©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Outside-in Integrin Signal Transduction
alphabeta(3)-(GP IIb-IIIa) TYROSINE PHOSPHORYLATION INDUCED BY PLATELET AGGREGATION (*)

(Received for publication, February 21, 1996; and in revised form, March 13, 1996)

Debbie A. Law Lisa Nannizzi-Alaimo David R. Phillips (§)

From the From COR Therapeutics, Inc., South San Francisco, California 94080

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

alphabeta(3)-(GPIIb-IIIa) is the most abundant integrin expressed on platelets and plays a critical role in platelet aggregation and normal hemostasis. In response to platelet stimulation by agonists such as thrombin, alphabeta(3) becomes a receptor for the adhesive proteins fibrinogen, von Willebrand factor, vitronectin, and fibronectin. Binding of extracellular matrix ligands allows the integrin to transmit a signal to the inside of the cell, but the exact mechanisms whereby integrins transduce these signals remain unclear. In this paper we demonstrate that the beta(3) subunit of alphabeta(3) was phosphorylated on tyrosine residues in response to thrombin-induced platelet aggregation. However, tyrosine phosphorylation was not observed when platelets were stimulated by thrombin in the presence of an inhibitor of aggregation. Phosphotyrosine was only detected when platelets were solubilized under protein-denaturing conditions. A peptide corresponding to residues 740-762 of the beta(3) cytoplasmic domain was capable of binding the signaling proteins SHC and GRB2. GRB2 binding occurred only when both tyrosine residues (Tyr-747 and Tyr-759) were phosphorylated. SHC binding also occurred to a peptide monophosphorylated at Tyr-759. The data suggest that tyrosine phosphorylation of an integrin beta subunit may be important in initiating outside-in signaling cascades by inducing association of signaling components directly with the integrin.


INTRODUCTION

Integrins form a widely distributed family of heterodimeric cell surface receptors that play critical roles in cell adhesion events(1) . Integrins function not only to recognize macromolecular ligands but also to transmit signals, a process known as outside-in integrin signaling. These signals can induce a spectrum of cellular responses such as cytoskeleton assembly, gene expression, cell division, and cell motility(2) . How ligand binding to integrins initiates signaling events is an unsolved problem in integrin biology. However, it is clear that the cytoplasmic domains of the integrin subunits play a critical role in the signaling process(3, 4, 5) .

Previous studies have established that the outside-in integrin signaling through alphabeta(3) is critical for platelet function. The alphabeta(3)-dependent aggregation of platelets causes increased tyrosine phosphorylation of signaling proteins (e.g. Syk and FAK(6, 7) ), increased Ca mobilization(8) , and increased cytoskeletal organization accompanying changes in cell shape(9) . Many of these activities also occur when alphabeta(3) is expressed and activated in nucleated cells, and mutations of this receptor have shown that the cytoplasmic domains are critical for function. Such experiments suggest a regulatory role for the alpha cytoplasmic domain in ligand binding, while the cytoplasmic domain of beta(3) is involved not only in ligand binding to alphabeta(3) but also in its signaling response(4, 5, 10) .

One of the principal processes by which alphabeta(3) appears to mediate the transfer of information from the extracellular to intracellular milieu is by tyrosine phosphorylation of cytoplasmic proteins, e.g. FAK, Syk, and cortactin(2) . Ligand occupancy of alphabeta(3) is a prerequisite for the full range of tyrosine phosphorylation events to occur(11) , implying an important role for outside-in integrin signaling in this process.

A primary mechanism by which cell surface receptors transduce signals is through direct phosphorylation of receptor subunits. Indeed, the cytoplasmic domain of beta(3) contains two tyrosine residues. Although previous studies have determined that the beta(3) cytoplasmic domain is phosphorylated on threonine and serine residues in response to thrombin stimulation(12) , phosphorylation of the beta(3) cytoplasmic tyrosine residues in response to platelet aggregation has not been documented. Three observations suggest this possibility. First, tyrosine 747 exists within a motif similar to tyrosines known to be phosphorylated in the epidermal growth factor and insulin receptors(13) . Second, tyrosine 747 of beta(3) exists in an NPXpY motif (where pY is phosphotyrosine), known to bind the phosphotyrosine binding (PTB) (^1)domain of signaling proteins(14, 15) . Finally, there is striking conservation of the tyrosine residues not only within the beta(1), beta(3), beta(5), and beta(6) integrin members but also between species(16) , suggesting a functional role for these residues.

The experiments described here have attempted to determine whether tyrosine phosphorylation of the cytoplasmic domain of beta(3) plays a role in signal transduction through alphabeta(3). In this study we have addressed the issue of the tyrosine phosphorylation state of beta(3) after platelet aggregation using an aggressive protein solubilization protocol. The data show that tyrosine phosphorylation of the integrin beta(3) subunit occurs during alphabeta(3) outside-in signaling and suggests a possible mechanism for integrin signal transduction.


EXPERIMENTAL PROCEDURES

Platelet Preparation and Stimulation

Blood was collected and resting platelets prepared as described(17) , except 0.6 unit/ml apyrase and 50 ng/ml prostaglandin I(2) (final concentrations) were present in collecting solution. Washed platelets at 8 times 10^8/ml were incubated for 1 h at 37 °C and then stimulated by addition of 0.1 unit/ml thrombin with stirring until aggregates were visible (40 s to 2 min). The peptide antagonist Mpr-RGDWP-Pen-NH(2) (where Mpr is p-methylbenzyl-3-thio-propionic acid and Pen is L-penicillamine) (18) (1 µM) was added just prior to thrombin addition, whereas 20 µg/ml LM609 blocking antibody was added 90 s prior to the addition of thrombin. For platelet lysates, resting or activated platelets were lysed at 2-4 times 10^8 platelets/ml (final lysis buffer either 1% Nonidet P-40 or 1% Brij 96, 137 mM NaCl, 20 mM Tris, pH 8, 2 mM EDTA, 1 mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride, 20 µM leupeptin, and 0.15 unit ml aprotinin).

Reagents

The anti-phosphotyrosine antibodies PY-20 and 4G10 were from Transduction Laboratories and Upstate Biotechnology Inc., respectively. Murine anti-human beta(3) monoclonal antibodies were raised against either a synthetic peptide corresponding to the cytoplasmic domain of beta(3) (C3a.19.5) or to alphabeta(3) (E8). The alpha(v)beta(3) antibody LM609 (19) was generously provided by David Cheresh. Anti-GRB2 antiserum was from Santa Cruz Biotechnology, and anti-SHC antiserum was from Transduction Laboratories. Horseradish peroxidase-conjugated secondary antibodies were sheep anti-mouse Ig (Amersham Corp.) and goat anti-rabbit IgG (Jackson Immunoresearch Laboratories). Control mouse IgGs were from Sigma. Peptides were synthesized by SynPep Corporation using solid phase Fmoc (N-(9-fluorenyl)methoxycarbonyl) chemistry. The following enzymes were used: p60, (Oncogene Science), partially purified p59, partially purified p56, purified p93 (Upstate Biotechnology Inc.), and baculovirus-expressed murine Syk (the kind gift of S. Harmer and A. L. DeFranco, UCSF, San Francisco).

Determination of beta(3) Phosphorylation State

Platelets were stimulated as described above and the reactions immediately terminated by the addition of Laemmli sample buffer containing sodium vanadate. For each reaction, 600 µl of platelets at 8 times 10^8/ml were treated and lysed immediately by the addition of 200 µl of 4 times sample buffer (to give a final concentration of 37 mM Tris, pH 6.8, 11.8% glycerol, 2.36% SDS, 2 mM sodium vanadate, and 0.002% bromphenol blue). Samples were boiled for 5 min, and 400 µl (i.e. the equivalent of 2.5 times 10^8 platelets) of each sample were loaded per well on the first dimension (7% non-reducing SDS-PAGE) followed by 5% reducing SDS-PAGE in the second dimension (as detailed in (20) ). This allowed for duplicate gels to be run for each experiment. In each case one gel was stained with Coomassie Blue, and for the other the proteins were transferred to nitrocellulose for immunoblotting.

Peptide Precipitations

Lyophilized peptides were dissolved at a concentration of 2 mg/ml in 0.1% trifluoroacetic acid, 50% acetonitrile prior to each experiment and then diluted as needed. Peptides (at final concentrations of 1 and 10 µM) were incubated with 1% Nonidet P-40 lysates from resting platelets for 90 min at 4 °C. The peptides and any associated proteins were then isolated using avidin-agarose beads (as described in (21) ), and the complexes were separated on gels and transferred to nitrocellulose for analysis by immunoblotting.

In Vitro Kinase Assays

Purified tyrosine kinases or immunoprecipitates were mixed with the relevant substrate for 10 min in a final volume of 20 µl of kinase assay buffer (20 mM Tris-HCl, pH 7.2, 10 mM MgCl(2), 10 mM MnCl(2)) with 5 µCi of -[P]ATP (Amersham Corp.). The substrates used were affinity-purified alphabeta(3) (5 µM, prepared as described in (22) ), peptide corresponding to the cytoplasmic domain of beta(3) (10 µM), and enolase (1.5 µM, Sigma). Reactions were stopped by the addition of 2 times Laemmli reducing sample buffer and boiled for 5 min. The samples were separated on SDS-PAGE gels and the radioactive bands visualized by autoradiography using Kodak X-Omat AR film.


RESULTS

The cytoplasmic domains of alphabeta(3) contain two tyrosines, both on beta(3) (Tyr-747 and Tyr-759 (13) ). In preliminary experiments we failed to detect tyrosine phosphorylation of beta(3) in either activated, non-aggregated platelets, in agreement with a previous report(12) , or activated, aggregated platelets. In these experiments, alphabeta(3) was isolated by immunoprecipitation following lysis of platelets in mild detergents containing vanadate. Reasoning that dephosphorylation of phosphotyrosine may have occurred during detergent solubilization (particularly as tyrosine phosphatases, such as PTP1B, are abundant in platelets(23) ), we examined the tyrosine phosphorylation state of beta(3) from control and thrombin-aggregated platelets using an aggressive strategy for protein solubilization. Using the experimental design detailed in procedures an 8-fold increase (n = 6) in tyrosine phosphorylation of beta(3) was observed in aggregated as compared with control platelets (see Fig. 1B). Increased tyrosine phosphorylation of other proteins was also observed, most notably FAK and Syk as previously documented(6, 7) . These increases were either abrogated (e.g. beta(3) and FAK) or blunted (e.g. Syk) by the addition of integrin antagonist peptide (Fig. 1B), demonstrating that phosphorylation occurred primarily in response to aggregation and was not due to thrombin-induced platelet stimulation. Platelets also express the alpha(v)beta(3) integrin but at very low levels, approximately 0.1-0.2% of the amount of alphabeta(3)(24) . To determine whether this integrin contributed to the observed phosphorylation of beta(3), the experiments were also performed in the presence of LM609, an antibody that blocks the ligand binding activity of alpha(v)beta(3). However, even at 20 µg/ml LM609, where binding to platelets could be observed by fluorescence-activated cell sorter, the same enhancement in the level of beta(3) tyrosine phosphorylation was observed as occurred in its absence (data not shown).


Figure 1: Aggregation-induced tyrosine phosphorylation of beta(3) in platelets. Nonreduced-reduced two-dimensional gel electrophoresis was carried out on lysates from platelets either resting, thrombin-aggregated, or treated with thrombin in the presence of a cyclic RGD peptide antagonist to block aggregation. A, Coomassie Blue staining of a gel to visualize the proteins present and to demonstrate the migration patterns and loading integrity of the proteins. The position of the beta(3) protein is indicated by the arrows. B, anti-phosphotyrosine (Anti-ptyr) immunoblot of a duplicate gel, which was transferred to nitrocellulose. beta(3) protein is again indicated by arrows and was confirmed by stripping and reprobing the immunoblot with the anti-beta(3) monoclonal antibody C3a.19.5. C, the spots obtained with the anti-beta(3) antibody lined up exactly with the spots on the anti-phosphotyrosine blot marked with the arrows. Molecular mass standards are indicated to the left. Densitometry was performed using ImageQuant software on a Molecular Dynamics densitometer, and the results were normalized for the amount of beta(3) present. An average 8-fold increase in the tyrosine phosphorylation of beta(3) from aggregated as compared with control platelets was observed (n = 6).



Phosphorylation of cytoplasmic tyrosine residues has been shown to be important in recruiting signaling molecules to a number of cell surface receptors. Because beta(3) was dephosphorylated in non-reducing detergent, synthetic peptides corresponding to residues 740-762 of beta(3) were utilized to identify signaling proteins that can become associated with the phosphorylated cytoplasmic domain of beta(3). The peptide containing two phosphorylated tyrosine residues (Tyr(P)-747 and Tyr(P)-759) was found to bind both GRB2 and SHC from platelet lysates (Fig. 2). GRB2 was not bound by peptides in which the two tyrosines were either not phosphorylated or in which only one was phosphorylated. Thus, GRB2 binding required phosphorylation of both tyrosines. Our data also showed that SHC was not bound by peptides in which tyrosines were not phosphorylated or when only Tyr-747 was phosphorylated. However, SHC was bound by the Tyr(P)-759 peptide (Fig. 2B), indicating that phosphorylation of only the more carboxyl tyrosine of beta(3) was sufficient for interacting with this signaling protein. We failed to detect the association of other signaling molecules such as FAK, Syk, and phosphatidylinositol 3-kinase with any of the beta(3) peptides (data not shown).


Figure 2: Binding of the signaling proteins SHC and GRB2 to peptides encompassing the tyrosine-phosphorylated beta(3) cytoplasmic region. A, single-letter amino acid sequence of peptides used in these studies. B and C, immunoblots of proteins precipitated by the indicated peptides. 0.5-1 mg of 1% Nonidet P-40 lysates from resting platelets were incubated with peptides followed by avidin-agarose precipitation. The resulting immunoblots were probed with anti-SHC (B) or anti-GRB2 (C) antibodies.



As a test of the physiological importance of GRB2 or SHC binding to phosphorylated beta(3), a peptide containing a naturally occurring mutation of beta(3) (S752P) was used. This single point mutation was identified in a patient with Glanzmann's thrombasthenia(25) . This autosomal dominant bleeding disorder is caused by platelets that fail to aggregate (26) and has been shown to prevent outside-in signaling through beta(3)(27) . The doubly tyrosine-phosphorylated mutant peptide was unable to precipitate GRB2 and showed a marked decrease in its ability to precipitate SHC (Fig. 2), suggesting that the Ser to Pro mutation in these patients alters protein structure such that interactions with signaling proteins cannot occur.

It was not possible to determine whether SHC or GRB2 bound to phosphorylated beta(3) during platelet aggregation, due to the rapid dephosphorylation of beta(3) in detergent lysis conditions required for immunoprecipitation experiments. However, as shown in Fig. 3, both proteins were tyrosine-phosphorylated in response to platelet aggregation and furthermore appeared to associate with several unidentified phosphoproteins (e.g. p120 and p170) in an aggregation-dependent manner.


Figure 3: SHC and GRB2 are tyrosine-phosphorylated in response to thrombin and co-immunoprecipitate other tyrosine-phosphorylated proteins. A, anti-phosphotyrosine (Anti-ptyr) immunoblot of the indicated immunoprecipitations (I.P), or total lysate, from control(-) and thrombin-aggregated (+) platelets. 3 mg of 1% Nonidet P-40 platelet lysates were immunoprecipitated with 10 µg of the indicated antibody (Ab) plus protein A-Sepharose. Immunoblots were first probed with a mix of PY-20 and 4G10 anti-phosphotyrosine antibodies (A) and then stripped and reprobed with either anti-SHC (B) or anti-GRB2 (C) antibodies to show loading integrity between control and thrombin-aggregated samples. Molecular mass standards are indicated to the left of each blot. The migrations of particular proteins are indicated by the arrows to the right of the blots. Asterisk in A and B indicates the position of Ig heavy chain.



The experiments above indicated that beta(3) was a substrate for a tyrosine kinase(s). A number of tyrosine kinases are present in platelets and indeed several have been implicated in integrin signaling, including Src, Fyn, and Syk(6, 11) . To determine whether any of these kinases could be involved in alphabeta(3) tyrosine phosphorylation, Src family tyrosine kinases p60, p59, and p56 as well as Syk were tested for their activities in phosphorylating full-length beta(3) and a peptide consisting of the cytoplasmic domain. Of these kinases p60 and Syk showed the greatest ability to phosphorylate beta(3) (Fig. 4A). p59 showed modest activity, whereas the partially purified p56 showed less activity. However, the p56 also showed a decreased ability to phosphorylate the control substrate enolase (Fig. 4A). That p59 and p56 could phosphorylate beta(3) was confirmed in experiments using kinases immunoprecipitated from platelet lysates (data not shown). In contrast, the SH2-containing tyrosine kinase p93, which has been implicated in signaling via cytokine receptors(28) , showed little ability to phosphorylate beta(3). Although increased kinase activity was observed with the beta(3) immune complex from thrombin-aggregated as compared with resting platelets (Fig. 4B), we were unable to detect direct association of Src, Fyn, Lyn, or Syk with alphabeta(3) using co-immunoprecipitation and immunoblotting techniques (data not shown).


Figure 4: In vitro phosphorylation of beta(3) and association of tyrosine kinase activity with beta(3) upon platelet aggregation. A, in vitro kinase assay demonstrating the ability of the indicated tyrosine kinases to phosphorylate either the beta(3) subunit of affinity-purified alphabeta(3) (no phosphorylation of alpha was observed), a peptide consisting of residues 715-762 of beta(3), or enolase. 4 units of p60 and p59, 12 units of p56, 50 ng of p93, and baculovirus-expressed Syk were used. Asterisk indicates the band corresponding to p93 autophosphorylation. The p56 appeared to be less active than the other Src kinases as shown by its reduced ability to phosphorylate the exogenous substrate enolase, as well as beta(3). B, kinase activity co-immunoprecipitating with beta(3) from control and thrombin-aggregated platelets. Anti-beta(3) (E8) or control antibody was used to precipitate protein from 0.5 mg of 1% Brij 96 platelet lysates. The resulting immunoprecipitates were subjected to an in vitro kinase assay, and phosphorylated proteins were visualized by autoradiography.




DISCUSSION

Platelet aggregation, a process mediated by alphabeta(3), is known to initiate an outside-in signal through alphabeta(3), as evidenced by increased phosphorylation of proteins such as Syk and FAK (Refs. 6 and 7 and Fig. 1). The present data demonstrate for the first time that the beta(3) integrin subunit of alphabeta(3) is tyrosine-phosphorylated in response to aggregation. We also found that SHC and GRB2, two proteins known to participate in the Ras signaling pathway(29) , which is active in platelets(30) , could associate with phosphorylated beta(3) cytoplasmic domain peptides. SHC and GRB2 were also phosphorylated during platelet aggregation. These data suggest that the initial event triggered by outside-in signaling via the alphabeta(3) integrin on platelets may in fact be the tyrosine phosphorylation of beta(3) and that the phosphorylated cytoplasmic domain has the capability of assembling signaling complexes, which also become tyrosine-phosphorylated.

Platelets express two integrins containing the beta(3) subunit: alphabeta(3) and alpha(v)beta(3). alphabeta(3) is abundant on platelets (50,000 molecules/platelet; 1% of total protein mass(31) ) and has been shown to mediate platelet aggregation(32) . In contrast, alpha(v)beta(3) is expressed at very low levels (50-100 molecules/platelet, 0.1-0.2% of the levels of alphabeta(3)(24) ), and it is unclear what role, if any, this integrin plays in platelet function. The presence of LM609, an antibody that blocks the ligand binding activity of alpha(v)beta(3)(19) , had no effect on the amount of beta(3) tyrosine phosphorylation observed, indicating that the beta(3) was phosphorylated as the alphabeta(3) integrin.

alphabeta(3) participates in both outside-in and inside-out signal transduction(32) . Inside-out signaling results in alphabeta(3) having enhanced affinity for fibrinogen and von Willebrand factor following platelet stimulation by agonists such as thrombin and ADP. Although the mechanism of inside-out signaling is not known, it most likely is mediated by membrane perturbation that affects a large number of receptors or is induced by a direct modification of all receptors, as the affinity state of all receptors is capable of being up-regulated. Outside-in signaling results when platelet stimulation occurs upon integrin ligation to a substrate that allows alphabeta(3) clustering, e.g. during platelet aggregation. Given the abundance of alphabeta(3) on platelets and the known transient nature of tyrosine phosphorylation of other cell surface signaling receptors (e.g. Ig-alpha and Ig-beta of the B cell receptor complex(33, 34) ), it is not unexpected that beta(3) tyrosine phosphorylation would occur on a small number of receptors and be transient and that dephosphorylation mechanisms would be essential and robust. This perhaps explains why tyrosine phosphorylation has not been observed previously for this integrin. In other cell systems, such as B lymphocytes, it has been possible to increase the amount of detectable B cell receptor tyrosine phosphorylation by performing stimulations at 0-4 °C(33) . Since this temperature does not allow for platelet aggregation, these conditions cannot be utilized to characterize beta(3) tyrosine phosphorylations during aggregation-induced alphabeta(3) signaling.

The alphabeta(3) integrin is not expected to have any intrinsic tyrosine kinase activity and, so far, we have been unable to detect the direct association of a tyrosine kinase(s) with this integrin. However, our in vitro data suggest that possible candidates include members of the Src family of tyrosine kinases or possibly Syk. Support for the role of these kinases in the phosphorylation of alphabeta(3)in vivo has been provided by Dorahy et al.(35) , who have demonstrated that Src and Lyn can be cross-linked to beta(3) in intact platelets by chemical cross-linking agents.

Although this is the first report of outside-in signaling inducing the tyrosine phosphorylation of beta(3), it should be noted that tyrosine phosphorylation of the beta(4) integrin subunit has been observed in response to integrin ligation(36) . beta(4) is novel among integrin subunits. Its cytoplasmic domain is 1,018 amino acids in length, bears no sequence homology to beta(3) or other integrin beta subunits, and contains an immune receptor tyrosine-based activation motif (ITAM)(37) . ITAMs are defined amino acid motifs containing two tyrosine residues as well as other critical amino acids. It is well documented that phosphorylated ITAMs play essential roles in signaling via T cell, B cell, and Fc receptor complexes (reviewed in (38) ).

The two tyrosines in beta(3) are separated by an 11-amino acid peptide sequence. This spacing is similar to that of the two tyrosines found in the ITAM domains of immune receptor complexes. Although the tyrosines of the beta(3) integrin subunit do not fulfil the ITAM sequence requirement of obligatory leucine or isoleucine residues +3 from the tyrosine residues, it becomes of interest to compare the interactions of the two phosphorylated receptor types. The tyrosine residues of ITAM domains are phosphorylated upon receptor ligation, which allows for the recruitment of cytoplasmic proteins. In particular, the direct binding to the tandem SH2 domains of the tyrosine kinases ZAP-70 and Syk has been documented(21, 39) . While we failed to detect the association of Syk with doubly phosphorylated beta(3) peptides, we did detect the association of two intracellular signaling proteins, GRB2 and SHC. SHC has been shown to associate with phosphorylated ITAMs from both the T cell and B cell receptor complexes(40, 41) . However, neither tyrosine in beta(3) exists in a GRB2 SH2 binding motif(42) . Also, GRB2 only contains a single SH2 domain while phosphorylations of both tyrosines were required for binding. Thus, it seems probable that binding of GRB2 is bridged by another protein, possibly one containing tandem phosphotyrosine binding domains. SHC can bind to phosphotyrosines through SH2 domain interactions as well as to NPXpY (where pY is phosphotyrosine) motifs via a PTB domain (14, 15) . Our data showed that SHC did not bind to monophosphorylated Tyr-747 peptide, which contains an NPXY motif. However, it did bind to the monophosphorylated Tyr-759 peptide (Fig. 2). The sequence surrounding Tyr-759 does not appear to be in a motif recognized by either the SH2 (42) or PTB domain (14, 15) of SHC. Although this may indicate that binding of SHC, too, is bridged by another protein, perhaps in a signaling complex distinct from that involving GRB2, we cannot rule out a direct association between SHC and beta(3). This suggestion derives from recent data showing that the proline may not be required in the motif recognized by proteins containing PTB domains (43) and that a hydrophobic residue in the -5 position promotes PTB(44) . This position on beta(3), residue 754, is a phenylalanine. Interestingly, we found that GRB2, as well as SHC, was tyrosine-phosphorylated in thrombin-aggregated platelets. To our knowledge, this is the first example of tyrosine phosphorylation of GRB2 during a signaling response. Since tyrosine phosphorylation does not appear to be necessary for GRB2 association with the Ras exchange factor SOS(45) , tyrosine phosphorylation of GRB2 may be involved in an as yet undescribed function of this adaptor protein.

The doubly phosphorylated beta(3) peptide containing the S752P point mutation failed to bind to GRB2 and showed markedly reduced binding with SHC. alphabeta(3) bearing the S752P mutation is known to be defective in outside-in signaling as Chinese hamster ovary cells expressing this receptor show reduced ability to spread on immobilized fibrinogen, form focal adhesions, and retract fibrin clots, compared with cells expressing wild type alphabeta(3)(27) . Our data suggest that this defect may be due to the inability, or reduced ability, of the S752P beta(3) cytoplasmic domain to recruit signaling proteins even if the tyrosines are phosphorylated. beta(3) endonexin, a protein of unknown function that was identified by its binding to the beta(3) cytoplasmic region in the yeast two-hybrid system, also has reduced ability to bind the beta(3) S752P cytoplasmic domain(46) .

These data provide a mechanism by which alphabeta(3) can mediate outside-in signaling. This mechanism surprisingly shares many of the overall features of growth factor and immune receptor-mediated signaling; receptor cross-linking leads to tyrosine phosphorylation of the receptor cytoplasmic domains, followed by the induced binding of signaling complexes to the phosphorylated receptor. The tyrosines in beta(3) do exist in sequences bearing striking homology to several other integrin beta subunits (e.g. 740-762, the carboxyl terminus, is 50% identical to beta(1), 36% to beta(5), and 54% to beta(6)(14) ). This suggests that tyrosine phosphorylation may also be of importance in outside-in signaling via these integrins. As yet, these homologous beta subunits have not been shown to be tyrosine-phosphorylated in response to integrin signaling, but beta(1) is constitutively tyrosine-phosphorylated in v-Src-transformed cells(47) . Although phosphorylation of beta(1) prevents the incorporation of alpha(5)beta(1) in focal adhesion sites(48) , it is not clear whether phosphorylated alpha(5)beta(1) contributes to cellular signaling processes. Taken together our data make it attractive to hypothesize that the tyrosine phosphorylation of integrin beta subunits is a general method for initiating outside-in signaling, allowing the phosphorylated beta subunits to recruit phosphotyrosine-binding cytoplasmic signaling molecules to the membrane, thereby activating intracellular signaling pathways.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: COR Therapeutics, Inc., 256 E. Grand Ave., South San Francisco, CA 94080. Tel.: 415-244-6884; Fax: 415-244-9270.

(^1)
The abbreviations used are: PTB, phosphotyrosine binding; PAGE, polyacrylamide gel electrophoresis; ITAM, immune receptor tyrosine-based activation motif.


ACKNOWLEDGEMENTS

We thank David Cheresh for the LM609 antibody, Anthony DeFranco and Stacey Harmer for the baculovirus-expressed Syk, Willy Teng, Mary Ann Naughton, and Bob Scarborough for providing the RGD inhibitor peptide and immunizing peptides, the COR antibody facility for making the anti-beta(3) monoclonal antibodies, and Leslie Parise for useful comments regarding the manuscript.


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