New Insights from IGF-IR Stimulating Activity Analyses: Pathological Considerations.

Insulin-like growth factor-I (IGF-I) and insulin-like growth factor-II (IGF-II) play a crucial factor in the growth, differentiation and survival of cells in health and disease. IGF-I and IGF-II primarily activate the IGF-I receptor (IGF-IR), which is present on the cell surface. Activation of the IGF-IR stimulates multiple pathways which finally results in multiple biological effects in a variety of tissues and cells. In addition, activation of the IGF-IR has been found to be essential for the growth of cancers. The conventional view in the past was that the IGF-IR was exclusively a tyrosine kinase receptor and that phosphorylation of tyrosine residues, after binding of IGF-I to the IGF-IR, started a cascade of post-receptor events. Recent research has shown that this view was too simplistic. It has been found that the IGF-IR also has kinase-independent functions and may even emit signals in the unoccupied state through some yet-to-be-defined non-canonical pathways. The IGF-IR may further form hybrids with the insulin receptors but also with receptor tyrosine kinases (RTKs) outside the insulin-IGF system. In addition, the IGF-IR has extensive cross-talk with many other receptor tyrosine kinases and their downstream effectors. Moreover, there is now emerging evidence that the IGF-IR utilizes parts of the G-protein coupled receptor (GPCR) pathways: the IGF-IR can be considered as a functional RTK/GPCR hybrid, which integrates the kinase signaling with some IGF-IR mediated canonical GPCR characteristics. Like the classical GPCRs the IGF-IR can also show homologous and heterologous desensitization. Recently, it has been found that after activation by a ligand, the IGF-IR may be translocated into the nucleus and function as a transcriptional cofactor. Thus, in recent years, it has become clear that the IGF-IR signaling pathways are much more complex than first thought. Therefore a big challenge for the (near) future will be how all the new knowledge about IGF-IR signaling can be translated into the clinical practice and improve diagnosis and treatment of diseases.


Introduction
The insulin-IGF system is formed by insulin, two insulin-like growth factors (IGF-I and IGF-II), four cell-membrane receptors (insulin receptor-A (IR-A), insulin receptor-B (IR-B), insulin-like growth factor-I receptor (IGF-IR) and insulin-like growth factor receptor-II (IGF-II-R)) and six IGF-binding proteins (IGFBP-1-6), several IGFBP-related proteins and IGFBP proteases [1][2][3][4]. All IGFBPs can bind both IGF-I and IGF-II (however with different binding affinity for some) [5]. Only the unbound forms of IGFs are thought to interact with the IGF-IR and the IGF-II receptor [6].
The IGF-I gene comprises a highly conserved sequence and contains six exons, which give rise to heterogeneous mRNA transcripts by a combination of multiple transcription initiation sites and alternative splicing [7]. These multiple transcripts code in humans for different precursor IGF-I polypeptides, namely the IGF-IEa, IGF-IEb and IGF-IEc isoforms, which also undergo posttranslational

IGF-I and the IGF-I Receptor
The IGF-IR is displayed on the cell surface and expressed by nearly all human tissues and cell types [5,9,17]. Surface density of the IGF-IR represents an important determinant of the magnitude of responses to IGF-I and the signaling pattern it provokes [18]. The IGF-IR is a heterotetrameric transmembrane protein composed of two alpha and two beta subunits which are linked by disulfide bonds [19,20]. The beta subunit of the IGF-IR consists of a short extracellular domain which is involved in linkage to the alpha subunits, a transmembrane domain and a cytoplasmic domain containing tyrosine kinase activity [21]. The beta subunit contains a consensus ATP-binding sequence and multiple tyrosine residues that are phosphorylated following ligand binding to the alpha subunit [21]. Binding of IGF-I or another ligand to the alpha subunit of the IGF-IR, induces a closer proximity of regions within the transmembrane domain resulting in autophosphorylation of three intracellular tyrosine residues (Tyr 1131 , Tyr 1135 , and Tyr 1136 ) within the beta subunit [21][22][23].
The conventional view was that the IGF-IR was exclusively a tyrosine kinase receptor and that the binding of IGF-I to the IGF-IR was essential to start the intracellular downstream signal cascade ( Figure 1). In this model, the activated receptor recruited and phosphorylated intracellularly substrates as the insulin receptor substrate proteins (IRSs) and SH2 containing collagen-related proteins (SHC) (Figure 1). Tyrosine phosphorylation of the IRSs in turn activated then the phosphatidylinositol 3-kinase (PI3K-Akt) pathway and its various biological responses, while tyrosine phosphorylation Figure 1. The Insulin-like Growth Factor-I (IGF-IR) is a transmembrane protein composed of two alpha (α) and two beta (β) subunits. The conventional view was that the IGF-IR was exclusively a tyrosine kinase receptor and that the binding of IGF-I to the IGF-IR started the intracellular downstream signal cascade. In this model IGF-I or IGF-II binding to the IGF-IR promotes tyrosine kinase activity and autophosporylation of the beta subunit of the IGF-IR. Intracellularly the activated IGF-IR receptor recruits phosphorylated substrates Insulin receptor substrates (IRSs) and SH2 containing collagen-related proteins (SHC). Tyrosine phosphorylation of IRSs and SHC proteins induces downstream signaling activation through the PI3K-AKT and Ras/Raf/MEK/Erk pathways. It was further thought that activation of the PI3K-AKT pathway had predominantly metabolic effects whereas activation of the Ras/Raf/MEK/Erk pathway had predominantly mitogenic effects.

The IGF-IR and Endocytosis
Many signaling receptors internalize via clathrin-coated pits [26]. Endocytosis of signaling receptors is widely recognized to confer control on cellular signaling responsiveness [27]. Ligandinduced activation typically increases receptor endocytic rate, and internalized receptors engage molecular sorting machineries that specify subsequent transport via divergent lysosomal and recycling routes [27]. These events, in turn, determine the degree to which cellular ligand responsiveness is attenuated ("down-regulated") or sustained ("re-sensitized") under conditions of prolonged or repeated ligand exposure [27].
The molecular basis for the close interactions between IGF-IR endocytosis and its signaling components is still poorly understood. Recently, it has been suggested that the ability of insulin receptor substrate-1 (IRS-1) to interact with the clathrin adapter protein AP2, which is essential for endocytosis, plays an important role in IGF-IR internalization [28]. Overexpression of IRS-I resulted in the accumulation of activated IGF-IR at the cellular membrane [28]. Conversely, knockdown of IRS-I induced faster internalization of IGF-IRs [28]. These data suggest that IRS-1 inhibits the recruitment of IGF-IR into clathrin-coated structures; the ability of IRS-I to bind to AP-2 avoids rapid endocytosis of the IGF-IR and prolongs its activity at the cell surface in HEK293T cells [28] ( Figure  2A). In contrast, accelerating IGF-IR endocytosis via IRS-1 depletion induces the shift from sustained to transient Akt signaling [28] ( Figure 2B). Thus, independent of its classic role as an adaptor in IGF- Figure 1. The Insulin-like Growth Factor-I (IGF-IR) is a transmembrane protein composed of two alpha (α) and two beta (β) subunits. The conventional view was that the IGF-IR was exclusively a tyrosine kinase receptor and that the binding of IGF-I to the IGF-IR started the intracellular downstream signal cascade. In this model IGF-I or IGF-II binding to the IGF-IR promotes tyrosine kinase activity and autophosporylation of the beta subunit of the IGF-IR. Intracellularly the activated IGF-IR receptor recruits phosphorylated substrates Insulin receptor substrates (IRSs) and SH2 containing collagen-related proteins (SHC). Tyrosine phosphorylation of IRSs and SHC proteins induces downstream signaling activation through the PI3K-AKT and Ras/Raf/MEK/Erk pathways. It was further thought that activation of the PI3K-AKT pathway had predominantly metabolic effects whereas activation of the Ras/Raf/MEK/Erk pathway had predominantly mitogenic effects.

The IGF-IR and Endocytosis
Many signaling receptors internalize via clathrin-coated pits [26]. Endocytosis of signaling receptors is widely recognized to confer control on cellular signaling responsiveness [27]. Ligand-induced activation typically increases receptor endocytic rate, and internalized receptors engage molecular sorting machineries that specify subsequent transport via divergent lysosomal and recycling routes [27]. These events, in turn, determine the degree to which cellular ligand responsiveness is attenuated ("down-regulated") or sustained ("re-sensitized") under conditions of prolonged or repeated ligand exposure [27].
The molecular basis for the close interactions between IGF-IR endocytosis and its signaling components is still poorly understood. Recently, it has been suggested that the ability of insulin receptor substrate-1 (IRS-1) to interact with the clathrin adapter protein AP2, which is essential for endocytosis, plays an important role in IGF-IR internalization [28]. Overexpression of IRS-I resulted in the accumulation of activated IGF-IR at the cellular membrane [28]. Conversely, knockdown of IRS-I induced faster internalization of IGF-IRs [28]. These data suggest that IRS-1 inhibits the recruitment of IGF-IR into clathrin-coated structures; the ability of IRS-I to bind to AP-2 avoids rapid endocytosis of the IGF-IR and prolongs its activity at the cell surface in HEK293T cells [28] (Figure 2A). In contrast, accelerating IGF-IR endocytosis via IRS-1 depletion induces the shift from sustained to transient Akt signaling [28] ( Figure 2B). Thus, independent of its classic role as an adaptor in IGF-I receptor signaling, IRS-1 has a role as an endocytic regulator of IGF-I receptor that ensures sustained IGF bioactivity, while IRS-1 degradation could be a trigger to internalize the IGF-IR [29].
Cells 2020, 9, x FOR PEER REVIEW 4 of 16 I receptor signaling, IRS-1 has a role as an endocytic regulator of IGF-I receptor that ensures sustained IGF bioactivity, while IRS-1 degradation could be a trigger to internalize the IGF-IR [29].  For the IGF-IR, ubiquitination also increases upon ligand binding [30]. The IGF-IR has been demonstrated to be a substrate for three ubiquitin ligases: Mdm2, (in human malignant melanoma cells), c-Cbl (HEK293 cells and human osteosarcoma cell lines U2OS and SAOS2) and Nedd4 (in mouse embryo fibroblasts). [30,31]. Mdm2 was originally described to control IGF-IR ubiquitination and thereby causing its degradation by the proteasome system [32]. Subsequently β-arrestins, known to be involved in the regulation of G-protein-coupled receptors (GPCRs), have also been identified as adaptor proteins to bring the oncoprotein Mdm2 to the IGF-IR in mouse embryo fibroblasts [33,34]. In addition, while removing the IGF-IR from the cell surface and inhibiting the "classical" kinase signaling pathway, β-arrestins may redirect the signaling wave through ERK [33] Ubiquitination may thus induce receptor internalization and degradation, but also enhance IGF-IR signaling [35] (which will be further addressed in the paragraph "the complexity of the post-receptor IGF-IR/IR pathways" below).

Structural Differences and Overlap between the IGF-IR and the IRs
It is hypothesized that the IGF-IR and IRs are created by gene duplication of common precursor receptor molecule [36]. Due to structural and functional homology, IGF-I and insulin can bind to (and activate) both IGF-IR and the IRs, as discussed above [37]. IGF-IR and IRs show 48% amino acid sequence homology [20]. Structural differences between the beta-subunit and kinase domains of the IGF-IR and the IRs leading to differences in substrate interactions may be (partly) responsible for IGF-I and insulin specificity as has been found in various cell types (rat-1 fibroblasts, murine skin keratinocytes and in NIH-3T3-fibroblasts) [38]. However, the signal transduction by the receptors may not be limited to its activation at the cell surface [39].
In addition to signaling through the classical tyrosine kinase pathways, it has been found that the IGF-IRs and IRs (in cells derived from C57Bl/6 mice) can emit signals in the unoccupied state through some yet-to-be-defined non-canonical pathways [40]. Boucher et al. demonstrated that cells lacking the IGF-IR and IR exhibit a major decrease in expression of multiple imprinted genes and microRNAs [40].
Although the IGF-IR and IRs have both distinct and overlapping functions, it has been suggested that in vivo specificity of the IGFs and insulin are at least in part reflected by the timing of the expression of the IGF-IR and IRs in target tissues in combination with ligand concentration and availability [41]. It has been further suggested that IGF-I and the IRs act as identical portals for the regulation of gene expression and that the differences between IGF-I and insulin effects are due to a modulation of the amplitude of the signal created by the specific ligand receptor interaction [41].

The IGF-IR and the IRs May Form Hybrids in the Human Body
In cells and tissues where both significant levels of the IGF-IRs and IRs are present, hybrids may be formed consisting of an alpha-beta subunit of the IGF-IR linked by disulfide bonds to an alpha-beta subunit of the IR [42] ( Figure 3A). They are formed in the endoplasmic reticulum before they reach the cell surface [43]. Two splice variants of the hybrid receptors exist for the IR because the IR is expressed (as above discussed) either with (IR-B) or without 12 amino acids encoded by exon 11 (IR-A) [44]. Thus both IR-A/IGF-IR (Hybrid A) and IR-B/IGF-IR (Hybrid B) receptors can be formed. Although the biological functions of these hybrid receptors is still unclear, it has been suggested that hybrid receptors may play a role in the overlapping functions of IGF-I and insulin [21]. Several studies (in baby hamster kidney cells, NIH3T3 cells overexpressing IGFR and CHO cells overexpressing IR-B) have suggested that cells by increasing the relative expression level of the IGF-IR above that of the IR lose their insulin sensitivity because hybrid receptors bind insulin with low affinity [45,46]. In addition, binding of insulin to the alpha-beta subunit of the IR which is part of a hybrid, may result in autophosphorylation of its ow beta subunit and, following transphosphorylation of the beta subunit of the IGF-IR, may result in a signal for growth, [9]. In contrast, when IGF-I binds to the alpha-beta subunit of the IGF-IR, this may activate the beta subunit of the IRs by the same mechanisms and thereby activate growth (IR-A) or metabolism (IR-B) [9]. Although this latter mechanism could explain why hybrids may stimulate metabolic functions when stimulated by IGF-I, most functional studies Cells 2020, 9, 862 6 of 17 have found that hybrid receptors behave more like IGF-IRs than IRs [47]. It has been hypothesized that this prioritization of hybrid receptors to IGF-I results from the ability of IGF-I to activate monomeric IGF-IR whereas, in contrast, dimerization of the IR has been considered necessary to induce a response to insulin [45].
Cells 2020, 9, x FOR PEER REVIEW 6 of 16 Although this latter mechanism could explain why hybrids may stimulate metabolic functions when stimulated by IGF-I, most functional studies have found that hybrid receptors behave more like IGF-IRs than IRs [47]. It has been hypothesized that this prioritization of hybrid receptors to IGF-I results from the ability of IGF-I to activate monomeric IGF-IR whereas, in contrast, dimerization of the IR has been considered necessary to induce a response to insulin [45]. It has been further hypothesized that IGF-IR/IR hybrids may affect tumor biology [48]. Specific downregulation of the IGF-IR by agents solely targeting the IGF-IR diminishes hybrid formation and this thereby enhances holo-IR formation [48]. An enhanced holo-IR formation results in an increase of insulin sensitivity [48]. As the IR, especially the IR-A, may also activate (post-receptor) signaling pathways involved in growth similar to the IGF-IR, the development of agents simultaneous targeting both IR-A and IGF-IR may be necessary to disrupt the malignant phenotype of cancers cells that are influenced by actions of the insulin-IGF system [48].
The IGF-IR may also heterodimerize with receptor tyrosine kinases (RTKs) outside the insulin-IGF system [49]. Heterodimerization of the IGF-IR with the EGFR is well-established [50] ( Figure 3B). Downstream signaling of both receptors converge via the canonical PI3K-Akt and ERK signaling pathways. Therefore inhibition of one receptor in these hybrids may shift the signaling pathway in favor of the other available counterpart receptor [49,51,52]. Thus the compensatory signaling may be bidirectional [53]. Moreover, evidence exists that IGF-IR can activate independently downstream EGFR pathways and this may subsequently result in EFGR tyrosine kinase inhibitor (TKI) resistance [52]. The IGF-IR signaling pathway shows also cross-talk with the growth hormone receptor (GHR), In cells and tissues where both significant levels of the IGF-IRs and IRs are present, hybrids may be formed consisting of an alpha-beta subunit of the IGF-IR linked by disulfide bonds to an alpha-beta subunit of the IR [42] (Figure 3A). They are formed in the endoplasmic reticulum before they reach the cell surface [43]. Two splice variants of the hybrid receptors exist for the IR because the IR is expressed (as above discussed) either with (IR-B) or without 12 amino acids encoded by exon 11 (IR-A) [44]. Thus both IR-A/IGF-IR (Hybrid A) and IR-B/IGF-IR (Hybrid B) receptors can be formed. Although the biological functions of these hybrid receptors is still unclear, it has been suggested that hybrid receptors may play a role in the overlapping functions of IGF-I and insulin [21]. Several studies (in baby hamster kidney cells, NIH3T3 cells overexpressing IGFR and CHO cells overexpressing IR-B) have suggested that cells by increasing the relative expression level of the IGF-IR above that of the IR lose their insulin sensitivity because hybrid receptors bind insulin with low affinity [45,46]. In addition, binding of insulin to the alpha-beta subunit of the IR which is part of a hybrid, may result in autophosphorylation of its ow beta subunit and, following transphosphorylation of the beta subunit of the IGF-IR, may result in a signal for growth, [9]. In contrast, when IGF-I binds to the alpha-beta subunit of the IGF-IR, this may activate the beta subunit of the IRs by the same mechanisms and Cells 2020, 9, 862 7 of 17 thereby activate growth (IR-A) or metabolism (IR-B) [9]. Although this latter mechanism could explain why hybrids may stimulate metabolic functions when stimulated by IGF-I, most functional studies have found that hybrid receptors behave more like IGF-IRs than IRs [47]. It has been hypothesized that this prioritization of hybrid receptors to IGF-I results from the ability of IGF-I to activate monomeric IGF-IR whereas, in contrast, dimerization of the IR has been considered necessary to induce a response to insulin [45].
It has been further hypothesized that IGF-IR/IR hybrids may affect tumor biology [48]. Specific downregulation of the IGF-IR by agents solely targeting the IGF-IR diminishes hybrid formation and this thereby enhances holo-IR formation [48]. An enhanced holo-IR formation results in an increase of insulin sensitivity [48]. As the IR, especially the IR-A, may also activate (post-receptor) signaling pathways involved in growth similar to the IGF-IR, the development of agents simultaneous targeting both IR-A and IGF-IR may be necessary to disrupt the malignant phenotype of cancers cells that are influenced by actions of the insulin-IGF system [48].

The Functional Relationship between Insulin/IGF Signaling and Discoidin Domain Receptors
In addition to its canonical role as a collagen receptor, it has recently been suggested that the discoidin domain receptor-1 (DDR1), a tyrosine kinase receptor, plays an important role in the regulation of the insulin-IGF system [60]. In contrast to most other RTKs, DDR1 is not activated by soluble growth factors but instead by various type of collagens [60]. While most other RTKs are fully activated in minutes, maximal activation of DDR1 occurs several hours after initial stimulation with collagen [60]. The DDR1 and the insulin-IGF system are linked by a feed-forward mechanism by which insulin and the IGFs induce DDR1 upregulation which in turn enhances expression and activity of the IRs and the IGF-IR [60]. The mechanisms by which DDR1 may affect downstream signaling of the IRs and IGF-IR are as yet not fully understood. It has been found that increasing DDR1 expression favors the expression of the more mitogenic IR-A isoform over the metabolic IR-B isoform and thus one of the functional consequences of this DDR1 upregulation may be increased IGF-II signaling through the IR-A [60]. This in turn may favor dedifferentiation and stem-like features [60]. It has been further hypothesized that inhibition of DDR1 may be a way to downregulate the tumor-inducing actions of the insulin/IGF system in human cancer cells while simultaneously inducing differentiation of cells without affecting the IR-B mediated metabolic actions [60]. In favor of this latter hypothesis, in vitro DDR1 silencing or downregulation blocked the IGF-2/IR-A autocrine loop in poorly differentiated thyroid cancer cells and induced cellular differentiation [61]. Although at present no clinical studies have shown that this strategy provides any clinical benefits for patients with tumors overexpressing the DRR1 and the insulin-IGF-I system, these results may help to develop novel therapeutic approaches for cancer.

The Complexity of the Post-Receptor IGF-IR/IR Pathways
In recent years, it has been become clear that the downstream complexity of the IGF-IR/IR pathways was grossly underestimated in the past. In this section we will discuss some important novel insights regarding this complexity. In the classical model, as discussed above, the IGF-IR was Cells 2020, 9, 862 8 of 17 traditionally described as a tyrosine kinase receptor with an ON/OFF (active/inactive) system. In this system IGF-I binding to the IGF-IR stabilized the ON state (active) and this exclusively mediated kinase-dependent signaling activation of both the PI3-AKT and ERK pathways [62,63] (Figure 4A). Ubiquitin-mediated receptor downregulation and degradation was originally described as a response to ligand/receptor interaction and thus inseparable from kinase signaling activation [62].
develop novel therapeutic approaches for cancer.

The Complexity of the Post-Receptor IGF-IR/IR Pathways
In recent years, it has been become clear that the downstream complexity of the IGF-IR/IR pathways was grossly underestimated in the past. In this section we will discuss some important novel insights regarding this complexity. In the classical model, as discussed above, the IGF-IR was traditionally described as a tyrosine kinase receptor with an ON/OFF (active/inactive) system. In this system IGF-I binding to the IGF-IR stabilized the ON state (active) and this exclusively mediated kinase-dependent signaling activation of both the PI3-AKT and ERK pathways [62,63] (Figure 4A). Ubiquitin-mediated receptor downregulation and degradation was originally described as a response to ligand/receptor interaction and thus inseparable from kinase signaling activation [62].  Almost 25 years ago, a study from Nobel Prize winner Dr. Robert Lefkowitz's laboratory reported that IGF-IR-dependent activation of the Mitogen-Activated Protein Kinase (MAPK) signaling pathway was inhibited by the Gαi-inhibitor pertussis toxin [64]. The last years there is emerging evidence that many RTKs can also utilize heterotetrameric G proteins to subserve some of their biological actions [65][66][67]. Recently, more extensive evidence has been published that the IGF-IR and IRs are also engaged in in G-protein coupled receptor (GPCR) signaling [62,63,[65][66][67]. As GPCR phosphorylation by GPCR-kinases (GRKs) governs interactions of the receptors with β-arrestins, Zheng et al. investigated the regulatory roles of the four widely expressed GRKs on IGF-IR signaling/degradation [68]. They found that lowering GRK5/6 abolished IGF-I-mediated ERK and AKT activation, whereas GRK2 inhibition increased ERK activation and partially inhibited AKT signaling [68]. In addition, β-arrestin-mediated ERK signaling was enhanced by overexpression of GRK6 and diminished by GRK2. Similarly, they demonstrated opposing effects of GRK2 and -6 on IGF-IR degradation: GRK2 decreased whereas GRK6 enhanced ligand-induced degradation [68]. GRK2 and GRK6 co-immunoprecipitated with IGF-IR and increased IGF-IR serine phosphorylation, promoting β-arrestin1 association. Thus this study demonstrated distinct roles for GRK2 and GRK6 in IGF-IR signaling through β-arrestin binding with divergent functional outcomes [68].
Based on the insight that IGF-IR may also "borrow" components of GPCR signaling, including β-arrestins and G-protein-coupled-receptor kinases (GRKs), a new paradigm has emerged for the IGF-IR [62]. In this new paradigm, the IGF-IR is considered to be a functional RTK/GPCR hybrid, which integrates the kinase signaling with some IGF-IR mediated canonical GPCR characteristics [62]. Binding of IGF-I to the IGF-IR thus not only leads to balanced phosphorylation-dependent Akt/ERK signaling intracellularly, but results simultaneously also in activation of signaling by G-proteins and β-arrestins [62] (Figures 4B and 5A).
β-arrestins and G-protein-coupled-receptor kinases (GRKs), a new paradigm has emerged for the IGF-IR [62]. In this new paradigm, the IGF-IR is considered to be a functional RTK/GPCR hybrid, which integrates the kinase signaling with some IGF-IR mediated canonical GPCR characteristics [62]. Binding of IGF-I to the IGF-IR thus not only leads to balanced phosphorylation-dependent Akt/ERK signaling intracellularly, but results simultaneously also in activation of signaling by Gproteins and β-arrestins [62] (Figures 4B and 5A). The IRs have been shown also to interact with Gproteins and β-arrestin-1 [69]. However, the IGF-IR and IRs engage in different G-proteins for downstream signaling [65]. This possibly provides a mechanism that is responsible for the signaling specificity of these two receptors [65]. Also another new paradigm, the paradigm of biased signaling, has been proposed for IGF-IR and IR signaling [67,70]. The paradigm of biased signaling also originates from the GPCR signaling field [71]. The regulatory process which was discovered as the means by which classical GPCRs "desensitized" or tuned off, has been also found to be active for the IGF-IR [71]. In this paradigm the ligand is biased towards a specific signaling i.e., the signal mediated by binding of a ligand to a receptor is no longer balanced but the ligand elicits one response of the ligand over another compared with the classical ligand [53,63]. The IGF-IR has been extensively studied as an anti-cancer target However, monotherapy trials with IGF-IR targeted antibodies, have, overall, been very disappointing [12]. The anti-IGF-IR antibody Figitumumab (CP-751,871; CP) was designed as an antagonist to prevent ligand-receptor interaction [72]. Although it was found that CP blocked the kinase cascade pathway (by blocking phosphorylation of IRS-1, PI3K and AKT), as with all anti-IGF-IR antibodies, The IRs have been shown also to interact with G-proteins and β-arrestin-1 [69]. However, the IGF-IR and IRs engage in different G-proteins for downstream signaling [65]. This possibly provides a mechanism that is responsible for the signaling specificity of these two receptors [65].
Also another new paradigm, the paradigm of biased signaling, has been proposed for IGF-IR and IR signaling [67,70]. The paradigm of biased signaling also originates from the GPCR signaling field [71]. The regulatory process which was discovered as the means by which classical GPCRs "desensitized" or tuned off, has been also found to be active for the IGF-IR [71]. In this paradigm the ligand is biased towards a specific signaling i.e., the signal mediated by binding of a ligand to a receptor is no longer balanced but the ligand elicits one response of the ligand over another compared with the classical ligand [53,63]. The IGF-IR has been extensively studied as an anti-cancer target However, monotherapy trials with IGF-IR targeted antibodies, have, overall, been very disappointing [12]. The anti-IGF-IR antibody Figitumumab (CP-751,871; CP) was designed as an antagonist to prevent ligand-receptor interaction [72]. Although it was found that CP blocked the kinase cascade pathway (by blocking phosphorylation of IRS-1, PI3K and AKT), as with all anti-IGF-IR antibodies, it simultaneously induced IGF-IR/β-arrestin-1 association with dual functional outcome: receptor ubiquitination and degradation and decrease in cell viability and β-arrestin-1-dependent ERK signaling activation [72].
Thus despite blocking all the "classical" tyrosine kinase-mediated effects of the IGF-IR, a blocking antibody directed against the IGF-IR may function as an IGF-IR/β-arrestin-1/ERK agonist and favor β-arrestin-1/ERK signaling [73] ( Figure 5B). Another example of biased signaling of the IGF-IR signaling is mediated by LL-37, a newly recognized bacterial peptide, which after binding to the IGF-IR may function as an IGF-IR agonist by increasing β-arrestin-1 signaling and electively activating the ERK pathway but without affecting simultaneously the PI3K-AKT pathway [74].
One should make a distinction between homologous and heterologous desensitization. Homologous desensitization occurs within a receptor system when it alters its own responsiveness, for example the loss of responsiveness (desensitization) that can occur upon binding of insulin to the IR [75]. It is considered to limit or restrain a cell's responses to certain stimuli; it leaves a cell (transiently) less-or unresponsive to a ligand that activates the desensitized receptor but not to ligands that activate other receptors. In contrast, in heterologous desensitization the responsiveness of one receptor system is regulated (positively or negatively) by activation of another receptor system (i.e., "cross-talk") [75]. For example, insulin after binding to the IR may induce heterologous desensitization of the signaling of the IGF-IR by downregulating β-arrestin-1 and inhibiting of IGF-I-stimulated MAP kinase phosphorylation [76]. However, it has been found that this latter effect could be substantially rescued by ectopic expression of wild-type β-arrestin-1, consistent with the view that the decrease in cellular β-arrestin-1 content is a major mechanism for the observed desensitization effects of insulin on IGF-IR mediated signaling [76].

The Nuclear Translocation of the IGF-IR and IRs and Its Significance
The IGF-IR and IRs not only function at the cell surface. When after binding of IGF-I to the IGF-IR and the IGF-I/IGF-IR complex has been internalized into the cell, there are three potential outcomes for the internalized IGF-IR: it can go back to the cellular surface, it can be degraded or it can go to the nucleus [77].
It has been documented that both the IGF-IR and IRs can be translocated to the nucleus [78,79]. Nuclear transport of IGF-IR is enhanced by IGF-I and IGF-II but only modestly by insulin [79]. This transport correlated directly with the magnitude of ligand-induced receptor phosphorylation of the IGF-IR with these ligands [79]. In addition, it has been found that ligand-mediated phosphorylation of the IGF-IR is essential for nuclear trafficking [78].
IGF-IR nuclear import and chromatin binding can be blocked by an IGF-IR kinase inhibitor, indicating that indeed IGF-IR kinase activity is required for the IGF-IR to enter the nucleus [79].
The IGF-IR can undergo both caveolin-and clathrin mediated endocytosis [80,81]. Consistent with clathrin-mediated endocytosis, nuclear IGF-IR translocation can be blocked by the inhibitors of clathrin-dependent endocytosis (dansylcadaverine and the dynamin-1 inhibitor dynasore), but not by caveolin-1 depletion [79]. Nevertheless, the exact mechanisms responsible for nuclear import of the IGF-IR and IRs are still unclear [78]. Sehat et al. found that the α subunit (native size, 120 kD) together with the β subunit (native size, 95 kD) was present in the nuclear fraction, suggesting that nuclear IGF-IR was an intact receptor [82]. Data of Aleksic et al. also suggested that full-length IGF-IR translocates to the nucleus [79].
Both the IGF-IR and IRs are present in the perinuclear and nucleolar area of the nucleus in a small ubiquitin-like modifier SUMOylated form [78]. Receptor SUMOylation occurs in a ligand dependent fashion and it has been demonstrated that SUMOylation plays a crucial role in the nuclear translocation of the IGF-IR [82,83] (Figure 6). The SUMO-modified IGF-IR is deSUMOylated after passage across the nuclear membrane [82]. Nuclear IGF-IR binds to enhancer regions and activates transcription [84]. It is able to autoregulate expression of its own gene leading to an increase in IGF-IR promoter activity and IGF-IR expression [78] (Figure 6). clathrin-mediated endocytosis, nuclear IGF-IR translocation can be blocked by the inhibitors of clathrin-dependent endocytosis (dansylcadaverine and the dynamin-1 inhibitor dynasore), but not by caveolin-1 depletion [79]. Nevertheless, the exact mechanisms responsible for nuclear import of the IGF-IR and IRs are still unclear [78]. Sehat et al. found that the α subunit (native size, 120 kD) together with the β subunit (native size, 95 kD) was present in the nuclear fraction, suggesting that nuclear IGF-IR was an intact receptor [82]. Data of Aleksic et al. also suggested that full-length IGF-IR translocates to the nucleus [79].
Both the IGF-IR and IRs are present in the perinuclear and nucleolar area of the nucleus in a small ubiquitin-like modifier SUMOylated form [78]. Receptor SUMOylation occurs in a ligand dependent fashion and it has been demonstrated that SUMOylation plays a crucial role in the nuclear translocation of the IGF-IR [82,83] (Figure 6). The SUMO-modified IGF-IR is deSUMOylated after passage across the nuclear membrane [82]. Nuclear IGF-IR binds to enhancer regions and activates transcription [84]. It is able to autoregulate expression of its own gene leading to an increase in IGF-IR promoter activity and IGF-IR expression [78] (Figure 6). Figure 6. Studies have confirmed that ligand-mediated phosphorylation of the IGF-IR is essential for nuclear trafficking. Following binding of IGF-I to the IGF-IR at the cell surface, the IGF-IR is transported into the cell and further translocated from the cytoplasm into the nucleus. SUMOylation (SUMO-1) in the cytoplasm of the IGF-IR also plays a crucial role in the nuclear translocation of the IGF-IR from the cytoplasm. When the SUMOylated IGF-IR translocates to the nucleus, it is thought to be involved in the transcriptional enhancement of specific target genes. Nuclear IGF-IR is able to autoregulate expression of its own gene leading to an increase in IGF-IR promoter activity and IGF-IR expression (left). Nuclear IGF-IR may also bind to Cyclin D1 (and additional) promoters with ensuing target gene activation (right). (Modified from Sarfstein & Werner. Minireview: nuclear insulin and insulin-like growth factor-1 receptors: a novel paradigm in signal transduction. Endocrinology, 2013; 154:1672-9).
It has been further suggested that nuclear IGF-IR has biological significance in cancer; prognosis was less good and survival was shorter in patients whose tumor showed intense and/or widespread nuclear IGF-IR [79]. It has been reported that nuclear IGF-IR is a feature of pre-invasive lesions and invasive cancers including prostate, renal and breast cancers, and an association between nuclear IGF-IR and adverse prognosis was identified in renal cancer [79]. Subsequent data did associate nuclear IGF-IR with proliferation, tumorigenicity, resistance to EGFR inhibition and clinical response to therapeutic anti-IGF-IR antibodies, which suggests that IGF-IR nuclear import has biological significance and may contribute directly to IGF-IR function [79,[85][86][87][88][89][90].
When the IGF-IR translocates to the nucleus, it is thought to be involved in the transcriptional enhancement of specific target genes [79,82,91]. It has been demonstrated that IGF-IR in the nucleus binds to the transcription factor lymphoid enhancer factor-1 (LEF1), leading to elevated protein levels of cyclin D1 and axin2 [84] (Figure 6). This might be an additional molecular mechanism by which IGF-IR promotes uncontrolled cell proliferation and contribute to the neoplastic transformation of cells [84].
When investigating the impact of IGF-IR levels on IGF-IR biosynthesis in estrogen receptor positive (ER+) and estrogen receptor depleted (ER-) breast cancer cells, it was found that in ER+ cell and ER-cells regulation of the IGF-IR gene and IGF-IR protein differed at the level of transcription; the IGF-IR protein was able to stimulate IGF-IR gene expression in ER-cells but not in ER+ cells [92]. Similarly to the IGF-IR, it was found that the IR was also translocated to the nucleus and to bind to the IGF-IR promoter. However, this was only observed in ER-cells but not in ER+ cells [92].
In addition, it has been found that transcription factors IGF-IR and IR display diametrically opposite activities in the context of IGF-IR gene regulation; in contrast to the IGF-IR, IR inhibited IGF-IR promoter activity [92]. Thus nuclear IGF-IR acted as a transcriptional activator of its own promoter, while nuclear IR functioned as a negative regulator of IGF-IR promoter activity [92]. Nevertheless, the authors of this latter paper concluded that the clinical implications of their findings-in particular the impact of IGF-IR/IR nuclear localization on targeted therapy-are at present unclear and require further investigation [92].

Conclusions
Although until recently the conventional view was that phosphorylation of tyrosine residues played a major role in the activation of the IGF-IR and initiated all downstream signaling, there is increasing evidence showing that this view was too simplistic and grossly underestimated the downstream complexity of the IGF-IR pathways. The IGF-IR has not only extensive cross-talk with many other receptors, but that the IGF-IR can be also considered as a functional RTK/GPCR hybrid, which integrates the kinase signaling with some IGF-IR mediated canonical GPCR characteristics. Like classical GPCRs the IGF-IR show homologous and heterologous desensitization. In addition, after activation by a ligand, the IGF-IR signaling can be translocated to the nucleus and function as a transcriptional cofactor. For example nuclear IGF-IR is able to autoregulate expression of its own gene.
Thus, it has become clear in recent years that the IGF-IR signaling pathway is far more complex than previously thought. It contains many points of regulation and shows signal divergence and cross-talk with many other signaling pathways at the receptor and post-receptor level. However, a big challenge for the (near) future will be how all this new knowledge about the IGF-IR signaling pathways can be translated into clinical practice and improve diagnosis and treatment of diseases.
Funding: This research received no external funding.