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Minireview: Integral Membrane Proteins that Function Coordinately with the Insulin-Like Growth Factor I Receptor to Regulate Intracellular Signaling

Department of Endocrinology and Metabolism, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599

Address all correspondence and requests for reprints to: David R. Clemmons, M.D., 6111 Thurston Bowles, CB 7170, Department of Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599. E-mail: dpm{at}med.unc.edu.

	Abstract

Top Abstract Introduction Integrin-Linked Signaling Transmembrane Adapter Proteins General Aspects of IGF-IR-Linked... Role of Integrins in... Mechanisms by which Ligand... Role of IAP References

 
Integral membrane proteins that are present on cell surfaces bind to extracellular ligands, and this binding influences multiple cellular processes. Three cell surface proteins, Vß3 integrin, integrin associated protein, and SHPS-1, have been shown to modulate both IGF-I receptor-linked signaling and cellular growth and migration responses that are stimulated by IGF-I. Ligand occupancy of these three proteins influences the recruitment of the phosphatase SHP-2 to the IGF-I receptor and thereby modulates the duration of IGF-I receptor tyrosine phosphorylation. In addition, changes in ligand occupancy of these three integral membrane proteins can regulate the transfer of SHP-2 phosphatase to downstream signaling molecules, which is also required for stimulation of cell migration and DNA synthesis by IGF-I. Determination of the spectrum of ligands for these three integral membrane proteins and the mechanisms by which each ligand functions to alter IGF-I signaling are important objectives of future research.

	Introduction

Top Abstract Introduction Integrin-Linked Signaling Transmembrane Adapter Proteins General Aspects of IGF-IR-Linked... Role of Integrins in... Mechanisms by which Ligand... Role of IAP References

 
THE RECEPTOR FOR IGF-I and -II is a member of the tyrosine kinase growth factor receptor family (1). Activation of this receptor and its intrinsic tyrosine kinase activity initiates a signaling cascade that involves multiple intracellular signaling pathways, such as the phosphatidylinositol 3-kinase (PI3K) and MAPK pathways, that are responsible for the diverse target actions of these growth factors, which include increases in cell division, cell size, protein synthesis, cell migration, and inhibition of apoptosis (2, 3, 4). Many of the signaling elements that are downstream from tyrosine kinase activation have been characterized, but several of the molecular events that occur before or concomitantly with kinase activation and whether these early events can influence the ultimate outcome of IGF-I-stimulated signal transduction have not been determined.

Although cell type-specific responses to the IGFs are determined in part by genetically preprogrammed variables, changes in the cellular environment function to alter signaling. These environmental cues allow cells to detect a number of inputs from several receptor-based signaling systems that function to alter their responsiveness to growth factor stimulation. One important cue is mediated by integrin receptors. Integrins are heterodimers that contain one - and ß-chain that bind to extracellular matrix (ECM) molecules and transduce signals to the intracellular environment (5). Integrin activation is usually analyzed by detaching cells from their ECM with trypsin then allowing them to reattach to defined ECM components. Although this is a disruptive event, much has been learned about integrin signaling using this model. Following reattachment, the composition of the ECM as well as the specific integrin that is activated determines the signaling event(s) that will ensue. Several examples in which this model system has been used have shown that activation of integrins often results in alteration of growth factor signaling, and specific examples have been described using the epithelial growth factor-, fibroblast growth factor-, platelet-derived growth factor-, and IGF-I-linked signaling systems (6, 7, 8, 9, 10). However, multiple cellular actions of growth factors such as stimulation of protein synthesis, increases in cell size, and inhibition of apoptosis do not involve cellular detachment and reattachment. Therefore, whether stably attached cells that have ECM ligands bound to their integrins can change cellular responsiveness to growth factors is an important question that has received limited investigation.

	Integrin-Linked Signaling

Top Abstract Introduction Integrin-Linked Signaling Transmembrane Adapter Proteins General Aspects of IGF-IR-Linked... Role of Integrins in... Mechanisms by which Ligand... Role of IAP References

 
Most integrins have a relatively short cytoplasmic domain. These domains do not contain intrinsic tyrosine kinase activity, but they do have binding sites for intracellular signaling molecules (11). After binding to their ECM ligands, integrin receptors can undergo tyrosine phosphorylation that subsequently facilitates binding of tyrosine kinases (12) such as focal adhesion kinase (FAK) and SRC kinase (13, 14). The phosphorylation of specific tyrosines contained in cytoplasmic domains can also result in changes in cytoskeletal protein localization and polymerization (15) as well as recruitment of binding partners to integrins that can directly interact with important signaling elements (15, 16). Another set of cell surface proteins that function coordinately with integrins to sense changes in extracellular environmental cues are the cadherins (17). VE, E, and N cadherins sense cell-cell contacts; following cell-cell contact at high density, they change their binding to and recruitment of molecules to integrin cytoplasmic domains (18).

In addition to integrin association with cytoskeletal proteins, the extracellular domains of integrins have been shown to not only bind to ECM proteins but also associate with cell surface membrane proteins. This association in some cases has been shown to alter integrin signaling (19). An important example is the association between integrin-associated protein (IAP) and the Vß3 integrin. IAP is a five-transmembrane domain protein with a long extracellular IgV-like domain (20). Upon appropriate stimulation, this protein can bind to Vß3 and increase its avidity for ligands (21, 22). In addition, IAP binding has been shown to alter the types of intracellular signaling molecules that are recruited to Vß3 (23). Importantly, the extracellular domain of IAP can also bind to ECM proteins, and this binding event may alter both its interaction with integrins as well as its ability to activate integrins. A well-characterized example of an ECM protein that binds to both IAP and Vß3 is thrombospondin-1 (TSP-1) (24). TSP-1 binds to these two proteins at different binding sites, and ligand occupancy of both proteins by TSP-1 results in increased activation of signaling molecules that associate with Vß3. Thus, ligand occupancy of IAP results in a stronger TSP-1-Vß3 bond and enhanced intracellular signaling by Vß3 (23, 24). This has been shown to be important in T cell adherence as well as T cell activation (25). Thus, integral membrane proteins that are associated with integrins can play an important role in modulating cellular responsiveness to integrin simulation that occurs following ligand occupancy. These proteins also have the potential to modulate signaling that is coupled between integrins and growth factor receptors.

	Transmembrane Adapter Proteins

Top Abstract Introduction Integrin-Linked Signaling Transmembrane Adapter Proteins General Aspects of IGF-IR-Linked... Role of Integrins in... Mechanisms by which Ligand... Role of IAP References

 
Adapter proteins are multidomain proteins that contain distinct domains that allow them to bring different types of signaling molecules into close approximation; therefore, they function as scaffolds for multiple intracellular signaling proteins. Insulin receptor substrate-1 (IRS-1) is an adaptor and recruits signaling molecules that are important for IGF-I receptor (IGF-IR) and insulin receptor-linked signal transduction (26). Some types of integral membrane proteins contain binding motifs within their intracellular domains that are similar to those contained in adapter proteins, and they can function as adapters in a manner similar to IRS-1. An excellent example of such a protein is SH2 domain-containing tyrosine phosphatase substrate 1 (SHPS-1), or signal regulatory protein , a transmembrane protein that contains a large extracellular region that contains one IgV and two IgC domains (27). This extracellular region has the potential to bind to extracellular ligands, and its function can be modified by differential ligand occupancy (28). The intracellular domain of this protein also contains two Src homology 2 recognition domains (29). When tyrosine residues in these two domains are phosphorylated they bind to intracellular signaling molecules that recognize these YXXL/V motifs. Therefore, SHPS-1 has the potential to recruit signaling molecules to the cytoplasmic interface of the cell membrane and focally concentrate them near molecular targets before downstream activation of intracellular signaling pathways (30). An excellent example of recruitment involving SHPS-1 and its ability to modulate growth factor receptor function is the recruitment of the tyrosine phosphatase, SHP-2. SHPS-1 is phosphorylated on tyrosine residues that are located within consensus sequences for Src homology 2 domain-containing proteins that bind in response to ligand occupancy of growth factor receptors. This results in recruitment of SHP-2 to SHPS-1 (30, 31).

Following recruitment, SHP-2 phosphatase activity is activated and it dephosphorylates SHPS-1. This has been shown to be followed by its association with the insulin, epithelial growth factor, or GH receptors (32, 33, 34). Overexpression of SHPS-1 has been shown to result in down-modulation of the tyrosine kinase activity of these receptors, presumably through SHP-2-mediated dephosphorylation (29). One important ligand of SHPS-1 that has been identified is IAP. Cell surface IAP can bind to the extracellular domain of SHPS-1 and modulate its ability to function as a self-recognition molecule (28). However, whether there are other ligands for SHPS-1 or IAP and whether these molecules can function to alter SHPS-1 phosphorylation or growth factor-linked intracellular signaling has not been reported. The purpose of this review is to summarize experimental results that pertain to the IGF system that suggest that it may be a model system for studying the interaction of all four proteins and thus develop a more comprehensive picture of IGF-IR-linked signaling.

	General Aspects of IGF-IR-Linked Signaling

Top Abstract Introduction Integrin-Linked Signaling Transmembrane Adapter Proteins General Aspects of IGF-IR-Linked... Role of Integrins in... Mechanisms by which Ligand... Role of IAP References

 
Almost all signaling studies that have analyzed IGF-I/IGF-IR functions have not taken into account the potential modifying effects of the differential ligand occupancy of other integral membrane protein-signaling partners. Nevertheless, these studies have yielded a great deal of information about the variables that are necessary to activate the IGF-IR tyrosine kinase and the immediate downstream events. In a manner that is typical for growth factor receptors, the IGF-IR tetramer is activated by ligand binding of the -subunits, and this results in a change in receptor conformation (35). Following this change, the tyrosine kinase domain is activated autocatalytically, and then it transphosphorylates three critical tyrosines within the cytoplasmic domain of the ßsubunit. Autoactivation of these tyrosines results in phosphorylation of an additional tyrosine residue (Y960) that is required for localization of the adaptor protein IRS-1 or IRS-2 (36) and for Shc binding (Fig. 1). The IRS proteins are multidomain proteins containing phosphotyrosine-binding domain and plextran homology domains as well as multiple sites for tyrosine phosphorylation (26). Following autoactivation, the IGF-IR phosphorylates IRS-1 on important recognition sites for both the p85 subunit of PI3K and Grb-2 (37). Efficient recruitment of Grb-2 and p85 to IGF-IR requires phosphorylation of IRS-1 or IRS-2. Sites for recruitment of other intracellular signaling molecules such as SHP-2, 14-3-3 proteins, and Shc are also phosphorylated by the receptor tyrosine kinase. Following recruitment and phosphorylation of p85 and Grb-2, both the PI3K and MAPK pathways are activated. Specifically recruitment of the p85 subunit results in recruitment of the p-85/p110 complex. The enzymatic activity within the p-110 subunit then converts membrane lipids such as 3,4 inositol phosphate to inositol triphosphate and to other active lipid-signaling molecules. These lipids combine with phosphoinositide-dependent kinase-1 to activate AKT and other downstream signaling components such as mammalian target of rapamycin, which is important for IGF-I stimulated protein synthesis. PI3K-coupled signaling has been linked to the ability of IGF-I to enhance glucose transport as well as its ability to inhibit apoptosis (38). IGF-I’s ability to stimulate cell migration is also mediated through this pathway, and this involves the coactivation of RAC-1 and GTP exchange factors (39).

View larger version (42K): [in this window] [in a new window]   Figure 1. Schematic diagram of IGF-IR-linked signaling (A) and integral membrane proteins that interact to modulate IGF-IR signaling (B).

Other pathways may be activated following IGF-IR activation, and they can modify signaling by both these primary pathways. These include activation of G proteins through G protein-coupled receptors. Recently it has been shown that two G protein subunits associate directly with IGF-IR (40). These factors can clearly interact with proteins such as RAC and RAS to modify signaling by RAF as well as RAC-1 activation by PI3K. Similarly, activation of protein kinase C has been shown to modify IGF signaling, and protein kinase C elements are important in activating downstream targets of PI3K (41). Although the major signaling components of these pathways have been identified, other intracellular proteins that might modify their action following IGF-IR activation are incompletely characterized. For example, the 14-3-3 proteins that are important in serine/threonine kinase activation are activated by the IGF-IR, but exactly where they intersect with activation of these two pathways and how they modify IGF-IR-linked pathway activation has been minimally characterized.

Phosphatases are also important for IGF-I-mediated signaling. SHP-2 phosphatase binds to the IGF-IR and dephosphorylates it in vitro (42, 43, 44). When cells are transfected with an SHP-2 mutant that binds to SHP-2 binding partners but contains no phosphatase activity, they have an attenuated migration response to IGF-I and attenuated MAPK activation. This suggests that in specific cell types (e.g. MCF-7 cells), this is the primary pathway for mediating migration and that SHP-2 dephosphorylation of some component of the MAPK pathway is necessary for IGF-I to stimulate this response (45). Thus, a reasonable picture of how IGF-I triggers intracellular signaling pathways and which pathways are linked to which biologic responses is known. However, the manner in which all of these signaling elements quantitatively influence the degree of pathway activation and how specific biologic responses are activated by different groups of signaling components have not been completely characterized.

	Role of Integrins in Mediating IGF-IR Activation

Top Abstract Introduction Integrin-Linked Signaling Transmembrane Adapter Proteins General Aspects of IGF-IR-Linked... Role of Integrins in... Mechanisms by which Ligand... Role of IAP References

 
Generally interactions between IGF-IR signaling and integrin receptors have been analyzed either by attempting to determine that there is direct binding of the two components using coimmunoprecipitation or showing a change in binding of a known interacting signaling intermediate. A second approach has been to use integrin receptor antagonists to determine the effects on growth factor receptor-linked functions, and a third has been to show that a kinase activated by one of the two receptor signaling systems (e.g. either growth factor receptor or integrin) can directly phosphorylate a signaling element that is linked to the cognate complementary receptor pathway. Examples of the first approach have primarily involved the Vß3 integrin. In pancreatic carcinoma cells that overexpressed the insulin receptor or Vß3, Schneller et al. (46) were able to demonstrate direct binding of Vß3 to the insulin receptor as well as the platelet-derived growth factor receptor. However, the consequences of this binding interaction in these cells were not analyzed, and the phenomenon was not extended beyond this single cell type. Similarly, this same laboratory showed an association between IRS-1 and Vß3 under specified conditions (47). They also showed that ligand occupancy of Vß3 enhanced this association. Other investigators have failed to demonstrate a direct physical interaction between Vß3 and the IGF-IR in various cell types (48). Nevertheless, these studies suggest that in some specialized cell types cooperative signaling between these two elements may occur by direct binding interaction.

Even though a direct binding interaction between Vß3 and the IGF-IR could not be demonstrated in primary smooth muscle cell (SMC) cultures, a secondary interaction was demonstrable. Using a competitive integrin receptor antagonist (e.g. the disintegrin echistatin), Jones et al. (48) showed that blocking ligand occupancy of Vß3 inhibited cell migration in response to IGF-I. Zheng and Clemmons (49) further demonstrated that prior incubation with echistatin resulted in inhibition of IGF-I-stimulated receptor phosphorylation as well as activation of IRS-1 and PI3K. Integrin antagonists that were competitive inhibitors of binding to the 5ß1 and IIß3 integrins had no effect. Therefore, this phenomenon appeared to require ligand occupancy of the specific integrin, Vß3. This finding was confirmed using a monoclonal antibody to Vß3 that was monospecific for this integrin (48).

A third example has been the activation of the signaling intermediates following integrin activation. These experiments have often been complicated by the fact that the deattachment/reattachment model of integrin activation has been used. However, using this model, it has been shown that cells freshly plated on a fibronectin matrix (which results in direct stimulation of Vß3) show increased expression of IRS-1 (50). Furthermore, plating of cells on a vitronectin-enriched matrix results in the association of FAK and IRS-1. Following this association, FAK is capable of phosphorylating IRS-1 on tyrosine residues, and this results in recruitment of p-85, GRB-2, and SHP-2 to phospho IRS-1. A similar approach was used with chondrocytes, wherein Loeser (51) demonstrated that blocking ligand occupancy of the 5ß1 integrin with an 5 specific antibody blocked the ability of IGF-I to inhibit apoptosis in this cell type. Similarly, plating chondrocytes on type I or II collagen followed by IGF-I stimulation resulted in the association of FAK with ß1 integrins as well as vinculin and paxcillin. IGF-I induced greater Shc expression in chondrocytes that were plated on type II collagen. Brooks et al. (52) demonstrated that an Vß5 antagonist inhibited tumor cell dissemination in vitro that could be induced by insulin. This was demonstrated using a concentration of insulin that was sufficient to stimulate the IGF-IR and migration of these tumor cells in vitro, and this response could be blocked with either an anti-IGF receptor antibody or anti-Vß5 antibody. In parallel studies, Chandrasekaran et al. (53) were able to demonstrate that the chemotactic effects of thrombospondin-1 for breast carcinoma cells that were mediated by the 3ß1 integrin could be potentiated by IGF-I. This response was blocked by blocking adhesion of thrombospondin to this integrin. Other investigators have also demonstrated that integrin ligation and IGF-IR-linked signaling activation is necessary for optimal cell migration responses in colon carcinoma cells and rabbit corneal endothelial cells (54).

Using the attachment/detachment/reattachment model system, it has been shown that plating cells on a fibronectin or vitronectin matrix, which results in activation of Vß3 or 5ß1, also results in activation of either FAK or SRC kinases (55). These kinases can directly phosphorylate the cytoplasmic domains of ß1 or ß3, resulting in recruitment of cytoskeletal proteins such as talin, vinculin, and actin as well as activation of receptor-linked downstream signaling molecules such as PI3K or MAPK (56). Therefore, direct activation of integrins following cell attachment has been postulated to potentiate growth factor signal transduction. Ivankovic-Dikic et al. (57) showed that FAK activation could be induced by the combination of plating cells on integrins and adding IGF-I. Likewise, plating cells on specific integrins was shown by Leung-Hagesteijn et al. (58) to modulate integrin-linked kinase induction and protein phosphatase 2C interaction in response to IGF-I.

	Mechanisms by which Ligand Occupancy of Vß3 Regulates IGF-IR

Top Abstract Introduction Integrin-Linked Signaling Transmembrane Adapter Proteins General Aspects of IGF-IR-Linked... Role of Integrins in... Mechanisms by which Ligand... Role of IAP References

 
To further elucidate the interaction between Vß3 integrin and IGF-IR activation, our laboratory has been investigating mechanisms that mediate the interaction between Vß3 activation and the IGF-IR-linked signaling systems. This is based on our observation made in stably attached cells that Vß3 antagonists completely block IGF-I-stimulated cell migration. Initially we were able to demonstrate that an interaction between the ß3 integrin and an unknown cytoplasmic protein-signaling element was necessary for IGF-I-stimulated migration (59). This was done by showing that cell-permeable peptides that contained the sequence within ß3 that was known to bind to cytoplasmic signaling molecules resulted in inhibition of this IGF-I-stimulated action. Furthermore, we were able to demonstrate that these cell-permeable peptides that inhibited the interaction of proteins with the two NXXY motifs within the ß3 integrin also attenuated the ability of IGF-I to stimulate IGF-IR phosphorylation. In an attempt to determine the molecular components that might interact with this region of ß3, we discovered that when cells were exposed to echistatin, at early time points after IGF-I stimulation of the IGF-IR, e.g. 3–5 min, there was no reduction in the ability of IGF-I to induce receptor autophosphorylation (60). However, at later time points, there was a marked diminution in IGF-IR autophosphorylation. Therefore, blocking ligand occupancy of Vß3 did not inhibit the induction of kinase activity, but it did accelerate the rate of receptor dephosphorylation.

Because the SHP-2 phosphatase had been shown in in vitro studies to bind to the IGF-IR, we examined its ability to bind to IGF-IR using these conditions. Prior exposure to echistatin resulted in an increase in the amount of SHP-2 bound to the receptor basally and 2–3 min after receptor stimulation and the time course of SHP-2 association correlated with this premature dephosphorylation response to IGF-I (61). To determine the molecular mechanism that might account for this effect, we examined the known SHP-2 recruiting molecule SHPS-1. IGF-I stimulated SHPS-1 phosphorylation, and this was associated with increased SHP-2 recruitment to SHPS-1. The SHP-2 that was recruited was activated and it subsequently dephosphorylated SHPS-1, thus making it available to be recruited to the IGF-IR. To prove that SHPS-1 phosphorylation was necessary for SHP-2 recruitment, we prepared an SHPS-1 mutant that had had the two tyrosines to which SHP-2 bound deleted. Cells expressing this mutant showed no recruitment of SHP-2 to SHPS-1 in response to IGF-I stimulation and recruitment to the IGF-IR was also blocked. To definitively demonstrate that SHP-2 was accounting for IGF-IR dephosphorylation, we used cells in which SHP-2 was expressed as a phosphatase defective mutant. Following IGF-I exposure, these cells showed SHP-2 recruitment to SHPS-1, but there was no recruitment of SHP-2 to IGF-IR, and dephosphorylation of IGF-IR did not occur presumably because of failure of SHP-2 transfer.

In summary, ligand occupancy of the Vß3 integrin results in the ability of SHP-2 to be recruited to SHPS-1 and IGF-IR in the correct temporal sequence (Fig. 1). Blocking ligand occupancy with echistatin results in premature recruitment of SHP-2 to the receptor by a mechanism that is independent of SHPS-1. Circumventing the SHPS-1 recruitment mechanism results in premature IGF-IR dephosphorylation and abortive signaling. These studies, however, do not rule out the possibility that SHP-2 also has to be transferred to downstream signaling elements for full IGF-I signaling to be forthcoming, and this possibility is currently being investigated.

Although SHPS-1 is a transmembrane protein, SHP-2 is localized primarily in the cytosol. Because the activation of the insulin receptor had been shown to phosphorylate SHPS-1 and recruit SHP-2 to the insulin receptor, it was concluded that SHPS-1 phosphorylation following IGF-I stimulation was acting to localize SHP-2 in the membrane. We postulated that ligand occupancy of the Vß3 integrin might be required to recruit SHP-2 to the membrane following growth factor receptor activation. We determined that echistatin exposure could block SHP-2 localization in the membrane fraction. To further analyze the role of ß3 and specifically the ß3 cytoplasmic domain, we prepared a mutant in which the two tyrosines in the ß3 cytoplasmic domain were converted to phenylalanines that could not be phosphorylated. This resulted in a complete inhibition of the ability of the cells to migrate in response to IGF-I (59). To further elucidate how this effect was mediated and whether the absence of ß3 phosphorylation altered SHP-2 recruitment to IGF-IR, we analyzed cells expressing this mutant. The ß₃ FF cells showed no recruitment of SHP-2 to IGF-IR in response to IGF-I (62). In addition, these cells showed no recruitment of SHP-2 to SHPS-1. Because SHPS-1 is an integral membrane protein, we analyzed the membrane localization of SHP-2 in the cells expressing the ß3 mutant. This showed that the amount of the SHP-2 localized in the membrane fraction under basal conditions was markedly reduced. Therefore, it appeared SHP-2 was unavailable to bind to SHPS-1 because it was not localized in the membrane compartment.

To further determine whether tyrosine phosphorylation of the wild-type ß3 protein was required for SHP-2 binding and its membrane localization, we used the tyrosine kinase inhibitor, herbimycin A. This blocked the phosphorylation of the ß3 integrin in response to IGF-I stimulation and more importantly blocked SHP-2 transfer to ß3 and SHP-2 membrane localization, thus definitively demonstrating that both are required. Therefore, in the presence of ligand occupancy of Vß3, IGF-I stimulates an unknown kinase to phosphorylate ß3, and this results in SHP-2 membrane localization. This is required for SHP-2 binding to SHPS-1 and transfer to IGF-IR, thus providing a direct link between integrin ligand occupancy and appropriate timing of dephosphorylation of the IGF-IR.

	Role of IAP

Top Abstract Introduction Integrin-Linked Signaling Transmembrane Adapter Proteins General Aspects of IGF-IR-Linked... Role of Integrins in... Mechanisms by which Ligand... Role of IAP References

 
IAP is a five-transmembrane domain protein that plays an important role in modulating the ability of IGF-I to signal. For IGF-I to adequately stimulate cell migration, it must increase the affinity of Vß3 for ligands. This 3- to 4-fold increase in affinity is not an immediate event but requires a 12-h exposure to IGF-I (46). Because IAP had been shown in other systems to enhance the affinity of Vß3 for ligands (24, 25), we determined that the amount of IAP associated with Vß3 in SMCs in the basal state was low, but following 12- and 24-h exposure to IGF-I, this association increased 6-fold (63). This increase in Vß3-IAP association was associated with a marked increase in the affinity of Vß3 for its ligands, and this could be blocked by a monoclonal antibody that was known to inhibit the association of IAP with Vß3. Importantly, this monoclonal antibody also inhibited the cell migration response to IGF-I. This suggested that some time-dependent event was required for IGF-I to stimulate IAP/Vß3 association. To determine the mechanism, we determined the lipid subdomain membrane compartmentalization of both Vß3 and IAP in the basal state in SMCs (63). It was shown that IAP was localized almost exclusively in membrane rafts, which are highly polar lipid domains of the plasma membrane. In contrast, Vß3 was localized primarily in the nonraft domains. Following 12–24 h of IGF-I exposure, IAP translocated from raft to nonraft domains. Approximately 70% of the IAP could be detected in that compartment, and it was directly associated with Vß3 following IGF-I stimulation. Thus, IAP and Vß3 are localized in different membrane subdomains and IGF-IR activation can alter their localization and association, thus resulting in a change in integrin avidity for ligands and ultimately integrin ligand occupancy (63).

To further elucidate the role of IAP, we examined the interaction between IAP and SHPS-1. The extracellular domain of IAP had been shown to associate with SHPS-1 when red blood cells associate with hepatocytes, and blocking this interaction was shown to block the uptake of erythrocytes by Kupfer cells (28). Thus, a direct interaction between these two proteins was shown to alter a cellular function. We were interested in determining whether IAP could associate with SHPS-1 in SMCs and whether this resulted in a modulation of IGF-IR-linked signaling. We demonstrated that SHPS-1 interacted directly with IAP in SMCs and stimulation of the IGF-IR resulted in an increased interaction (64). Next we demonstrated that this interaction was required for SHPS-1 phosphorylation and SHP-2 transfer to SHPS-1 following IGF-I stimulation. This was shown by expressing a mutant in which the extracellular domain of IAP had been deleted and therefore could not bind to SHPS-1. These cells did not migrate in response to IGF-I and did not transfer SHP-2 to the IGF-IR. To determine that this was also a functionally intact mechanism in nontransfected cells, a monoclonal antibody to IAP that blocks the IAP-SHPS-1 interaction was used. The monoclonal antibody blocked IGF-I-stimulated transfer of SHP-2 to IGF-IR and inhibited IGF-I-stimulated cell migration. Thus, a direct interaction between two cell surface integral membrane proteins IAP and SHPS-1 is required for IGF-I to stimulate SHP-2 transfer to the IGF-IR and presumably for the transfer of SHP-2 to downstream molecules (Fig. 1). This SHP-2 transfer appears to be required for IGF-I to stimulate biologic actions such as cell division, protein synthesis, and cell migration.

In summary, four integral membrane proteins, the IGF-IR, Vß3, SHPS-1, and IAP function coordinately to control cellular responses to IGF-I (Fig. 1). Each protein has important functions that can be altered by ligand occupancy. Therefore, determination of the various extracellular ligands that bind to each protein and how changes in ligand occupancy alter IGF-I signaling and target cell actions are important goals of future research.

	Acknowledgments

 
The authors thank Ms. Laura Lindsey for her help in preparing the manuscript.

	Footnotes

 
This work was supported by NIH Grants HL-56850 and AG-02331.

Abbreviations: ECM, Extracellular matrix; FAK, focal adhesion kinase; IAP, integrin-associated protein; IGF-IR, IGF-I receptor; IRS, insulin receptor substrate; PI3K, phosphatidylinositol 3-kinase; RAC, related to A and C kinase; SHP, SH2 domain protein; SHP-2, SH2 domain-containing tyrosine phosphatase; SHPS-1, tyrosine phosphatase SHP-2 substrate 1; SMC, smooth muscle cell; TSP, thrombospondin.

Received October 25, 2002.

Accepted for publication January 13, 2003.

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Top Abstract Introduction Integrin-Linked Signaling Transmembrane Adapter Proteins General Aspects of IGF-IR-Linked... Role of Integrins in... Mechanisms by which Ligand... Role of IAP References

 

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