIS) using ΔGαx HEK hH1-4R cells was compared to the mean AUC60 of HEK hH1-4R cells, in which all G proteins were present (100% control, Figure 10). This approximation was reasonable, because mostly the expression of the different receptor subtypes was comparable (Table 1). The non − specific binding was subtracted from the total binding to receive the specific binding. Specific binding was normalized to the buffer value (100%) and the corrected non − specific binding value (0%). Each point represents mean ± SEM of at least three independent experiments, each performed in triplicate.
When comparing the HIS induced DMR responses of ∆Gα s/l, 12/13 HEK hH 1 R with that of HEK hH 1 R cells by visual inspection, there was no discernible difference ( Figure 9B). Consequently, the E max values for HIS using ∆Gα s/l, 12/13 HEK hH 1 R were not significantly different from the control cell line HEK hH 1 R ( Figure 10A). Of note, in Section 2.4.1 we discussed that the binding capacity of [ 3 H]MEP was by factor 10 lower in ∆Gα x HEK hH 1 R cells than in HEK hH 1 R cells. Apparently, this difference had no impact on the signal amplitude and the E max value, supporting the hypothesis that the absence of the Gα proteins impaired the binding of [ 3 H]MEP to the hH 1 R [99,100]. The absence of the Gα s protein in ∆Gα s/l HEK hH 1 R cells caused the pEC 50 value for HIS to significantly increase to 7.96 + 0.09 compared to HEK hH 1 R cells (pEC 50 = 7.43 + 0.05), an effect also observed in ∆Gα 12/13 HEK hH 1 R cells (pEC 50 = 7.78 + 0.05). By contrast, the absence of the Gα q/11 protein in ∆Gα q/11, six HEK hH 1 R cells lowered the signal amplitude (E max = 46 ± 38%; Figure 10A) and slightly altered the time course of the signal ( Figure 9B). Moreover, the pEC 50 value for HIS in ∆Gα q/11, six HEK hH 1 R cells was significantly lower in both cell lines (∆Gα q/11 HEK hH 1 R pEC 50 = 6.38 ± 0.02, ∆Gα six HEK hH 1 R pEC 50 = 6.63 ± 0.15) than with HEK hH 1 R cells ( Figure 10B). We still hypothesize that the presence of Gα q/11 stabilized the active state of hH 1 R in HEK cells and that this effect is further enhanced in the absence of other Gα proteins.
The lack of Gα proteins in ∆Gα x HEK hH 2 R cells did not alter the time course of the DMR response ( Figure 9B). Despite the lack of the Gα s protein in ∆Gα s HEK hH 2 R cells, stimulation with HIS evoked a robust DMR response, similar to that observed with HEK hH 2 R cells. Consequently, the E max value of ∆Gα s HEK hH 2 R cells was not significantly different compared to HEK hH 2 R cells ( Figure 10A). This was unexpected, as we observed a significant decrease in E max in our experiments with CTX to mask Gα s . Stimulation of ∆Gα q/11 HEK hH 2 R cells with HIS showed a decrease in the E max value to 84 ± 14% ( Figure 10A) compared to HEK hH 2 R cells. The pEC 50 value determined for HIS in ∆Gα s/l, q/11 HEK hH 2 R cells remained in the same range as in HEK hH 2 R cells ( Figure 10B). In Section 2.3.1 it was considered that Gα 12/13 protein might be responsible for the HIS induced DMR at HEK hH 2 R cells. This hypothesis was affirmed as the signal amplitude of the DMR response to HIS in ∆Gα 12/13 HEK hH 2 R cells was considerably lower compared to that of HEK hH 2 R cells ( Figure 9B). The corresponding E max value determined in ∆Gα 12/13 HEK hH 2 R cells amounted to 10.0 ± 0.8% ( Figure 10A) relative to HEK hH 2 R cells. In ∆Gα six HEK hH 2 R cells, which lack the Gα 12/13 protein too, the E max value was also reduced significantly to 20 ± 2.0%. In addition to E max , the HIS pEC 50 value in both cell lines wassignificantly reduced (∆Gα 12/13 HEK hH 2 R pEC 50 = 5.77 ± 0.46; ∆Gα six HEK hH 2 R pEC 50 = 6.01 ± 0.04) compared to HEK hH 2 R cells (pEC 50 = 6.57 ± 0.05; Figure 10B). We interpreted this as an indication that Gα 12/13 might stabilize the active state of the hH 2 R and is essential for hH 2 R mediated signal transduction in HEK cells. Further studies are necessary to substantiate or rule out the involvement of other cellular constituents, such as Gα z .  [63], the Gαq/11 (ΔGαq/11 HEK) [44], the Gα12/13 (ΔGα12/13 HEK) [64] or six Gα proteins (ΔGαs/l, q/11, 12/13 = ΔGαsix HEK) [41] were stably transfected with hH1−4Rs. The knocked−out Gα protein is marked with a red "X". HEK hH1−4R cells, expressing all four G protein classes were used as reference. (B) The ΔGαx HEK hH1-4R cells either lacking the Gαs/l (ΔGαs/l), Gαq/11 (ΔGαq/11), Gα12/13 (ΔGα12/13) or Gαs/l, q/11, 12/13 (ΔGαsix) proteins were stimulated with indicated HIS concentration and the DMR response was recorded for 60 min. Depicted are representative DMR traces, which were corrected for the buffer. Each trace represent mean ± SEM of a representative experiment performed in triplicate. The ∆Gα x HEK cells lacking either the Gα s/l (∆Gα s/l HEK) [63], the Gα q/11 (∆Gα q/11 HEK) [44], the Gα 12/13 (∆Gα 12/13 HEK) [64] or six Gα proteins (∆Gα s/l, q/11, 12/13 = ∆Gα six HEK) [41] were stably transfected with hH 1−4 Rs. The knocked − out Gα protein is marked with a red "X". HEK hH 1−4 R cells, expressing all four G protein classes were used as reference. (B) The ∆Gα x HEK hH 1-4 R cells either lacking the Gα s/l (∆Gα s/l ), Gα q/11 (∆Gα q/11 ), Gα 12/13 (∆Gα 12/13 ) or Gα s/l , q/11 , 12/13 (∆Gα six ) proteins were stimulated with indicated HIS concentration and the DMR response was recorded for 60 min. Depicted are representative DMR traces, which were corrected for the buffer. Each trace represent mean ± SEM of a representative experiment performed in triplicate. When comparing the HIS induced DMR responses of ΔGαs/l, 12/13 HEK hH1R with that of HEK hH1R cells by visual inspection, there was no discernible difference ( Figure 9B). Consequently, the Emax values for HIS using ΔGαs/l, 12/13 HEK hH1R were not significantly different from the control cell line HEK hH1R ( Figure 10A). Of note, in Section 2.4.1 we discussed that the binding capacity of [ 3 H]MEP was by factor 10 lower in ΔGαx HEK hH1R cells than in HEK hH1R cells. Apparently, this difference had no impact on the signal amplitude and the Emax value, supporting the hypothesis that the absence of the Gα proteins impaired the binding of [ 3 H]MEP to the hH1R [99,100]. The absence of the Gαs protein in ΔGαs/l HEK hH1R cells caused the pEC50 value for HIS to significantly increase to 7.96 + 0.09 compared to HEK hH1R cells (pEC50 = 7.43 + 0.05), an effect also observed in ΔGα12/13 HEK hH1R cells (pEC50 = 7.78 + 0.05). By contrast, the absence of the Gαq/11 protein in ΔGαq/11, six HEK hH1R cells lowered the signal amplitude (Emax = 46 ± 38%; Figure 10A) and slightly altered the time course of the signal ( Figure 9B). Moreover, the pEC50 value for HIS in ΔGαq/11, six HEK hH1R cells was significantly lower in both cell lines (ΔGαq/11 HEK hH1R pEC50 = 6.38 ± 0.02, ΔGαsix HEK hH1R pEC50 = 6.63 ± 0.15) than with HEK hH1R cells ( Figure 10B). We still hypothesize that the presence of Gαq/11 stabilized the active state Figure 10. Effect of G protein knock-out on the efficacy and potency of HIS at hH 1-4 Rs. E max and pEC 50 values determined for HIS in ∆Gα x HEK hH 1-4 R cells. (A) Bar chart of E max values determined for HIS in HEK hH 1-4 R cells (wt, grey) and in ∆Gα s/l HEK, ∆Gα q/11 HEK, ∆Gα 12/13 HEK, ∆Gα six HEK cells each stably expressing hH 1-4 Rs, respectively. The E max max values were calculated using AUC 60 at the highest HIS concentration (10 µM for hH 1,2,4 R and 100 µM for hH 3 R) and normalized to the mean AUC 60 from HEK hH 1-4 R cells at the corresponding receptor subtype (100%) and to the corresponding buffer value (0%) determined in ∆Gα x HEK hH 1-4 R cells. (B) Scatter plot of the pEC 50 values in HEK hH 1-4 R cells (wt, grey) and in ∆Gα s/l HEK, ∆Gα q/11 HEK, ∆Gα 12/13 HEK, ∆Gα six HEK cells each stably expressing hH 1-4 Rs, respectively. The pEC 50 were determined by plotting the AUC 60 against the respective HIS concentration. (A,B) Data presented are means ± SEM of at least three independent experiments each performed in triplicate. Statistical difference relative to the control was analyzed by one-way ANOVA followed by Dunnett's multiple comparison test. Significance levels are indicated by asterisks (* p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001).
Unlike HEK hH 3 R cells (all G proteins present) in which the DMR signal increased steadily but slowly after addition of HIS (Figure 2A), the DMR signal in all ∆Gα x HEK hH 3 R cells increased rapidly, showing a peak within 10 min upon stimulation with HIS ( Figure 9B). Interestingly, such a time course was not observed in any of the experiments in HEK hH 3 R cells with Gα protein modulators ( Figure 5B). The E max values of HIS in ∆Gα q/11 HEK hH 3 R, ∆Gα 12/13 HEK hH 3 R cells and ∆Gα s/l HEK hH 3 R cells significantly declined to 40 ± 6.4%, 49 ± 5.5% and 69 ± 11%, respectively, compared to the 100% control (HEK hH 3 R cells; Figure 10A). In ∆Gα six HEK cells, the Gα q/11, s/l, 12/13 proteins were knocked-out, so it can be assumed that among the common Gα proteins, only the Gα i/o was expressed. Upon stimulation of these cells with HIS, a weaker DMR response was detected with an E max value of 34 ± 3.7% compared to HEK hH 3 R cells ( Figure 10A). As the Gα i/o signaling pathway is almost exclusively considered as physiologically relevant for the hH 3 R, we did not expect a complete suppression of the signal in ∆Gα six HEK hH 3 R cells. However, before assigning this response solely to the Gα i/o protein, it should be noted that other G proteins, such as Gα z should be considered. Strikingly, the pEC 50 value determined for HIS in all ∆Gα x HEK hH 3 R cells was significantly higher ( Figure 10B) compared to that determined with HEK hH 3 R cells.
Different to the hH 3 R, the time course of the HIS induced DMR response recorded using ∆Gα x hH 4 R cells (Figure 9) agreed well with that of HEK hH 4 R cells (100% control). The lack of Gα q/11 in ∆Gα q/11 HEK hH 4 R cells led to a significant decrease in the DMR signal to 54 ± 3.3% ( Figure 10A), whereas knock-out of Gα s (∆Gα s HEK hH 4 R) showed a weaker impact on the DMR response, reducing the E max to 86 ± 13% relative to the control. This was surprising because a much more pronounced suppression was observed after treatment with CTX. In ∆Gα 12/13 HEK hH 4 R cells, the E max value was suppressed to 62 ± 6.7% compared to HEK hH 4 R cells; therefore, it can be concluded that the Gα 12/13 pathway seems to be involved in the signal transduction of the hH 4 R in HEK cells. The knock-out of the three subclasses of Gα proteins in ∆Gα six hH 4 R cells reduced the E max value to 58 ± 7.3% compared to HEK hH 4 R cells, being in good agreement with results determined with the Gα i/o modulator PTX. However, as the other three G proteins classes have been shown to be essentially involved in the signaling of the hH 4 R (see Section 2.3.1), we expected a more pronounced reduction of the signal. Perhaps the cells compensate for the lack of targeted G proteins by enhanced expression of either Gα i/o or other (G) proteins are involved in the signal transduction process.

Pharmacological versus Molecular Biological Approach to Silence Gα Protein
The contribution of Gα proteins to the DMR response elicited by HIS at the hH 1-4 Rs stably expressed in HEK cells was investigated either by a classical pharmacological (G protein modulators) or by a molecular biological (Gα protein knock-out) approach. In the pharmacological approach, HEK hH 1-4 Rs cells were pre-treated with Gα protein modulators FR, CTX and PTX to silence either Gα q/11 , Gα s or Gα i/o proteins, respectively (Section 2.3). In the molecular biological approach, the Gα q/11 , Gα s , Gα 12/13 proteins were knocked out individually or in combination (knock-out of Gα q/11, s/l, 12/13 ) using the CRISPR/Cas9 technology (Section 2.4). The focus of this section was to highlight the similarities and discuss the differences of the results obtained with these two approaches.
Silencing of the Gα q/11 signaling pathway either by 1.00 µM FR in HEK hH 1-4 R cells or by knocking out Gα q/11 in ∆Gα q/11 HEK hH 1-4 R cells have shown agreement in terms of E max and pEC 50 values for HIS. At the hH 1 R, for example, the time course of DMR traces were similarly altered by both approaches compared to the control (HEK hH 1 R cell w/o; Figure 11) and the E max for HIS was significantly reduced (∆Gα q/11 HEK hH 1 R E max = 46 ± 3.8; HEK hH 1 R + 1 µM FR E max = 41 ± 9.5) relative to HEK hH 1 R cells (100% w/o modulator). Moreover, both procedures to silence the Gα q/11 have led to a significant decrease in the pEC 50 determined for HIS (∆Gα q/11 HEK hH 1 R pEC 50 = 6.38 ± 0.02; HEK hH 1 R + 1 µM FR pEC 50 = 6.60 ± 0.29) relative to HEK hH 1 R control cells (pEC 50 = 7.43 ± 0.05). A similar effect was observed for HIS with ∆Gα six HEK hH 1 R cells, which also lack the Gα q/11 protein. We expected such a pronounced perturbation of the E max and the pEC 50 value, as hH 1 R is predominantly described as a Gα q/11 coupled receptor [57]. It was surprising that for the hH 2 R, silencing of the Gα q/11 signaling pathway by both approaches (FR and Gα q/11 knock-out) had almost no impact on the DMR response (kinetics, E max and pEC 50 ), as it is commonly accepted that the Gα q/11 protein is considerably involved in the signal transduction of the hH 2 R [56]. We cannot confirm this in the DMR assay using HEK cells. By contrast, deactivation of the Gα q/11 signaling pathway either by FR or knock-out affected the E max value for HIS at hH 3,4 Rs ( Figure 11). In HEK hH 3,4 Rs, 1.00 µM FR reduced the E max to 57 ± 5.4% and 47 ± 4.5%, respectively ( Figure 6A) and in ∆Gα q/11 HEK hH 3,4 R cells the E max was diminished to 40 ± 6.4% and 54 ± 3.3%, respectively ( Figure 10A; compared to untreated HEK hH 3,4 R cells). Moreover, at hH 3 R, the pEC 50 value for HIS increased significantly by both approaches (HEK hH 3 R + 1 µM FR pEC 50 = 7.20 ± 0.05, ∆Gα q/11 HEK hH 3 R cells pEC 50 = 7.28 ± 0.08) relative to HEK hH 3 R cells (pEC 50 = 6.49 ± 0.06). By contrast, in both systems the pEC 50 determined for HIS at the hH 4 R was not significantly altered. As both approaches led to the same consequences, we are convinced that the results are not an artifact and conclude that Gα q/11 contributed to the DMR signaling of the hH 3,4 Rs in HEK cells, even though the limited literature suggests the opposite [91,101]. We consider this as an intriguing starting point for further investigations. Figure 11. G protein inhibition using a classical pharmacological concept (G protein modulator) or by a molecular biological approach (Gα protein knock-out cells). DMR traces recorded for HIS in HEK hH1-4R cells in the absence (w/o modulator) or presence of G protein modulators (100 ng/mL PTX, 100 ng/mL CTX, 1 µM FR, 20 µM gallein (gal) and combination of 10 ng/mL PTX + 10 ng/mL CTX + 1 µM FR) or in Gα protein knock-out cells (ΔGαs/l HEK, ΔGαq/11 HEK, ΔGα12/13 HEK and ΔGαsix HEK cells) stably transfected with hH1-4Rs. In the case of the hH1,2,4Rs the cells were stimulated with 10 µM HIS, whereas in the case of the hH3R the cells were stimulated with 100 µM HIS. All traces shown were corrected for the assay buffer and represent means ± SEM of at least three independent experiments, each performed in triplicate.
Unlike Gαq/11, we found differences between CTX and knocking-out Gαs ( Figure 11). To be more specific, in experiments using HEK hH1-4Rs cells pre-treated with 100 ng/mL CTX, we concluded that Gαs was markedly involved in the hH1-4R mediated signal transduction process in HEK cells throughout. For example, for the hH1R the Emax was dramatically reduced to 23 ± 4.9% ( Figure 6A). Moreover, the Emax of HIS determined in HEK hH3,4R cells was significantly reduced to 54 ± 7.6% or 35 ± 7.9%, respectively ( Figures 5  and 6A). By contrast, we observed that knock-out of Gαs/l in ΔGαs/l HEK hH1-4R cells had a weaker effect on the Emax value. In ΔGαs/l HEK hH1,3,4R cells, the Emax value amounted to 115 ± 23%, 69 ± 12% and 86 ± 13% of control responses respectively ( Figure 10A), suggesting that Gαs plays a supporting role in the HIS induced DMR response. Various explanations can be considered to address this discrepancy in Emax between the two approaches. On the one hand, it seems possible that HEK cells have "adapted" their repertoire of expressed Gα proteins to compensate for the lack of Gαs in ΔGαs/l HEK hH1-4R cells so that the Emax remained unaffected. On the other hand, it is conceivable that, in addition to Gαs, CTX may have off-target effects that were relevant for the generation of the DMR signal in HEK hH1-4R cells, which led to a decrease in Emax. Elucidation of the difference in results between the pharmacological and molecular biological approaches for Gαs modulation should be pursued in the future.
Regarding the Gαi/o signaling pathway, in the experiments with PTX we found that Gαi/o was directly involved in the hH1R mediated DMR response in HEK cells, as the Emax was reduced to 50 ± 9.3% in the presence of 100 ng/mL PTX ( Figure 6A). Alternatively, in ΔGαsix hH1R cells, which among the canonical Gα proteins only express Gαi/o, HIS elicited a DMR response with a corresponding Emax of 33 ± 0.5% ( Figure 10A). As we ruled out that Gα12/13 and Gαz play a role in the hH1R mediated DMR response (Figure 7), we conclude that the residual 33% represent the interaction of the hH1R with the Gαi/o, in accordance with literature [57]. The hH3,4Rs are considered as Gαi/o selective receptors [57], however, according to our experiments, we conclude that Gαi/o was not exclusively involved in the manifestation of the DMR signal in HEK hH3,4R cells. Namely, pretreatment of HEK hH3,4R cells with 100 ng/mL of PTX led to a dramatic decrease in Emax, and the pEC50 was Figure 11. G protein inhibition using a classical pharmacological concept (G protein modulator) or by a molecular biological approach (Gα protein knock-out cells). DMR traces recorded for HIS in HEK hH 1-4 R cells in the absence (w/o modulator) or presence of G protein modulators (100 ng/mL PTX, 100 ng/mL CTX, 1 µM FR, 20 µM gallein (gal) and combination of 10 ng/mL PTX + 10 ng/mL CTX + 1 µM FR) or in Gα protein knock-out cells (∆Gα s/l HEK, ∆Gα q/11 HEK, ∆Gα 12/13 HEK and ∆Gα six HEK cells) stably transfected with hH 1-4 Rs. In the case of the hH 1,2,4 Rs the cells were stimulated with 10 µM HIS, whereas in the case of the hH 3 R the cells were stimulated with 100 µM HIS. All traces shown were corrected for the assay buffer and represent means ± SEM of at least three independent experiments, each performed in triplicate.
Unlike Gα q/11 , we found differences between CTX and knocking-out Gα s (Figure 11). To be more specific, in experiments using HEK hH 1-4 Rs cells pre-treated with 100 ng/mL CTX, we concluded that Gα s was markedly involved in the hH 1-4 R mediated signal transduction process in HEK cells throughout. For example, for the hH 1 R the E max was dramatically reduced to 23 ± 4.9% ( Figure 6A). Moreover, the E max of HIS determined in HEK hH 3,4 R cells was significantly reduced to 54 ± 7.6% or 35 ± 7.9%, respectively ( Figures 5 and 6A). By contrast, we observed that knock-out of Gα s/l in ∆Gα s/l HEK hH 1-4 R cells had a weaker effect on the E max value. In ∆Gα s/l HEK hH 1,3,4 R cells, the E max value amounted to 115 ± 23%, 69 ± 12% and 86 ± 13% of control responses respectively ( Figure 10A), suggesting that Gα s plays a supporting role in the HIS induced DMR response. Various explanations can be considered to address this discrepancy in E max between the two approaches. On the one hand, it seems possible that HEK cells have "adapted" their repertoire of expressed Gα proteins to compensate for the lack of Gα s in ∆Gα s/l HEK hH 1-4 R cells so that the E max remained unaffected. On the other hand, it is conceivable that, in addition to Gα s , CTX may have off-target effects that were relevant for the generation of the DMR signal in HEK hH 1-4 R cells, which led to a decrease in E max . Elucidation of the difference in results between the pharmacological and molecular biological approaches for Gα s modulation should be pursued in the future.
Regarding the Gα i/o signaling pathway, in the experiments with PTX we found that Gα i/o was directly involved in the hH 1 R mediated DMR response in HEK cells, as the E max was reduced to 50 ± 9.3% in the presence of 100 ng/mL PTX ( Figure 6A). Alternatively, in ∆Gα six hH 1 R cells, which among the canonical Gα proteins only express Gα i/o , HIS elicited a DMR response with a corresponding E max of 33 ± 0.5% ( Figure 10A). As we ruled out that Gα 12/13 and Gα z play a role in the hH 1 R mediated DMR response (Figure 7), we conclude that the residual 33% represent the interaction of the hH 1 R with the Gα i/o , in accordance with literature [57]. The hH 3,4 Rs are considered as Gα i/o selective receptors [57], however, according to our experiments, we conclude that Gα i/o was not exclusively involved in the manifestation of the DMR signal in HEK hH 3,4 R cells. Namely, pretreatment of HEK hH 3,4 R cells with 100 ng/mL of PTX led to a dramatic decrease in E max , and the pEC 50 was reduced in HEK hH 3 R cells compared to controls ( Figure 6B). Additionally, in the presence of the Gα q/11 protein modulator FR, the E max was significantly reduced for both receptor subtypes ( Figure 6B). Moreover, it was not possible to completely abolish the HIS triggered DMR in HEK hH 3,4 R cells with a modulator cocktail comprising FR, CTX and PTX (Figure 7). In addition to the canonical Gα proteins, we observed that the Gα 12/13 proteins might be involved in the signal transduction process of the hH 3,4 Rs, as the E max in ∆Gα 12/13 HEK hH 3,4 R cells decreased by about 55% compared with HEK hH 3,4 R cells ( Figure 10A). However, we cannot exclude that the Gα z might also be involved. In the case of the hH 2 R, the modulation of Gα i/o by 100 ng/mL PTX had a weaker effect on the HIS induced DMR response (E max = 77 ± 4.1%) compared to the hH 1,3,4 Rs (E max 51-32%; Figure 6A). Moreover, we failed to suppress the HIS induced DMR response by more than 40% with Gα protein modulators FR, CTX and PTX (Figure 7), and most of the DMR signal was abolished in ∆Gα 12/13 HEK hH 2 R and ∆Gα six HEK hH 2 R cells, both of which lack the Gα 12/13 protein ( Figure 10A). We conclude that Gα q/11 , Gα s , and Gα i/o played a minor role in the generation of the HIS DMR signal in HEK hH 2 R cells, and that Gα 12/13 proteins must have been involved. It has already been described in the literature that the hH 2 R is capable to interact with the Gα 12/13 protein [59,60], however, it was unexpected that the involvement of Gα 12/13 would exceed the contribution of Gα q/11 , Gα s , and Gα i/o .
In summary, we successfully established a DMR assay for the entire histaminergic receptor family stably expressed in HEK cells, providing an opportunity to monitor the functions of HRs and its ligands in real-time. High S/B-ratios above 100 for HIS and 24 for inverse agonists facilitate investigations on signaling pathways of hH 1-4 Rs and might be beneficial for further investigations, e.g., with respect to inverse agonism and functional bias of HR ligands. We took advantage of the integrative nature of the DMR assay to investigate the involvement of endogenously expressed G proteins in the signaling transduction processes mediated by hH 1-4 Rs. However, in view of the physiological relevance of the results, experiments with cells or tissues which endogenously express the receptors are pending. For example, using modulatory tools such as PTX, CTX and FR, the impact of ligands on the signaling pathway of the receptor can be studied as well, which is particularly interesting with respect to ligand induced signal bias. At this point, it should be noted that apart from G proteins, the recruitment of β-arrestin also plays an important role in the signal transduction processes of GPCRs [24] and consequently also for HRs [102][103][104]. Interestingly, although investigations on the mechanistic details of β-arrestin activation are available, there is also evidence that no β-arrestin mediated signaling was observed in absence of functional G proteins [41]. The DMR assay could be a valuable approach to investigate the contribution of β-arrestins to a holistic response of HRs. Pharmacological tools (e.g., biased ligands, protein inhibitors) in combination with a molecular biological approach (e.g., cells lacking (either) Gα proteins and/or β-arrestins) might be helpful to gain new insights into the interaction of G proteins and β-arrestins [41]. Moreover, several polymorphisms were discovered for HRs [105] which are under investigation to be associated with diseases such as heart failure (H 2 R [106]) or allergic rhinitis (H 4 R [107]) and correlated with the effectiveness of drugs (H 1 R [108], H 3 R [109], H 4 R [110]). Thus, the DMR assay might be a valuable tool to characterize such polymorphisms of HRs, especially focusing on the differences in the signaling pathways between receptor variants.
Although our studies still leave some open questions, we are convinced that the presented work provides valuable information for further investigation on signal transduction mechanisms of the HR family.

Radioligand Binding
All radioligand binding experiments (saturation and competition) were performed using suspensions of live HEK hH 1-4 R and ∆Gα x HEK hH 1-4 R cells. The cells were cultivated in DMEM supplemented with 10% FBS + P/S and 600 µg/mL G418 until 90-100% confluency was reached. On the day of the assay, the cells were detached by trypsinization (0.05% trypsin, 0.02% EDTA in PBS, at 37 • C for 2-4 min), harvested by centrifugation (800× g at rt for 5 min) and resuspended in L-15 medium devoid of additional supplements. The number of cells was determined using a hemocytometer (Neubauer, improved) and the cell density was adjusted to 1.0 × 10 6 cells/mL.
Before dispensing the cell suspension, all (radio)ligand dilutions were prepared 10fold concentrated in L-15 medium and dispensed (10 µL/well) in 96 well plates (PP microplates, Greiner Bio-One, Frickenhausen, Germany). Total binding was determined in the presence of L-15 medium (10 µL/well), and the non-specific binding was assessed in the presence of a competitor: for hH 1 R diphenhydramine (DPH), for hH 2 R famotidine (FAM), for hH 3,4 Rs histamine (HIS), each at a final concentration of 10 µM. For saturation binding experiments, serial dilutions of the following radioligands were prepared ( Subsequently, the cell suspension was added to the (radio)ligands (80 µL/well) to reach a final assay volume of 100 µL/well. After an incubation period of 60-120 min, the cells were harvested by filtration using a Brandel 96 sample harvester and the radioactivity was determined by liquid scintillation counting as described previously [112].
Data was analyzed using the GraphPad Prism 8 or 9 software (San Diego, CA, USA). Specific binding was calculated by subtracting the non-specific binding from the total binding. For saturation binding experiments binding data was plotted against the free radioligand concentration (nM) and best fitted to a one site saturation binding model (one site-total and non-specific binding; one site-specific binding) yielding K d values.
Receptor expression was quantified using the extrapolated B max values, specific activity (a s ) of the radioligands and the cell number seeded per well and is indicated as specific binding sites per cell.
For competition binding experiments, the specific binding was plotted against the −log(concentration ligand) and analyzed applying the four parameters logistic equation (log(modulator) vs. response-variable slope (four parameters)) yielding the pIC 50 values, which were individually converted to pKi values using the Cheng-Prusoff equation [113].

DMR Assay
The DMR assay was essentially performed as described [38] with the following modifications: The cells were cultured in DMEM supplemented with 10% FBS, 2 mM L-glutamine, P/S, and 600 µg/mL G418 until 90-100% confluency. The day before the assay, the cells were detached by trypsinization (0.05% trypsin, 0.02% EDTA in PBS, at 37 • C for 2-4 min), harvested by centrifugation (800× g, RT, 5 min,) and subsequently resuspended in DMEM supplemented with 10% FBS + P/S w/o G418. The cell density was adjusted to 1 × 10 6 cells/mL and the cell suspension was dispensed (90 µL/well) into an uncoated label-free 96 well plate (Cat. No. 5080, Corning B.V. Life Sciences, Amsterdam, Netherlands). Subsequently, the cells were spun down at 600× g for 1 min and allowed to attach in a humidified atmosphere containing 5% CO 2 at 37 • C overnight. On the day of the measurement, the cells were gently washed twice with assay medium (HBSS containing 20 mM HEPES). After the last washing step, the final volume was adjusted to 90 µL/well with assay medium and the plate was centrifuged at 600× g for 1 min. The cells were allowed to equilibrate at 37 • C for at least 2 h in an EnSpire multimode plate reader (PerkinElmer, Rodgau, Germany), before the baseline was recorded every minute for 5-10 min. Immediately after the baseline record, the compounds (10 µL/well; 10-fold concentrated in assay medium) were added and the response was recorded every minute for 60 min.
For experiments with the G-protein modulators PTX and CTX the cells were pretreated with the modulator at the respective final concentration (1.00, 10.0 or 100 ng/mL) overnight and the assay was performed as described above. In the case of FR900359 and gallein the cells were incubated with the modulator (FR900359 1.00, 0.10 or 0.01 µM; gallein 20.0 µM) for 30 min before the baseline record. Afterwards the assay was performed by analogy with the procedure described above.
The time course data is presented as resonance wave-length shift in pm relative to the last data point before the test compounds were added at time zero. Data were analyzed using the GraphPad Prism 8 and 9 software (San Diego, CA, USA). For analysis, the data were corrected for the baseline drift by subtracting the mean values of the buffer control. Subsequently, the area under curve (AUC) was calculated individually for each well defining the first 5-10 values as baseline. For the estimation of the S/B ratios the modulus of the AUC was used according to the following equation.
Corresponding to the signal deflection (positive or negative) the positive or the negative AUC was used for the construction of concentration response curves. The AUCs were normalized to the maximum response elicited by the highest histamine concentration (100% control) and assay medium (0% control) and plotted against the logarithmic ligand concentration. The pEC 50 values were calculated by applying the four parameters logistic equation (log(agonist) vs. response-variable slope (four parameters)). Real-time DMR traces are presented from representative experiments (mean ± SEM) with each trace reflecting the average of three technical replicates. Each experiment was performed at least three times to obtain at least three independent biological replicates.

Statistical Analyses
Statistical differences were analyzed using either the student t test (two-tailed) or oneway ANOVA followed by Dunnett's or Tukey's multiple comparisons test, as indicated in the corresponding Figures/Tables. All calculated p-values are two-sided and considered as statistically significant when lower than 0.5 indicated as * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001, **** p ≤ 0.0001. All calculations were performed using the GraphPad Prism 8 or 9 software (San Diego, CA, USA).

Data Availability Statement:
The data presented in this study are available on request from the first author.