Vol. 265, No. 4, Issue of February 5, pp. 2168-2172, 1990 Printed in U.S. A.

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1990 by The American Society for Biochemistry and Molecular Biology, Inc.

Integrin (cx&)-Ligand Interaction IDENTIFICATION OF A HETERODIMERIC RGD BINDING SITE ON THE VITRONECTIN RECEPTOR*

(Received for publication, July 11, 1989)

Jeffrey W. Smith* and David A. Cheresh From the Department of Immunology, Scripps Clinic and Research Foundation, La Jolla, California 92037

The vitronectin receptor mediates cell adhesion to the extracellular matrix proteins vitronectin, fib- rinogen, von Willebrand factor, and thrombospondin in an RGD-dependent manner. We previously demon- strated the direct interaction between the vitronectin receptor and an RGD-containing peptide by photo- affinity labeling the receptor with 12SI-sulfosuccinim- idyl-2-(p-azido-salicylamido)-1,3’-dithiopropionate (SASD)-GRGDSPK (Smith, J. W., and Cheresh, D. A. (1988) J. Biol. Chem. 263, 18726-18731). In that report, we identified amino acid residues 61-203 of the &subunit as proximal to the ligand binding site. Here we demonstrate that “‘1-SASD-GRGDSPK affin- ity labels the a-subunit of the receptor at least two distinct sites within the region encompassing residues 139-349. Both of these regions are within the putative divalent cation binding region of the o-subunit. Collec- tively, our results suggest that discrete amino-terminal domains of both subunits of the receptor contribute to the structure of the ligand binding domain and fur- thermore that the ligand and divalent cation binding domains are spatially and functionally linked.

Cell adhesion is mediated by cell surface receptors for the extracellular matrix. Many of these receptors are members of the integrin gene family (1, 2). The integrins are expressed on the cell surface as heterodimers of cy- and P-subunits, and each cell has a specific repertoire of receptors that define its adhesive capabilities. Receptors in the integrin family are associated with several significant biological events including hemostasis (3) and developmental processes that require tis- sue remodeling including gastrulation and neural crest cell migration (4, 5). The integrins are also apparently of great importance in a pathological sense because they have been implicated in tumor invasion and metastasis (6, 7). Integrin- mediated cell surface interaction with extracellular matrix proteins is not limited to vertebrates. For example, Drosophila development is markedly dependent upon proper integrin function (8). In addition, some microbes have developed “in- tegrin-like” receptors that bind the host extracellular matrix and potentiate infection (9).

We have been examining the vitronectin receptor (integrin

* This work was supported by National Institutes of Health Grants CA45726 and CA50286. This is Scripps Publication 5896. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

4 Supported by a fellowship from the Cancer Research Institute, New York.

01& or VNR),’ which is a member of the p-3 integrin subfam- ily (1). VNR is expressed on a variety of cells including endothelial, melanoma, osteosarcoma, and smooth muscle (10-13). Although it was originally named for its ability to bind vitronectin, it has since been demonstrated that VNR also mediates cell adhesion to fibrinogen, von Willebrand factor (11, 14), and, more recently, thrombospondin (13). It is likely that the ability of VNR to recognize multiple ligands is a function of its ability to bind the tripeptide sequence RGD, which is contained within the primary amino acid sequence of these adhesive proteins (2). This property is common to many integrins and has facilitated their purifica- tion by affinity chromatography on RGD-containing matrices (15). The biological effects of RGD-containing peptides have led to considerable interest in their potential therapeutic applications. In fact, studies have shown that these peptides are capable of inhibiting tumor metastasis and microbial invasion (6, 9).

Given the physiological significance of RGD recognition, we have focused our efforts on defining the location of the RGD binding domain within the primary amino acid sequence of VNR. This has been technically difficult because VNR binds to RGD peptides with relatively low affinity (16) and because heterodimeric integrity is required for ligand recog- nition (11, 17, 18). We recently demonstrated the direct interaction between VNR and a photoaffinity derivative of a small RGD peptide, ‘251-SASD-GRGDSPK (19). When incu- bated with VNR and subsequently photolyzed, ‘251-SASD- GRGDSPK affinity-labeled both subunits of the receptor. However, the kinetics of incorporation of affinity label into each subunit were consistent with the existence of a single RGD binding site on VNR (19). We hypothesized that closely opposed regions of both subunits contribute to the three- dimensional structure of the RGD binding domain, thus ex- plaining the affinity labeling of each subunit. In that report we also determined that amino acid residues 61-203 of the fi- subunit were affinity-labeled by ‘251-SASD-GRGDSPK. Since this region is homologous on several RGD binding integrins, it probably constitutes a portion of the ligand binding domain.

Here, we report the identification of the region of the (Y- subunit that is affinity-labeled by ‘251-SASD-GRGDSPK. The affinity label can be localized to at least two distinct sites on the a-subunit between residues 139 and 349. These regions encompass the three amino-terminal putative divalent cation binding domains. The photoaffinity-labeled domains and the putative cation binding sites of VNR are highly homologous to other integrins, including platelet glycoprotein IIb and the a-subunit (01~) of the fibronectin receptor. The results pre-

’ The abbreviations used are: VNR, vitronectin receptor; SASD, sulfosuccinimidy12-(p-azido-salicylamido)-1,3’-dithiopropionate; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electropho- resis; LFA-1, leukocyte function-associated molecule 1; gp, glycopro- tein.

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The Integrin RGD Binding Domain 2169

sented here and in our previous report (19) indicate that specific amino-terminal domains of each subunit of VNR contribute to the structure of a divalent cation-dependent heterodimeric ligand binding site.

EXPERIMENTAL PROCEDURES Materials-Synthetic peptides were purchased from Telios Phar-

maceuticals. SASD was obtained from Pierce Chemical Co. P-Amino- 2-methyl-1,3-propanediol, acetone, triethylamine, and acetic acid were from Aldrich. Immobilon filters were obtained from Millipore. Nan51 (lMS.30) was purchased from Amersham Corp. Chymotrypsin and V8 protease were purchased from Boehringer Mannheim. X- Omat-XARB x-ray film was purchased from Kodak.

Purification and Photoaffinity Labeling of the Vitronectin Recep- tor-Vitronectin receptor was purified from human placenta by se- quential affinity chromatography on Mab LM 609-Sepharose and wheat germ 1ectinSepharose as previously described (19). The photo- affinity derivative of the peptide GRGDSPK was synthesized by coupling the peptide to SASD as previously described (19). Purified receptor was affinity-labeled by incubating VNR (200 rg/ml in phos- phate-buffered saline + 0.1% Nonidet P-40, 1.0 mM MgCl*, 0.5 mM MnCl*) with 7.0 X lo6 cpm of ‘251-SASD-GRGDSPK (5.4 x 10e7 M) for 1 h in the dark. The sample was photolyzed with UV light in a Rayonette light box for 10 min.

Digestion and Amino Acid Sequence Analysis of Photouffinity- labeled cu-Subunit-Photoaftinitv-labeled o-subunit was nurified bv preparative SDS-PAGE and electroelution (20). The yield of (Y sub”- unit from electroelution was typically 60%, which is within the reported range for a protein with a relatively high M, (20). Electro- luted material was quantitatively precipitated by incubation in ace- tone:triethylamine:acetic acidwater (85:5:5:5 by volume). Electroe- luted o-subunit (25 pg) was resuspended in ammediol sample buffer (21) and digested with 5.5 pg of Staphylococcus aurew V8 protease for 18 h at-37 “C. The resultant peptides were separated by SDS- PAGE (21) and electronhoreticallv transferred to Immobilon. Com- parison’of the radioac&ty loaded on the gel with that recovered on the Immobilon blot by y counting the radioactive bands indicated that 71% of the radioactivity was recovered (2729 cpm recovered from 3843 cpm loaded). Alternatively, 20 pg of cross-linked a-subunit were purified by SDS-PAGE, excised from the gel, and placed in the well of an ammediol-buffered gel (21). This gel slice was overlaid with 4 pg of chymotrypsin in ammediol sample buffer and electrophoresed until the dye front reached the bottom of the stacking gel. At this point the power was turned off for 50 min to allow digestion to occur (22). Subsequently, electrophoresis was continued, and the peptides were transferred to Immobilon membranes. The recovery of radioac- tivity from affinity-labeled (Y subunit was determined by y counting and was 83% (2822 cpm recovered from 3400). In two preliminary experiments in which similar digests were detected by autoradiogra- phy of dried gels, rather than by blotting, the recovery of radioactive peptides was detected by y counting gel slices. In this case, the recovery of radioactive peptides was nearly quantitative, indicating that the somewhat lower yield on Immobilon blots was due to a lack of quantitative electrophoretic transfer. Immobilon blots were ex- posed to x-ray film at -70°C to identify affinity-labeled peptides.

Amino-terminal amino acid sequence analysis of affinity-labeled peptides was performed as described (23) with an Applied Biosystems model 470A protein sequenator. On-line phenylthiohydantoin analy- sis was performed with the 03pth program. Initial yields were lo- 50% as described (23, 24), and repetitive yields were 75-90% depend- ing on the residue being resolved. It should be mentioned that only a small percentage of each of the affinity-labeled peptides is actually covalently linked to the affinity label. As we have previously docu- mented, this is because only 0.4% of the added affinity label becomes incorporated into VNR upon photolysis (19). This “tracer” method- ology is appropriate for separation and identification of affinity- labeled peptides by SDS-PAGE, since the SASD moiety does not alter the migration of peptides on SDS gels. However, further sepa- ration of co-migrating peptides for sequence analysis by methods such as reversed-phase high pressure liquid chromatography is not feasible, since insufficient amounts of peptide are actually covalently linked to the SASD moiety in order to purify and sequence.

RESULTS

Amino Acid Sequence of the a-subunit of VNR Proximal to the Ligand Binding Site-Purified VNR was photoaffinity-

labeled with 1251-SASD-GRGDSPK (Fig. 1, lane 1) in order to identify regions of the a-subunit proximal to the ligand binding site. We previously demonstrated that the interaction between both subunits of VNR and ‘251-SASD-GRGDSPK was highly specific (19). No affinity label was associated with the light chain of the cr-subunit, so the heavy chain was purified by preparative SDS-PAGE under reducing condi- tions. This also served to remove the RGD peptide from the receptor by cleaving the disulfide bond in SASD, leaving the receptor radiolabeled proximal to the ligand binding site. This material was subjected to digestion with chymotrypsin by the method of Cleveland et al. (22), yielding a highly reproducible peptide map in which two prominent affinity-labeled bands of 26.5 kDa (peptide A) and 25 kDa (containing peptides B + C from sequence analysis, see below) are visualized (Fig. 1, lane 2). A 55kDa peptide, containing less radiolabel, was also visualized, but when smaller amounts of a-subunit were di- gested this radiolabel could be chased into the two low molec- ular weight bands. Therefore, attention was focused on the 26.5 and 25-kDa peptides. These bands were subjected to automated Edman degradation and the results are shown in Table I (and schematically in Fig. 2). The amino-terminal amino acid sequence and relative mass of the 26.5-kDa peptide (peptide A) indicated that it began at residue 36 of the a- subunit and extended to the chymotryptic clip site, tyrosine, at position 224.

Two sequences, designated as peptides B and C, were re- solved in the 25-kDa band. The amino-terminal amino acid sequence of peptide B began at residue 225 of the a-subunit and based on its relative mass, its end point was near residue 450. The amino terminus of peptide C corresponded to residue 295, indicating that it extended to residue 515. Although it was impossible to determine which of these peptides was affinity-labeled, it is likely that both were since they have residues 295-450 in common. The results from amino acid sequence analysis of affinity-labeled chymotryptic peptides were consistent with two distinct sites of affinity labeling, because there was no overlapping region between peptides A and B, but since the relative mass of peptides cannot be determined at the single residue level by SDS-PAGE, it was possible that peptide A actually extended slightly beyond the amino terminus of peptide B. If the a-subunit was affinity- labeled exclusively in this overlapping region, then all of

MrxlO-3 MrxlO-3 MrxlO-3

160- WCC 92- 69-

9% QL 46- -P 30-

21- 14.3%

1 2 3 FIG. 1. Proteolytic digestion of photoaffinity-labeled a-sub-

unit. Purified VNR was photoaffinity-labeled as described under “Experimental Procedures” (lone I ). The a-subunit (20 rg) was excised and subjected to digestion with 4 rg of chymotrypsin accord- ing to the method of Cleveland et al. (22) (lane 2). Alternatively, 25 pg of affinity-labeled o-subunit were electroeluted and subjected to digestion with 5.5 rg of S. aureus V8 protease for 18 h at 37°C (lane 3). Both digests were electrophoretically blotted to Immobilon filters and exposed to x-ray film. The corresponding autoradiograms are shown. Arrows point to the radiolabeled peptides that were subjected to NHP-terminal amino acid sequence analysis. These are referred to as A-H in accordance with the notation in Table I and Fig. 2. Peptide D (lane 2, arrowhead) was visualized with Coomassie Blue but was not radiolabeled.

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S. aureus V8 pi-otease

The Integrin RGD Binding Domain

TABLE I Amino acid sequence of affinity-labeled peptides

The affinity-labeled peptides sbown in Fig. 1 were subject to amino acid sequence analysis while bound to lmmobilon filters. The residues resolved by this analysis are shown in lower case letters, and the actual amino acid sequence of the receptor in the corresponding region (27) is shown below this in upper case letters. Cysteine is not determined by this method (*). Positions where no residue was resolved are denoted as x in the sequence. The proposed end point of each peptide was determined based on its M. and the cumulative molecular weights of each amino acid in its sequence.

NH,- COOH- Amino acid Protease Peptide M, terminal terminal sequence

residue residue resolved

Chymotrypsin A 26,500 36 224 l-l-v-g-a-p-k L-L-V-G-A-P-K

B 25,000 225 450 s-v-a-v-x-d S-V-A-V-G-D

C 25,000 295 515 i-g-a-p I-G-A-P

D 75,000 436 860 r-a-r-p-v-i-t-v unlabeled R-A-R-P-V-I-T-V

E 25,500 139 349 y-a-p-*-r-s-q-d Y-A-P-C-R-S-Q-D

F 21,000 191 349 i-v-s-k-y-d-p-n-v-y I-V-S-K-Y-D-P-N-V-Y

G 4,500 139 167

H 4,500 312 349

y-a-p-*-r-x-q-d Y-A-P-C-R-S-Q-D

v-g-q-v-s-v-x-l-q-r V-G-Q-V-S-V-S-L-Q-R

FIG. 2. Schematic model of proteolytically digested a-sub- unit. A schematic model of the a-subunit of VNR is shown. The membrane-spanning region is shaded black, and the putative divalent cation binding domains are cross-hatched. The chymotryptic and V8 protease-generated peptides are depicted as lines below the appropri- ate region of the subunit. The peptides are labeled A-H using the same notation as Fig. 1 and Table I. The sites of affinity labeling were identified by defining the smallest common region of the affinity- labeled peptides from both digests (denoted as Affinity Labeled Re- gion). The position of the 78-kDa unlabeled chymotryptic peptide is shown for reference.

affinity label associated with peptides A and B could be accounted for by a single site of affinity labeling at or near position 225. This possibility was discounted because results from V8 protease digestion demonstrated that the a-subunit was affinity-labeled at a site at least 58 residues distant to residue 225 (since either peptide G or H must contain affinity label, see below). Thus, peptide mapping with chymotrypsin indicates that the a-subunit was affinity-labeled at two dis- tinct sites near the amino terminus of the molecule.

Interestingly, a nonlabeled peptide of 78 kDa (peptide D) was also reproducibly generated by chymotrypic digestion and was identified by staining the blot with Coomassie Blue. Its amino-terminal sequence corresponded to residues 436-443, and the relative mass indicated that it extended to the car-

boxy1 terminus of the heavy chain of the a-subunit. This demonstrated that no affinity label was contained within the region extending from residues 436 to 860.

To confirm and further localize the site of affinity label, cross-linked a-subunit was electroeluted, digested with S. aweus V8 protease, and the resulting peptides were separated by SDS-PAGE and blotted to Immobilon. Autoradiography revealed three radiolabeled bands with relative masses of 25.5, 21, and 4.5 kDa (Fig. 1, lane 3). These peptides were also subjected to Edman degradation. Amino-terminal sequence analysis of the 25.5-kDa band (peptide E) showed that it began at residue 139, and its proposed end point, based on relative mass, was one of the V8 clip sites at position 349, 351, or 353 (denoted as 349 below). The amino-terminal residue of the 21-kDa band (peptide F) corresponded to resi- due 191, and its proposed end point was also residue 349. Two sequences were resolved from the 4.5-kDa band (peptides G and H). The amino-terminal sequence and relative mass of peptide G indicated that it encompassed residues 139-167. Peptide H encompassed residues 312-349. The sequences of peptides F, G, and H were subsets of the larger V8-generated peptide (peptide E, residues 139-349); therefore, results from the V8 digestion indicate that all of the affinity label resides within this region. These results were entirely consistent with the results from chymotryptic digestion. In addition, analysis of the two peptide maps for regions in common revealed that two or more affinity-labeling sites exist within the region encompassing residues 139-349. The simplest explanation of the data would be that one site resides between residues 139 and 224 and another exists between residues 225 and 349. However, it is also conceivable that affinity labeling occurred to the carboxyl side of residue 224 and also within the region encompassed by peptide H (residues 312-349). Although we cannot unambiguously delineate between these two possibili- ties, it is clear that two sites of labeling exist between residues 139 and 349.

The Affinity-labeled Site on the a-Subunit of VNR Has

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The Integrin RGD Binding Domain 2171

FIG. 3. The photoaffinity-labeled region of the a-subunit of VNR is homologous to other integrins. The amino acid sequence homology between gpIIb, the a-subunits of fibronectin receptor (FNR) and LFA-1, and the photoaffinity-labeled region of the (Y- subunit of VNR is shown. The sequence alignment proposed by Larson et al. (29) was used except that the region corresponding to VNR residues 142-157 was aligned according to Fitzgerald et al. (27). The degree of homology between the four proteins is shown above the sequences. Asterisks denote positions where at least three of the sequences contain the same residue, and periods denote positions where two of the sequences have the same residue. The putative divalent cation binding sites are in reuerse tone, and the position of the LFA-1 I domain is noted. As discussed under “Results,” two possibilities exist for the exact placement of the two sites of affinity labeling between residues 139 and 349. We have chosen to depict the position of the first (- ) and second (- - -) affinity-labeling sites according to the simplest hypothesis (see “Results”).

Significant Homology with the a-Subunits of the Fibronectin Receptor and Platelet Integrin Glycoprotein Ilb-IIZa-Like VNR, the fibronectin receptor (~1&) and platelet glycoprotein IIb-IIIa bind their target ligands in an RGD-dependent man- ner (25, 26). Therefore, we compared the primary amino acid sequence of the affinity-labeled site of the a-subunit of VNR with the corresponding regions on the a-subunits of these integrins (Fig. 3). These proteins share an overall homology of 36-45% (27), which can largely be accounted for by several regions where the homology approaches identity. In fact, three of the four putative divalent cation binding sites of VNR (28) are located within the affinity-labeled region of the a-subunit, and these sites contain sequences which are nearly identical between VNR, the fibronectin receptor, and gpIIb (Fig. 3). The significance of this common structural motif is under- scored by our results demonstrating its proximity to the ligand binding site. In contrast, the a-subunit of leukocyte function- associated molecule 1 (LFA-1) has significant structural dif- ferences in this region of the molecule. In particular, LFA-1 lacks the first putative divalent cation binding site that VNR,

the fibronectin receptor, and gpIIb contain and also contains a 200-amino acid insertion, termed an “I” domain (29). It is interesting to note that the I domain on the LFA-1 a-subunit directly corresponds to the amino-terminal region of the affinity-labeling site on the a-subunit of VNR.

DISCUSSION

The results presented here lead to several salient conclu- sions regarding the location and structure of the ligand bind- ing domain of VNR. Since we previously determined that residues 61-203 of the P-subunit of VNR were proximal to the ligand binding site (19) and others have demonstrated that the same region is proximal to the ligand binding site of platelet glycoprotein IIb-IIIa (30), in this report we focused on identification of the region of the a-subunit proximal to the ligand binding site. This was considerably more difficult since only a small portion (lo-20%) of the total affinity label was associated with this subunit (19, 30). Peptide mapping and amino acid sequence analysis of a-subunit, affinity-la- beled with lZ51SASD-GRGDSPK, revealed that all of the radiolabel associated with this subunit was localized at two distinct sites in the region encompassing residues 139-349.

The observation that VNR was photoaffinity-labeled at three distinct sites, two on the a-subunit and one on the fi, by ‘*“I-SASD-GRGDSPK may be explained by several hy- potheses. As previously discussed, the bound RGD peptide may exist in more than one conformation (19, 31) resulting in different orientations of the photoactivatable aryl azide and three sites of affinity labeling, two on the a-subunit and one on the /3. The RGD binding site may be a flexible structure in which the three affinity-labeled regions are subject to rearrangements, moving them in and out of proximity to bound RGD peptide. Alternatively, the receptor may exist in two distinct conformations, each of which is competent to bind the RGD peptide. Each competent conformation might share the same region of the P-subunit (residues 61-203) but would consist of distinct regions of the a-subunit. This is an attractive hypothesis because the region of the a-subunit that was affinity-labeled is composed of seven structurally homol- ogous repeats (27,29). These repeats may be able to subsitute for one another at the ligand binding site and act as inter- changeable “cassettes” in the ligand binding domain. This would provide a receptor, like VNR, with the ability to fine- tune the structure of its ligand binding pocket for specific conformations of the RGD sequence and may explain how one receptor can mediate cell adhesion to more than one matrix protein.

The regions of VNR involved in ligand binding are highly homologous to platelet glycoprotein IIb-IIIa and the fibronec- tin receptor. These adhesion receptors also bind their target ligands in an RGD-dependent manner. The amino acid se- quence homology of the a-subunits of these receptors (gpIIb, o(“, and CQ,) is between 36 and 45%, but in the region that is affinity-labeled it is significantly higher. In fact, the sequences of the putative divalent cation binding sites approach identity, and several other regions have conservative amino acid sub- stitutions.

However, this similarity at the ligand binding sites does not preclude a difference in ligand recognition by the integrins. For example, we recently found that integrins on endothelial cells recognize a site on fibrinogen distinct from the site recognized by platelets (32). More specifically, we have deter- mined that purified gpIIb-IIIa and VNR recognize distinct, but structurally similar, sites on fibrinogen.’ VNR binds ex-

*J. W. Smith, Z. M. Ruggeri, T. J. Kunicki, and D. A. Cheresh, manuscript in preparation.

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2172 The Integrin RGD Binding Domain

elusively to the RGD sequence at fibrinogen residues 572- 574. However, gpIIb-IIIa does not require this sequence for binding to fibrinogen. This difference in ligand recognition is probably a result of the tertiary structure of the ligand binding domain of these two integrins since they have identical /?- subunits, and the data presented here indicate that the region of the a-subunits proximal to the RGD binding site has significant homology at the level of primary sequence.

The integrin gene family also includes receptors associated with leukocyte adhesion. Recently Larson et al. (29) proposed a subclassification of integrin a-subunits based on the pres- ence of an inserted domain near the amino terminus of the molecule. The term I denotes the presence of an inserted or interactive domain on the a-subunits of LFA-1, MAC-l, and p150,95 (33-35), all of which are involved in leukocyte adhe- sion (36). Interestingly, this domain is inserted directly into a region of the a-subunit homologous to that which is involved in RGD recognition by VNR. This insertion apparently has profound effects on the ligand binding properties of the LFA- 1 molecule since its target ligand, ICAM-1, does not contain an RGD in its primary sequence (37). LFA-1 is also function- ally distinct from other integrins because it mediates cell-cell interactions rather than cell-extracellular matrix interactions (37). These observations are consistent with the hypothesis that the I domain is an “interactive” domain responsible for ligand recognition (29). However, evidence has also been presented that MAC-l, which contains an I domain, binds to its target ligand, C3bi, through the RGD sequence (38). There- fore, the presence of an I domain may give a receptor the ability to interact with ligands which do not contain the RGD sequence, but insertion of this domain apparently does not exclude interaction with the RGD sequence.

The functional significance of the region of the integrin (Y- subunits identified here is also underscored by the observation that exon 8 of the a-subunit of Drosophila position-specific antigen-2 is subject to alternative splicing during development (39). This region corresponds to residues 181-218 of the (Y- subunit of VNR, which is within the affinity-labeled region. This splicing event may change the structure of the ligand binding site and the ligand specificity of position-specific antigen-2.

Photoaffinity labeling indicates that the architecture of the integrin RGD binding domain may be structurally far more complex than the tripeptide sequence it evolved to interact with. The results presented here, and previously (19), provide a rigorous demonstration that three distinct regions of VNR are proximal to the ligand binding site. Although the precise three-dimensional structure of the integrins awaits elucida- tion by x-ray and nuclear magnetic resonance techniques, our results demonstrate that discrete amino-terminal domains of both subunits contribute to ligand recognition and also sug- gest that the functional relationship between divalent cation and ligand binding may be a result of the spatial proximity of these two binding domains. These results provide a structural

basis for integrin-RGD interaction and a framework for de- sign and interpretation of future studies aimed at determining the precise mechanism of integrin-extracellular matrix inter- action.

Acknowledgments-We wish to thank Sally Irwin for superb tech- nical assistance. We also wish to acknowledge Tim Burke for his expertise and advice in amino acid sequence analysis. We thank Deborah Vestal and Zaverio Ruggeri for critical review of this man- uscript. We also thank Lynne Kottel for preparation of this manu- script.

i: 3.

4.

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9.

10. 11. 12.

13.

14.

15.

16.

17. 18.

19. 20.

21.

22.

23. 24.

25.

26. 21.

28.

29.

30.

31. 32.

33. 34.

35.

36. 31.

38.

39.

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J W Smith and D A Chereshbinding site on the vitronectin receptor.

Integrin (alpha v beta 3)-ligand interaction. Identification of a heterodimeric RGD

1990, 265:2168-2172.J. Biol. Chem. 

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