In those proteins, an essential metal ion is bound by a metal ion-dependent adhesion site (MIDAS). The MIDAS is presented at the apex of a larger protein module called an I domain. The metal ligands in the MIDAS can be separated into three distantly spaced clusters of oxygenated residues. These three coordination sites also appear to exist in the integrin ␤3 and ␤5 subunits. Here, we examined the putative metal binding site within ␤3 and ␤5 using site-directed mutagenesis and ligand binding studies. We also investigated the fold of the domain containing the putative metal binding site using the PHD structural algorithm. The results of the study point to the similarity between the integrin ␤ subunits and the MIDAS motif at two of three key coordination points. Importantly though, the study failed to identify a residue in either ␤ subunit that corresponds to the second metal coordination group in the MIDAS. Moreover, structural algorithms indicate that the fold of the ␤ subunits is considerably different than the I domains. Thus, the integrin ␤ subunits appear to present a MIDAS-like motif in the context of a protein module that is structurally distinct from known I domains.Integrins are ␣␤ heterodimers that mediate cell adhesion (1, 2). Integrins participate in development and tissue remodeling and are linked to several diseases. The integrins bind to many adhesive and extracellular matrix proteins. The focal points of this study are the ␣v␤3 and ␣v␤5 integrins, both of which recognize the Arg-Gly-Asp (RGD) 1 tripeptide motif. The ␣v␤3 integrin binds to at least nine adhesive proteins and has two important biological functions. First, ␣v␤3 mediates the adhesion of osteoclasts to the bone surface (3), an event often considered to be the first step in bone resorption (4). Second, the ␣v␤3 integrin is expressed on the surface of angiogenic endothelial cells, where it is required for cell survival and further vessel development (5-7). It has been suggested that inhibitors of the ␣v␤3 integrin could be applied as antagonists of osteoporosis and tumor angiogenesis. The biological function of the ␣v␤5 integrin is less clear. This integrin can mediate cell adhesion to vitronectin. The ␣v␤5 integrin is also required for the internalization of adenovirus (8, 9), and it may be associated with angiogenesis (7).All integrins require divalent cations to bind their ligands. An important clue to the structural basis for ion binding was revealed by the crystal structures of the I domains from the integrin ␣ L and ␣ M subunits (10, 11). Each I domain spans approximately 200 residues and is homologous to an "inserted" domain in a number of other proteins including von Willebrand factor (12). In ␣ L and ␣ M , the I domain is necessary and sufficient for ligand contact. These I domains contain a metal binding site called a MIDAS (metal ion-dependent adhesion site). This ion binding site consists of five liganding residues that can be separated into three groups. Each group of coordinating residues is located at separate positions wit...

Many integrin adhesion receptors bind ligands containing the Arg-Gly-Asp (RGD) peptide motif. Most integrins exhibit considerable specificity for particular ligands and can distinguish among the many conformations of RGD. In this study we identify the domain of the integrin ␤ subunit involved in determining ligand binding specificity. Chimeras of ␤3 and ␤5, the most homologous integrin ␤ subunits, were expressed with ␣v on the surface of human 293 cells. The ligand binding phenotype of each chimera was assessed using the ligands Fab-9 and fibrinogen, both of which have a binding preference for ␣v␤3. The results of the study show that when exons C and D of the ␤3 subunit (residues 95-233) are substituted into ␤5, the chimera gained the ability to bind Fab-9 with an affinity close to that of wild-type ␣v␤3. This chimera was able to mediate cell adhesion to fibrinogen. Furthermore, the swap of only a 39-residue segment of this larger domain, ␤3 residues 164 -202, into the backbone of ␤5 enabled the chimeric integrin to bind soluble Fab-9. This small domain is highly divergent among the integrin ␤ subunits, suggesting that it may play a role in determining ligand selection by all integrins.Integrins are transmembrane ␣␤ heterodimers that are responsible for most of the physical contacts between cells and the extracellular matrix. Integrins are involved in a number of tissue remodeling events including embryogenesis, angiogenesis, wound repair, and bone resorption (1-5). Integrins are also causally linked to pathological conditions such as tumor progression, thrombosis, inflammation, and osteoporosis (6 -8).The integrin protein family contains 13 ␣ and 9 ␤ subunits. These subunits can cross-pair to form heterodimers with different expression patterns and distinct ligand binding profiles (9, 10). Each integrin binds to only a limited series of ligands, ensuring that cell adhesion and migration are precisely regulated. Such precision is key to tissue remodeling.Based on their ligand recognition specificity, integrins can be divided into two broad sub-families. About one-half of the integrins bind to ligands that lack any consensus binding motif.The remaining integrins bind to the RGD 1 consensus sequence. Importantly though, even the integrins that bind to RGD display a great deal of specificity for matrix ligands (11,12). Some of this specificity is determined by contacts that are ancillary to the RGD sequence (13,14), but much of the specificity is determined by the shape of the RGD motif and the residues in its immediate vicinity. Thus, to understand the structural basis of matrix selection, it is important to understand how integrins distinguish the many conformations of RGD.The goal of the present study was to identify domain(s) within the integrin ␤ subunit that confer ligand binding specificity. As a model system, we compared the ligand binding domains of the two most homologous integrins, ␣v␤3 and ␣v␤5. The ␤3 and ␤5 subunits have 56% identity at the amino acid level (15, 16). Although both ␣v␤3 and ␣v␤5 recogn...

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