A Naturally Occurring Extracellular R-! Clasp Contributes to Stabilization of ! 3 Integrins in a Bent , Resting Conformation †

Control of RIIb! 3 and Rv! 3 integrin activation is critical for cardiovascular homeostasis. Mutations that perturb association of integrin R and ! subunits in their transmembrane and cytoplasmic regions activate the integrin heterodimer, suggesting that a low-affinity or “off” conformation is the default state, likely corresponding to the bent conformation seen in the crystal structure of Rv! 3. In this bent structure, a segment of Rv (301-308) and ! 3 (560-567) are juxtaposed. Here we provide evidence that these regions of Rv/RIIb and ! 3 function as a novel extracellular clasp to restrain activation. Synthetic peptides representing the RIIb and ! 3 clasp regions promote integrin activation as judged by cell adhesion, cell spreading, and exposure of epitopes for three ! 3 LIBS antibodies. Mutation of the clasp region of Rv or ! 3 results in a constitutively activated integrin, confirming the role of the extracellular clasp in restraining integrin activation. Molecular dynamics simulations of the Rv! 3 structure yield a refined model for the Rv! 3 clasp and provide plausible explanations for the effects of the activating mutations. Control of integrin activity is of crucial importance in platelet aggregation (1), leukocyte adherence and trafficking (2) (3), angiogenesis (4), and apoptosis (5). Crystal structures of integrin R chain domains (2) and the structure of the entire extracellular domain of Rv! 3 (6, 7) have provided new hypotheses for integrin regulation. Multiple mechanisms contribute to regulation of integrin activation. This is not unanticipated given the critical role of integrins in so many important biological scenarios and the disastrous consequences of inappropriate activation of platelet or leukocyte integrins. Two general mechanisms regulate the functional state of integrins. Conformational changes of the R! dimer are clearly involved in transitions from lowto high-affinity states (8), usually judged by the binding of soluble ligands or the exposure of binding sites for mAbs that recognize an activated conformation stabilized by ligands or a “ligandinduced binding site” (anti-LIBS mAbs) (9-11). Integrin dimers competent for ligand binding may also be clustered resulting in a high “avidity” state that increases binding to multivalent, usually immobilized ligands (12-14). Early studies of RIIb! 3 activation suggested that association of juxtamembrane cytoplasmic ion pairs opposite each other in the R and ! subunit tails could restrain activation (15) (16). This notion of a cytoplasmic “clasp” of the R and ! subunits was strengthened by the effects of additional mutations in the juxtamembrane regions of RIIb and ! 3 that resulted in constitutive activation (17). Further, the addition of non-native, coiled-coil dimerizing peptides to the cytoplasmic tailsofRand! subunitsconstrainedactivation (18,19). A role for the TM domains of R and ! subunits in integrin activation has also been proposed based on mutations that constitutively activate RIIb! 3 (17, 20). These data suggest a model in which a specific R-! TM helix interface contributes to stabilizing the off state, likely acting in concert with the juxtamembrane clasp in the cytoplasmic tails. The publication of the crystal structure of free and RGDbound extracellular domains of Rv! 3 (6, 7) gave rise to an entirely new model for activation. The bent or genuflected integrin seen in the crystal structure suggested that massive conformational changes of the entire Rv! 3 extracellular domain must accompany integrin activation, if indeed the fully active integrin were to assume the extended, upright conformation expected from earlier EM studies (21-24). A growing body of data supports the idea that the bent structure seen in the crystals of Rv! 3 is likely the physiologically relevant “off” or low-affinity state and an extended, erect integrin represents the fully active state (8, 13, 14, 25, 26). Particularly revealing was an experiment in which non-native Cys residues were inserted in the R and ! subunits of Rv! 3 (or the homologous position of RIIb) at locations that were within disulfide bond distance in the bent structure but were predicted to be very far apart in an extended structure (RvG307 > C, and ! 3-R563 > C). When the mutant R and ! subunits were expressed in cells, they formed R! dimers that were inactive, even in Mn2+, until mild reducing agent was applied, or either one of the mutant R or ! subunits was replaced with the wild-type (non-Cys) construct (8). This † Supported by NIH Grant HL54390 (W.A.F.) and by the NSF sponsored UAB ADVANCE program (Y.S.). * To whom inquires should be addressed. Phone: 314-362-3348. Fax: 314-362-7183. E-mail: frazier@biochem.wustl. edu. ‡ Washington University School of Medicine. § The University of Alabama at Birmingham. 1 Abbreviations: 4N1K, thrombospondin-1 peptide with sequence KRFYVVMWKK; LIBS, ligand-induced binding site; RGD peptide, peptide with sequence GRGDSP; TM, transmembrane; Vn, vitronectin; WT, wild type. Biochemistry 2008, 47, 11616–11624 11616 10.1021/bi8015108 CCC: $40.75  2008 American Chemical Society Published on Web 10/09/2008 experiment demonstrates that the bent structure can exist on the cell surface and is inactive. Not addressed is whether these juxtaposed regions of the R (G307) and ! (R563) subunits normally have a role in regulating integrin activation by forming an extracellular clasp. Here we test this hypothesis. EXPERIMENTAL PROCEDURES Reagents, Cell Lines, and Peptides. Human K562 erythroleukemic cells (ATCC: CCL-243), stably expressing Rv! 3 integrin (27), HEK 293 cells (ATCC: CRL1573), and C32 human melanoma cells (ATCC: CRL-1585) were grown as described (28). Ligand-induced binding site (LIBS) antibodies, LIBS1 and LIBS6, were generously provided by Dr. Mark Ginsberg (Scripps Research Institute) (9), and the LIBS antibody D3 was a gift from Dr. Lisa Jennings (The University of Tennessee, Memphis, TN) (11). Peptides were synthesized, purified, and verified by mass spectrometry as previously described (29). All other reagents were purchased from Sigma-Aldrich unless otherwise stated. Adhesion and spreading assays of C32 cells on Vn were performed as described (30). Collection of human blood was performed under an approved protocol of the Washington University School of Medicine Human Studies Committee. Washed platelets were prepared as described (31) and stored on ice for 1-48 h or treated as described below. Binding of anti! 3 mAb AP3 and anti-LIBS mAbs LIBS1, LIBS6, and D3 to cells and platelets under indicated conditions was quantified using a Coulter EPICS flow cytometer. Data were analyzed using WinMDI software. Preparation and Expression of Mutants of RV and ! 3. Fulllength cDNA clones of human Rv and ! 3 integrin subunits were provided by Dr. Scott Blystone. Restriction fragments containing the mutation sites were subcloned into Bluescript BSKS+ for PCR mutagenesis using overlapping primers containing the mutant bases. Details are provided in Supporting Information. After confirmation by DNA sequencing, the restriction fragment containing the desired mutation was reassembled in pCDNA3 for Rv (G418 selection) and either pREP10 or pBLY100 for ! 3 (hygromycin selection). Initial tests of the ! 3 mutants were performed by transfecting them into human ovarian carcinoma clone OV10 which expresses WT Rv (largely as Rv! 5) but no ! 3 (32). For cotransfections of both subunits, HEK 293 cells were used (33) (34). Expression was determined by flow cytometry with mAbs L230 (Rv) and AP3 (! 3). LIBS binding was determined as above and normalized to AP3 binding or the binding of the LIBS mAb in the presence of excess RGDS peptide and Mn2+ to yield an activation index. Molecular Dynamics Simulations. The reported crystal structure of Rv! 3 (PDB ID: 1JV2) (6) was subjected to energy minimization and equilibration using GROMACS version 3.3 (35). Details of the method are provided in Supporting Information.