Identification of a Region in the Integrin β3 Subunit That Confers Ligand Binding Specificity

Abstract

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...

Processing of Integrin αv Subunit by Membrane Type 1 Matrix Metalloproteinase Stimulates Migration of Breast Carcinoma Cells on Vitronectin and Enhances Tyrosine Phosphorylation of Focal Adhesion Kinase

“…Fab-9 Binding to ␣ v ␤ 3 -expressing Cells-Fab-9 binding was performed as reported previously (43). Cells (3 ϫ 10 6 /ml) were incubated with increasing concentrations of 125 I-Fab-9 at 14°C for 70 min.…”

Mechanisms and Consequences of Affinity Modulation of Integrin αVβ3 Detected with a Novel Patch-engineered Monovalent Ligand

“…Analysis of Cell-surface Integrin Expression-Cells were suspended in incubation buffer (137 mM NaCl, 2.7 mM KCl, 3.3 mM NaH 2 PO 4 , 3.8 mM HEPES, 1 mM MgCl 2 , 5.5 mM glucose, and 1 mg/ml bovine serum albumin, pH 7.4) and incubated for 30 min on ice with a monoclonal antibody (10 g/ml) specific for ␣ V ␤ 3 (LM609), ␣ IIb ␤ 3 (D57) (34), or ␣ V ␤ 5 (P1F6) (40). After washing, the cells were incubated for another 30 min on ice with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (H ϩ L chain-specific; BIOSOURCE International), washed again, and analyzed on a FACSCalibur flow cytometer (Becton Dickinson, Mountain View, CA) (23).…”

Foot-and-Mouth Disease Virus Receptors: Comparison of Bovine α V Integrin Utilization by Type A and O Viruses

“…While the consensus binding motif of some integrins is unknown, at least eight integrins recognize and bind to ligands via an RGD sequence (63). Interestingly, despite recognizing this tripeptide sequence, these integrins bind different extracellular matrix ligands (40,69). While the reasons for this specificity are obscure, there is evidence that ligand sequences either flanking the RGD motif or located in other regions of the ligand are responsible for binding specificity (12,54).…”