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Vß3 Integrin Linked Signaling Pathways: Cellular Responses to Changes in Multiple Signaling Inputs
Department of Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599
Address all correspondence and requests for reprints to: David R. Clemmons, Division of Endocrinology, University of North Carolina, CB 7170, Chapel Hill, North Carolina 27599. E-mail: endo{at}med.unc.edu.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONINTEGRIN ACTIVATIONDIRECT INTERACTIONS OF INTEGRINS...INDIRECT ACTIVATION OF INTEGRINS...INTERACTIONS OF INTEGRIN AND...{alpha}Vß3 INTEGRIN...COOPERATIVE INTERACTION BETWEEN...{alpha}Vß3 INTEGRINS...INCREASES IN LIGAND OCCUPANCY...MECHANISM OF...REFERENCES |
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Vß3, that is expressed in smooth muscle cells modulates IGF-I actions. Ligand occupancy of Vß3 is required for IGF-I to stimulate cell migration and division. Src homology 2 containing tyrosine phosphatase (SHP-2) is a tyrosine phosphatase whose recruitment to signaling molecules is stimulated by growth factors including IGF-I. If Vß3 ligand occupancy is inhibited, there is no recruitment of SHP-2 to Vß3 and its transfer to downstream signaling molecules is blocked. Ligand occupancy of Vß3 stimulates tyrosine phosphorylation of the ß3-subunit, resulting in recruitment of SHP-2. This transfer is mediated by an insulin receptor substrate-1-related protein termed DOK-1. Subsequently, SHP-2 is transferred to another transmembrane protein, SHPS-1. This transfer requires IGF-I receptor-mediated tyrosine phosphorylation of SHPS-1, which contains two YXXL motifs that mediate SHP-2 binding. The transfer of SHP-2 to SHPS-1 is also required for recruitment of Shc to SHPS-1. Ligand occupancy of Vß3 results in sustained Shc phosphorylation and enhanced Shc recruitment. Shc activation results in induction of MAPK. Inhibition of the Shc/SHPS-1 complex formation results in failure to achieve sustained MAPK activation and an attenuated mitogenic response. Thus, within the vessel wall, a mechanism exists whereby ligand occupancy of the Vß3 integrin is required for assembly of a multicomponent membrane signaling complex that is necessary for cells to respond optimally to IGF-I. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONINTEGRIN ACTIVATIONDIRECT INTERACTIONS OF INTEGRINS...INDIRECT ACTIVATION OF INTEGRINS...INTERACTIONS OF INTEGRIN AND...{alpha}Vß3 INTEGRIN...COOPERATIVE INTERACTION BETWEEN...{alpha}Vß3 INTEGRINS...INCREASES IN LIGAND OCCUPANCY...MECHANISM OF...REFERENCES |
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In addition to IGFBPs, extracellular matrix (ECM) proteins have been shown to play a role in modulating cellular responses to IGF-I (9, 10). Thus, ECM proteins, such as types I and IV collagen, fibronectin, thrombospondin-1 (TS-1), and osteopontin have been shown to modulate the response of various cell types to IGF-I stimulation (9, 10, 11, 12). Changes in the abundance of these proteins have been shown not only to modify cellular adherence that can alter IGF-I signaling but also to actively stimulate signal transduction through their binding to a specific class of cell surface receptors, termed integrins. Integrins are heterodimers that consist of one - and one ß-subunit (13). Vertebrates express 18 different -subunits and eight ß-subunits. These subunits assemble into 24 distinct integrin heterodimers. Generally, the types of heterodimers that are expressed and their relative abundance varies among different cell types. This review will focus on the response of vascular smooth muscle cells (SMC) to IGF-I and the role that ligand occupancy of smooth muscle cell integrins plays in modulating this response.
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INTEGRIN ACTIVATION |
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TOPABSTRACTINTRODUCTIONINTEGRIN ACTIVATIONDIRECT INTERACTIONS OF INTEGRINS...INDIRECT ACTIVATION OF INTEGRINS...INTERACTIONS OF INTEGRIN AND...{alpha}Vß3 INTEGRIN...COOPERATIVE INTERACTION BETWEEN...{alpha}Vß3 INTEGRINS...INCREASES IN LIGAND OCCUPANCY...MECHANISM OF...REFERENCES |
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1ß1, 5ß1, Vß3, 2ß1, 3ß1, 5ß1, Vß5, and 1ß6 integrins (14). In SMC, integrins have been shown to be important mediators of cell adhesion, migration, ECM assembly, ECM contraction, and cellular replication (15). Integrin ligand occupancy can also regulate the state of cellular differentiation as well as the ability of cells to resist apoptosis (13, 16). Unlike tyrosine kinase-containing receptors, such as the IGF-I receptor, integrins do not contain intrinsic tyrosine kinase activity. In response to ligand occupancy, integrins signal through alternative mechanisms. These involve a conformational change in the integrin that results in a change in its activation state. The change in activation has been assessed by showing evidence of polymerization, clustering, or the surface exposure of different antibody binding epitopes (14, 15). Similarly, phosphorylation of key residues within the cytoplasmic domains of the ß-subunit is often associated with binding of specific proteins to integrins in response to changes in ligand occupancy (17). Integrin cytoplasmic domains can bind constitutively to cytoskeletal components such as talin, so that changes in integrin conformation and activation can result in changes in cytoskeletal protein function. Changes in talin binding to integrin cytoplasmic tails results in a reorganization of actin and myosin filaments, and this leads to major changes in cell shape as well as locomotion (18). After conformational changes or phosphorylation of specific residues, integrin cytoplasmic domains bind to signaling molecules, and the display of signaling molecules can change as a function of integrin activation (19). Signaling molecules can assemble into multicomponent signaling complexes that have been shown to include small GTPase proteins such as, Rho, Rac, and Cdc42 as well as components of the MAPK pathway such as Shc and Grb-2 (20). Tyrosine kinases such as focal adhesion kinase (FAK) and Src and proteins that have been shown to modify the activity of these kinases, such as p130 CAS, also bind to activated integrins (21). The extracellular domains of integrins also have been shown to bind to specific proteins that can modulate integrin function. These include the urokinase type plasminogen activator and receptor, integrin-associated protein (IAP), and tetraspanin or CD9 (22, 23, 24). Direct integrin activation either by stimulatory ligands or changes in ion concentrations such as calcium or magnesium has been shown to activate protein complex assembly, which can result in activation of an integrin-linked signaling pathway (outside in signaling) (25). For example, the phosphatidylinositol-3 (PI-3) kinase pathway can be activated in this manner. It is important to point out, however, that most experiments wherein outside in signaling was used to stimulate integrin activation do not control for changes in ligand occupancy of the growth factor receptors so that the contribution role of growth factor receptor occupancy in regulating integrin activation is unknown. |
DIRECT INTERACTIONS OF INTEGRINS AND GROWTH FACTOR RECEPTORS INFLUENCE SIGNALING |
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TOPABSTRACTINTRODUCTIONINTEGRIN ACTIVATIONDIRECT INTERACTIONS OF INTEGRINS...INDIRECT ACTIVATION OF INTEGRINS...INTERACTIONS OF INTEGRIN AND...{alpha}Vß3 INTEGRIN...COOPERATIVE INTERACTION BETWEEN...{alpha}Vß3 INTEGRINS...INCREASES IN LIGAND OCCUPANCY...MECHANISM OF...REFERENCES |
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Vß1 integrin after cell attachment has been shown to result in activation of the epidermal growth factor (EGF) receptor in the absence of EGF binding] (26), the paradigm that has been most extensively evaluated is the direct association between integrins and growth factor receptors in response to growth factor stimulation. Stimulation of the Vß3 integrin on endothelial and/or SMC showed that this integrin could associate with platelet-derived growth factor (PDGF), insulin, or vascular endothelial growth factor (VEGF) receptors (27, 28). Similarly, the EGF receptor has been shown to interact directly with 5ß1 and 6ß4 integrins (29). In general, direct activation of the EGF receptor by integrins (in the absence of EGF) requires integrin-mediated clustering such as that which occurs in response to integrin activation by ECM proteins after cell attachment. Direct physical association of IGF-I receptors and integrins has been demonstrated, although it is cell-type specific. Coprecipitation of the IGF-I receptor and the 6 integrin after dual ligand stimulation was demonstrated for lens epithelial cells (30) and for 5ß1 or 1ß1 integrins and the IGF-I receptor in chondrocytes (31). |
INDIRECT ACTIVATION OF INTEGRINS BY GROWTH FACTOR STIMULATION |
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TOPABSTRACTINTRODUCTIONINTEGRIN ACTIVATIONDIRECT INTERACTIONS OF INTEGRINS...INDIRECT ACTIVATION OF INTEGRINS...INTERACTIONS OF INTEGRIN AND...{alpha}Vß3 INTEGRIN...COOPERATIVE INTERACTION BETWEEN...{alpha}Vß3 INTEGRINS...INCREASES IN LIGAND OCCUPANCY...MECHANISM OF...REFERENCES |
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Vß3 and Vß5 (32). Similarly, TGFß binding to its receptor has been shown to result in enhanced expression of 5ß1 integrin on cell surfaces (33). IGF-I has been shown to enhance the affinity for Vß3 integrin for ligands without change in receptor number (34). Ligand occupancy of the hepatocyte growth factor receptor results in phosphorylation of the cytoplasmic domain of ß4, which results in Shc and PI-3 kinase recruitment (35). Hepatocyte growth factor receptor occupancy can also induce integrin clustering, thus resulting in changes in signaling complex assembly. Therefore, there are multiple ways in which dual activation of growth factor receptors and integrins may cooperatively interact to enhance cellular responses. |
INTERACTIONS OF INTEGRIN AND GROWTH FACTOR RECEPTOR SIGNALING PATHWAYS |
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TOPABSTRACTINTRODUCTIONINTEGRIN ACTIVATIONDIRECT INTERACTIONS OF INTEGRINS...INDIRECT ACTIVATION OF INTEGRINS...INTERACTIONS OF INTEGRIN AND...{alpha}Vß3 INTEGRIN...COOPERATIVE INTERACTION BETWEEN...{alpha}Vß3 INTEGRINS...INCREASES IN LIGAND OCCUPANCY...MECHANISM OF...REFERENCES |
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6ß1 integrin by laminin (37). This localization is required for PDGF to stimulate cell proliferation because it allows the recruitment of PI-3 kinase, AKT and FAK to rafts after PDGF exposure. Alternatively, specific signaling complexes can be assembled in association with integrin cytoplasmic tails that are not dependent on their localization to specific membrane compartments. For example, FAK activation after changes in integrin conformation leads to the association of a Src family kinase, p130 CAS and PI-3 kinase, resulting in an enhancement in the ability of growth factors to activate PI-3 kinase when it is associated with this complex. |
Vß3 INTEGRIN AND GROWTH FACTOR RECEPTOR COOPERATIVE INTERACTION IN VASCULAR CELLS
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TOPABSTRACTINTRODUCTIONINTEGRIN ACTIVATIONDIRECT INTERACTIONS OF INTEGRINS...INDIRECT ACTIVATION OF INTEGRINS...INTERACTIONS OF INTEGRIN AND...{alpha}Vß3 INTEGRIN...COOPERATIVE INTERACTION BETWEEN...{alpha}Vß3 INTEGRINS...INCREASES IN LIGAND OCCUPANCY...MECHANISM OF...REFERENCES |
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3ß1 integrin activity in response to IGF-I (40). Similarly, increased FAK activation in response to IGF-I has been shown in cells that are plated on specific combinations of integrins. |
COOPERATIVE INTERACTION BETWEEN Vß3 INTEGRIN AND IGF-I RECEPTOR SIGNALING
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TOPABSTRACTINTRODUCTIONINTEGRIN ACTIVATIONDIRECT INTERACTIONS OF INTEGRINS...INDIRECT ACTIVATION OF INTEGRINS...INTERACTIONS OF INTEGRIN AND...{alpha}Vß3 INTEGRIN...COOPERATIVE INTERACTION BETWEEN...{alpha}Vß3 INTEGRINS...INCREASES IN LIGAND OCCUPANCY...MECHANISM OF...REFERENCES |
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Vß3 integrin is capable of binding a variety of ligands that are present in vascular tissue such as osteopontin, thrombospondin, and vitronectin. Changes in ligand occupancy of Vß3 have been shown to directly influence fibroblast growth factor (FGF) signaling in endothelial cells and IGF-I signaling in SMC. Disruption of the ligand occupancy of Vß3 with a specific monoclonal antibody, LM609, has been shown to inhibit FGF-induced signaling in vascular endothelium and to inhibit FGF-stimulated angiogenesis (41). Similarly, exposure of human SMC to this antibody was shown to inhibit IGF-I-stimulated cell migration. The importance of Vß3 ligand occupancy was further supported in studies in which exposure of SMC to disintegrin antagonists, such as echistatin, a small peptide that binds directly to Vß3 and inhibits vitronectin-stimulated Vß3 functions, resulted in blocking IGF-I-stimulated SMC migration and division (34). The importance of these observations was reinforced in in vivo studies wherein it was shown that infusion of echistatin into developing atherosclerotic lesions in a pig model resulted in inhibition of IGF-I-stimulated IGFBP-5 synthesis and attenuation of lesion formation (42). That blocking Vß3 ligand occupancy was altering IGF-I signaling was shown by studies in which it was determined that exposure to echistatin resulted in a reduced ability of IGF-I to stimulate IGF-I receptor phosphorylation and attenuated activation of IRS-1 and PI-3 kinase by the IGF-I receptor (43). In contrast, antagonism of 5ß1 receptor ligand occupancy resulted in no change in these parameters. These findings were confirmed using the specific anti-Vß3 integrin monoclonal antibody. Unlike some other cell types, direct association between the Vß3 integrin and the IGF-I receptor in SMC has not been demonstrated after IGF-I stimulation. |
Vß3 INTEGRINS AND SHP-2 PHOSPHATASE
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TOPABSTRACTINTRODUCTIONINTEGRIN ACTIVATIONDIRECT INTERACTIONS OF INTEGRINS...INDIRECT ACTIVATION OF INTEGRINS...INTERACTIONS OF INTEGRIN AND...{alpha}Vß3 INTEGRIN...COOPERATIVE INTERACTION BETWEEN...{alpha}Vß3 INTEGRINS...INCREASES IN LIGAND OCCUPANCY...MECHANISM OF...REFERENCES |
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Vß3 integrin ligand occupancy has also been shown to regulate the activity of protein tyrosine phosphatases. These enzymes dephosphorylate tyrosine residues, and these dephosphorylation reactions can result in either activation or inactivation of growth factor signaling complexes. Blocking ligand occupancy of the Vß3 integrin was shown initially to result in aberrant transfer of Src homology 2 containing tyrosine phosphatase (SHP-2) to the IGF-I receptor (44). Transfer of this phosphatase to the receptor normally occurs 20 min after IGF-I stimulation, causing a progressive decrease in the amount of tyrosine receptor phosphorylation and subsequent attenuation of MAPK and PI-3 kinase activation. In the presence of the disintegrin echistatin, however, SHP-2 was transferred to the receptor 5 min after exposure to IGF-I causing premature dephosphorylation, and attenuation of IGF-I receptor mediated downstream signaling (45). Because others have shown that sustained growth factor receptor and MAPK activation is required for an optimal cell growth response (46, 47), this is one potential mechanism by which exposure to the disintegrin echistatin could result in attenuation of IGF-I signaling and biological actions. These findings strongly suggest that IGF-I signaling is modulated by the cooperative interaction of ligand-activated Vß3 and IGF-I receptor-linked signaling events that work in concert to extend the duration of IGF-I receptor activation by regulating the translocation of SHP-2 phosphatase.
To better understand how failure to transfer SHP-2 results in attenuated MAPK and PI-3 kinase signaling, it is useful to review the classical IGF-I receptor-linked signaling pathway. The IGF-I receptor is a heterotetramer that consists of two -subunits that contain the ligand binding domains and two ß-subunits that contain the tyrosine kinase activity. After ligand binding, the receptor undergoes a conformational change resulting in the activation of the tyrosine kinase, which results in transphosphorylation of the opposite ß-subunit on specific tyrosine residues. These phosphotyrosines then bind to adaptor molecules such as Shc and IRS-1 (Fig. 1
). Phosphorylation of these proteins has been shown to lead to activation of the PI-3 kinase and MAPK signaling pathways (48). After IRS-1 association and phosphorylation, it binds to the p85 regulatory subunit of PI-3 kinase, which recruits the p110 catalytic subunit to the plasma membrane resulting in activation of its enzymatic activity. MAPK activation can result from IRS-1 recruitment of Grb2 and son of sevenless to the plasma membrane resulting in activation of Ras/Raf signaling, which is followed by MAPK kinase (MEK) and MAPK activation or alternatively this pathway can be activated by phosphorylation of Shc, which then recruits Grb2, thus resulting in Ras activation. In SMC, inhibitor studies have shown that activation of PI-3 kinase is absolutely required for stimulation of cell migration and contributes to full activation of cellular proliferation (49). Similarly, although the role of MAPK is predominant for stimulating cellular proliferation, it also plays a role in activating cell migration principally through activating small GTPase proteins such as Rac or Rho that are necessary for full activation of cell motility (50).
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INCREASES IN LIGAND OCCUPANCY OF Vß3 ENHANCE IGF-I SIGNALING AND ACTIONS IN SMOOTH MUSCLE CELLS
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TOPABSTRACTINTRODUCTIONINTEGRIN ACTIVATIONDIRECT INTERACTIONS OF INTEGRINS...INDIRECT ACTIVATION OF INTEGRINS...INTERACTIONS OF INTEGRIN AND...{alpha}Vß3 INTEGRIN...COOPERATIVE INTERACTION BETWEEN...{alpha}Vß3 INTEGRINS...INCREASES IN LIGAND OCCUPANCY...MECHANISM OF...REFERENCES |
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Vß3 due to the availability of ligands. At high culture density, there is abundant ligand available, and this is reflected in increased constitutive activation, e.g. tyrosine phosphorylation of the ß3-subunit of the Vß3 is enhanced (51). The addition of an increased amount of a soluble ligand such as vitronectin results in further enhancement of ß3 phosphorylation. If purified vitronectin is added to low-density cultures in which little basal ß3 phosphorylation can be detected, phosphorylation of the ß3-subunit is immediately stimulated and this is sustained for at least 2 h after vitronectin exposure. Two potential tyrosine phosphorylation sites (i.e. 773 and 785) are located within the cytoplasmic domain of the ß3-subunit. One of the tyrosines, 773, is contained within an NPXY motif making it capable of binding to phosphotyrosine binding (PTB) domain-containing proteins (52). Activation of these tyrosines by ligand occupancy results in sustained MAPK activation and enhanced SMC growth in response to IGF-I. The importance of tyrosine phosphorylation of the ß3-subunit for IGF-I signaling was confirmed by preparing cells in which these two tyrosines were mutated to phenylalanine. Cells expressing the mutant did not recruit SHP-2 to the plasma membrane or to downstream signaling molecules (51). Furthermore, these cells did not proliferate or migrate in response to IGF-I. MAPK was activated, but the response to IGF-I was not sustained. This effect is specific for tyrosine 773 because blocking SHP-2 transfer to that specific site resulted in impaired IGF-I activation of MAPK (53). The importance of SHP-2 transfer to ß3 for IGF-I signaling was confirmed by exposure of cells to a SHP-2/ß3 blocking peptide. Inhibition of SHP-2 transfer resulted in attenuation of IGF-I stimulated PI-3 and MAPK activation as well as inhibition of IGF-I stimulated SMC activation and proliferation. Therefore, ligand-induced stimulation of ß3 phosphorylation leads to SHP-2 transfer to the plasma membrane, and this event is dependent on ß3 tyrosine phosphorylation.
Binding of Activated SHP-2 to Vß3 Is Mediated by the Adapter Protein DOK-1
The transfer of SHP-2 to the ß3-subunit also requires the activation of Vß3 or the IGF-I receptor. SHP-2 binds to phosphorylated ß3. It is bound to ß3 basally in high-density cultures that have constitutive ß3 phosphorylation. In low-density cultures, the addition of either vitronectin or IGF-I results in stimulation of ß3 phosphorylation and subsequent recruitment of SHP-2 to phosphorylated ß3 (51). Although SHP-2 binding to ß3 is tyrosine phosphorylation-dependent, SHP-2 does not contain a PTB domain, and therefore it is incapable of binding directly to ß3. Moreover, stimulation of ß3 phosphorylation alone is not adequate to confer SHP-2 transfer to ß3. An intracellular protein that contains a PTB domain, DOK-1, has been shown to bind directly to the tyrosine phosphorylated NPXY sequence in ß3 (52). In addition to its PTB domain, DOK-1 also contains multiple YXXL motifs within its C-terminal domain. When these tyrosines are phosphorylated, they are capable of binding SH-2 domain containing proteins. We have determined that one of these motifs (Y337XXL) mediates SHP-2 association with DOK-1 (53). After exposure of SMC cultures to IGF-I, DOK-1 is tyrosine phosphorylated and binds to SHP-2. Exposure of SMC to a cell-permeable peptide that contained this Y337XXL sequence inhibited SHP-2 binding to DOK-1. Similarly, expression of a mutant DOK-1 that had tyrosine 337 substituted with phenylalanine eliminated SHP-2 binding. More importantly, each of the manipulations inhibited SHP-2 transfer to ß3, suggesting that DOK-1 was mediating SHP-2/ß3 association (Fig. 2A). To confirm that DOK-1 could mediate SHP-2 transfer to ß3, we prepared a mutant form of DOK-1 in which arginines 207 and 208 (the residues that had been shown to mediate ß3 binding) were substituted with alanines. The mutant DOK-1 did not associate with ß3 and did not transfer SHP-2 to ß3 after IGF-I stimulation. Therefore, inhibition of either SHP-2 binding to DOK-1 or DOK-1 binding to ß3 results in failure of SHP-2 to bind to ß3 in response to IGF-I. Thus, DOK-1 is an important linker protein that can mediate the transfer of SHP-2 to the plasma membrane after stimulation by either ligand occupancy of ß3 or IGF-I receptor stimulation.
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Vß3 Membrane-Associated Complex to SHPS-1 after IGF-I StimulationVß3 to the IGF-I receptor or whether other molecules whose activity could be modified by the IGF-I receptor were involved. Initial studies showed that blocking SHP-2 transfer to IRS-1 had no effect on MAPK activation. A second possibility was that the single chain transmembrane protein SHPS-1 was involved. SHPS-1 contains three Ig-like motifs in its extracellular domain and a cytoplasmic tail with four YXXL/I/V motifs. When two of these motifs are tyrosine phosphorylated, SHP-2 can bind to them through its SH-2 domains. Stimulation of SHPS-1 phosphorylation had been shown to recruit SHP-2 to SHPS-1, and when SHP-2 transfer to SHPS-1 was blocked, MAPK activation by insulin was attenuated (54). Therefore, our studies focused on the role of SHPS-1 in mediating SHP-2 transfer and its role in MAPK activation. SHPS-1 phosphorylation and SHP-2 transfer to SHPS-1 were stimulated when SMC cultures were exposed to IGF-I (45). If SHP-2 transfer to ß3 was blocked, however, either by inhibiting ß3 tyrosine phosphorylation or blocking DOK1 transfer of SHP-2 to ß3, there was no SHP-2 transfer to SHPS-1 although SHPS-1 phosphorylation was maintained (51). Instead, SHP-2 was aberrantly transferred to IGF-I receptor. Thus, transfer of SHP-2 to SHPS-1 requires that SHP-2 is first recruited to Vß3 and that SHPS-1 is phosphorylated in response to IGF-I. Disruption of SHP-2 transfer to the plasma membrane by pretreating cells with disintegrin antagonists or anti Vß3 antibodies also impairs SHP-2 transfer SHPS-1 despite IGF-I stimulation (44). In all cases wherein SHP-2 transfer to SHPS-1 is blocked, this is associated with failure to properly activate MAPK and PI-3 kinase in response to IGF-I. Other investigators have shown that PI-3 kinase activation can be attenuated after failure to appropriately transfer SHP-2 to downstream signaling molecules; therefore our observations regarding SHP-2 transfer are consistent with previously published data (55, 56).
SHPS-1 Provides a Scaffold for Localizing SHP-2 and Shc and Formation of This Complex Is Necessary for IGF-I Induced MAPK Activation
One potential role of SHPS-1 is that of a scaffolding protein for the assembly of multicomponent signaling complexes. Because our studies had shown that Shc activation was required to achieve optimal MAPK activation, we asked whether Shc localized to SHPS-1 after IGF-I stimulation. This was demonstrated by showing that IGF-I stimulated Shc association with SHPS-1. We further determined that in the presence of a specific peptide that blocked Shc association with SHPS-1, the Shc phosphorylation in response to IGF-I was markedly inhibited. This was accompanied by reduced MAPK activation and an attenuated cell growth response to IGF-I. Importantly, this peptide did not block SHP-2 association with SHPS-1, indicating that although SHP-2 association with SHPS-1 was also required for signaling, it was not sufficient. When SHP-2 transfer is blocked however, Shc binding to SHPS-1 cannot be detected. Therefore, at least two roles for SHPS-1 have been defined by our studies. The first is that in response to ligand occupancy of the IGF-I receptor, SHPS-1 undergoes tyrosine phosphorylation and binds to SHP-2 (Fig. 2B
). SHP-2 is then transferred to appropriate downstream signaling molecules such as PI-3 kinase, which is required for their activation. Second, the coassociation of SHP-2 and Shc with SHPS-1 appears to be required for normal Shc activation in response to IGF-I receptor activation. This activation of Shc is necessary for sustained MAPK activation and an optimal mitogenic response of SMC to IGF-I. The molecular mechanism by which SHP-2 and Shc interact cooperatively while localized on SHPS-1 to form this signaling complex requires further study. The identity of the tyrosine kinase that is phosphorylating Shc within this complex is unknown. Preliminary data indicate that it may be a Src family kinase. Exposure of SMC to Src family kinase inhibitors results in attenuation of MAPK signaling and Shc activation in response to IGF-I. Similarly, use of a mutant form of Shc that does not bind to c-Src results in attenuation of Shc activation in response to IGF-I as well as reduced MAPK activation. SHP-2 is also required for optimal Src activation because expression of mutant forms of SHP-2 that do not bind to c-Src results in decreased Src and Shc activation (57, 58). Thus, SHP-2 binding to Src, which occurs in response to IGF-I, may be required for Shc and subsequently MAPK activation, and SHP-2 transfer to SHPS-1 may be required to localize Src within the SHPS-1 signaling complex.
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MECHANISM OF Vß3 ACTIVATION
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TOPABSTRACTINTRODUCTIONINTEGRIN ACTIVATIONDIRECT INTERACTIONS OF INTEGRINS...INDIRECT ACTIVATION OF INTEGRINS...INTERACTIONS OF INTEGRIN AND...{alpha}Vß3 INTEGRIN...COOPERATIVE INTERACTION BETWEEN...{alpha}Vß3 INTEGRINS...INCREASES IN LIGAND OCCUPANCY...MECHANISM OF...REFERENCES |
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Vß3 in mediating IGF-I signaling, two types of experimental approaches have been used. First, the relative importance of different binding domains on Vß3 and on the vitronectin molecule itself in mediating positive signaling through Vß3 has been identified. Specifically, we have determined that the heparin binding domain of vitronectin, which had been reported to bind to the Vß5 integrin, also mediates vitronectin binding to Vß3 (59). ECM proteins that contain RGD (arginine-glycine-asparginine) sequences are known to bind to integrin receptors through this recognition domain. Vitronectin contains an RGD sequence that mediates its binding to Vß3. To determine the relative importance of each of these two domains in mediating the cooperative signaling between Vß3 and IGF-I receptor, we prepared synthetic peptides that each contained one of these regions of vitronectin. Addition of each of the peptides to SMC cultures with IGF-I gave quite different results. Exposure of cells to the heparin binding domain peptide resulted in activation of Vß3 phosphorylation and recruitment of SHP-2 to the plasma membrane (60). More importantly, however, it resulted in sustained IGF-I receptor phosphorylation in response to IGF-I as well as sustained MAPK activation and an enhanced mitogenic response. These responses were similar to the response of SMC to exposure to intact vitronectin. In contrast, addition of a synthetic peptide that bound to the RGD binding domain on Vß3 did not enhance these properties. Furthermore, it resulted in premature recruitment of SHP-2 to the receptor and premature receptor dephosphorylation, similar to the pSMC response to echistatin. Addition of the RGD peptide with the heparin binding domain peptide resulted in a partial attenuation of the ability of the heparin binding peptide to enhance IGF-I stimulated receptor phosphorylation and sustained MAPK activation, suggesting that this site might be playing a partially antagonistic role.
To confirm the importance of the heparin binding domain within the intact protein, SMC were exposed to vitronectin and an antibody that had been prepared against the domain of the ß3-subunit that binds to the heparin binding domain of vitronectin. In the presence of this antibody, the duration of IGF-I receptor phosphorylation and MAPK activation were attenuated and were similar in intensity and duration to cells that had not been exposed to exogenously added vitronectin. This strongly suggests that binding through this domain is necessary for these enhanced responses. To further delineate the role of the RGD domain, cells were exposed to echistatin and/or the RGD peptide or synthetic molecules known to bind to the RGD binding site on Vß3. Exposure of each of these ligands for periods greater than a 7-h exposure resulted in reduced ß3 phosphorylation and SHP-2 recruitment to Vß3. This decrease in ß3 phosphorylation was not due directly to activation of a phosphatase but rather to proteolytic cleavage. Binding of ligand to the RGD binding site on Vß3 activates calpain, which then cleaves the ß3-subunit, resulting in failure to recruit SHP-2 to the membrane on IGF-I stimulation (61). Because the binding site for the DOK-1/SHP-2 complex has been eliminated, SHP-2 transfer to ß3 and subsequently to SHPS-1 is impaired, resulting in failure to appropriately transfer this protein to the IGF-I receptor and to downstream signaling molecules; thus disintegrin antagonists and RGD binding site ligands appear to modulate the effect of heparin binding domain ligands by accelerating ß3 cleavage and inhibiting SHP-2 transfer. Whether they impair other markers of ß3 activation such as integrin clustering and/or recruitment of signaling molecules to specific membrane microdomains in response to IGF-I stimulation remains to be investigated.
An additional tool for studying the role of extracellular proteins in regulating IGF-I actions has come from the analysis of the role of integrin binding partners that bind to Vß3 on integrin signaling. One important binding partner of Vß3 is IAP, a five-transmembrane domain protein that modulates the ability of IGF-I to induce a 3- to 4-fold increase in the affinity of Vß3 for its ligands (62). IAP has been shown to enhance Vß3 affinity for ligands. After a 12-h exposure to IGF-I (63), the amount of IAP associated with Vß3 increased 6-fold. Expression of a mutant form of IAP that did not bind to Vß3 was associated with failure of IGF-I to stimulate an increase in Vß3/IAP association and, more importantly, failure of IGF-I to stimulate an increase in Vß3 affinity for vitronectin and to increase cell migration. These results were confirmed by using a monoclonal antibody (B6H12) that has been shown to inhibit IAP/Vß3 association. This monoclonal antibody also inhibited the ability of IGF-I to stimulate cell migration.
IGF-I stimulated the association of IAP with Vß3 by changing its membrane microdomain compartmentalization. In the basal state, IAP is associated almost exclusively with membrane rafts. After IGF-I exposure, IAP is slowly translocated from raft domains to nonraft domains where most of the Vß3 resides. Thus, IGF-I modulates integrin avidity for ligands by stimulating the translocation of IAP to the nonraft membrane compartment where it can associate with Vß3.
To further elucidate the role of IAP on IGF-I action, we determined whether it interacted with SHPS-1. We were able to demonstrate that the extracellular domain of IAP associated with SHPS-1 and that exposure to the anti-IAP monoclonal antibody, B6H12, blocked this interaction. We demonstrated that IAP bound directly to SHPS-1 and that in quiescent SMC disruption of the IAP/SHPS-1 interaction using B6H12 resulted in prevention of IGF-I from stimulating SHPS-1 phosphorylation, and SHP-2 transfer to SHPS-1 (64). Despite sustained IGF-I receptor phosphorylation, there was minimal phosphorylation of Shc and MAPK as well as impaired cell migration and proliferation responses to IGF-I. Overexpression of a deletion mutant of IAP that could not bind to SHPS-1 also showed impaired SHP-2 transfer and impaired cell migration and cell division responses to IGF-I. Therefore, IGF-I stimulation of SHPS-1 phosphorylation and the subsequent transfer of SHP-2 to SHPS-1 requires IAP association with SHPS-1. Inhibition of this interaction with a monoclonal antibody or by mutagenesis inhibits SHP-2 transfer and is associated with failure of SMC to respond to IGF-I with increases in MAPK activation and cell division. This further emphasizes the important role of SHP-2 transfer to SHPS-1 in activation of downstream signaling molecules such as Shc that are necessary for normal IGF-I responsiveness. Data published for other growth factor receptor-linked signaling systems (65, 66) suggest that integrin clustering and the appropriate assembly of a signaling complex within microdomains of the plasma membrane might be involved in the mechanism by which IAP/SHPS-1 interaction modulates SHP-2 transfer and Shc activation.
TS-1 is a pericellular protein that interacts with multiple cell surface receptors. It binds to both Vß3 and IAP. In the presence of increased concentrations of TS-1 or a synthetic peptide that contains the region of TS-1 that binds to IAP, there is an alteration in the SHPS-1/IAP interaction, which results in a delay in SHP-2 transfer to the IGF-I receptor prolonging receptor phosphorylation and enhanced MAPK activation in response to IGF-I (67). Therefore, altering the timing of SHP-2 transfer to both SHPS-1 and to the IGF-I receptor by changes in the IAP/SHPS-1 interaction can result in enhanced activation of signaling molecules such as MAPK and potentially Shc and enhancement of IGF-I actions.
There has been a great deal of interest in actions of IGFBPs that are mediated through their direct interaction with cellular proteins rather than by binding to IGF-I. IGFBP-5 is a form of IGFBP that is secreted by SMC and is abundant in ECM. Recently, we have shown that IGFBP-5 binds to thrombospondin and inhibits its interaction with IAP. This leads to a decrease in the ability of TS-1 to prolong IGF-I receptor phosphorylation and to enhance IGF-I-stimulated protein synthesis and cell migration (68). Therefore, TS-1 may be one of the cell surface proteins through which IGFBP-5 acts to alter cellular responses to IGF-I.
In summary, IGF-I receptor-linked signaling in arterial SMC is highly regulated by ligand occupancy of the Vß3 integrin. These two signaling systems intersect by controlling the ability of activated Vß3 to regulate SHP-2 transfer to the plasma membrane and thus alter the optimal assembly of signaling molecules on the transmembrane protein, SHPS-1 (Fig. 2C). Ligand occupancy of SHPS-1 by proteins such as IAP can also modulate SHP-2 transfer, and disruption of the SHPS-1/IAP interaction leads to attenuation of IGF-I signaling. Vß3 is a very versatile molecule for regulating IGF-I signaling because stimulation of its domain that binds to heparin binding motifs in ECM proteins such as vitronectin results in enhancement of IGF-I actions, whereas stimulation of RGD recognition domain results in their attenuation. Both of these mechanisms involve changes in the transfer of SHP-2 to SHPS-1 and to other downstream signaling molecules as well as their activation in response to IGF-I stimulation. Future studies should be able to build on these observations to construct a molecular map that will identify the multiple components of the membrane-associated signaling complexes that are assembled in response to Vß3 and IGF-I receptor activation and how differential activation of these two signaling systems alters their assembly and subsequent actions.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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First Published Online November 4, 2004
Abbreviations: ECM, Extracellular matrix; EGF, epidermal growth factor; FAK, focal adhesion kinase; FGF, fibroblast growth factor; IAP, integrin-associated protein; IGFBP, IGF binding protein; IRS-1, insulin receptor substrate-1; PDGF, platelet-derived growth factor; PI-3, phosphatidylinositol-3; PTB, phosphotyrosine binding; RGD, arginine-glycine-asparginine; SHP-2, Src homology 2 containing tyrosine phosphatase; SMC, smooth muscle cells; TS-1, thrombospondin-1; VEGF, vascular endothelial growth factor.
Received for publication September 22, 2004. Accepted for publication October 29, 2004.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONINTEGRIN ACTIVATIONDIRECT INTERACTIONS OF INTEGRINS...INDIRECT ACTIVATION OF INTEGRINS...INTERACTIONS OF INTEGRIN AND...{alpha}Vß3 INTEGRIN...COOPERATIVE INTERACTION BETWEEN...{alpha}Vß3 INTEGRINS...INCREASES IN LIGAND OCCUPANCY...MECHANISM OF...REFERENCES |
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