Ligand binding to integrin alpha(v)beta(3) requires tyrosine 178 in the alpha(v) subunit.
Integrin alpha(v)beta(3) has been implicated in angiogenesis and other biological processes. However, the ligand-binding sites in alpha(v), a non-I-domain alpha subunit, remain to be identified. Recently in alpha(IIb), the other partner of the beta(3) subunit, several discontinuous residues important for ligand binding were identified in the predicted loops between repeats 2 and 3 (W3 4-1 loop) and within repeat 3 (W3 2-3 loop). Based on these findings, alanine-scanning mutagenesis in 293 cells was used to investigate the role of these loops (cysteine [C]142-C155 and glycine [G]172-G181) of alpha(v) in ligand binding. Wild-type alpha(v)beta(3) was able to bind soluble fibrinogen following integrin activation either by 0.5 mM manganese dichloride (MnCl(2)) or a mutation of beta(3) threonine (T)562 to asparagine. However, mutation of tyrosine (Y)178 to alanine in the predicted G172-G181 loop of alpha(v) abolished fibrinogen binding, and alanine (A) substitutions at adjacent residues phenylalanine (F)177 and tryptophan (W)179 had a similar effect. Cells expressing Y178Aalpha(v) also failed to bind to immobilized fibrinogen. Moreover, the Y178A mutation abolished the binding of WOW-1 Fab, a monovalent ligand-mimetic anti-alpha(v)beta(3) antibody, and the expression of beta(3) ligand-induced binding sites (LIBS) induced by arginine-glycine-aspartic acid-tryptophan (RGDW). In sharp contrast to the data obtained with alpha(IIb), none of the mutations in the predicted W3 4-1 loop in alpha(v) impaired ligand binding. These results implicate alpha(v) Y178 in ligand binding to alpha(v)beta(3), and they suggest that there are key structural differences in the adhesive ligand-binding sites of alpha(v)beta(3) and alpha(IIb)beta(3).
Honda
S
S
Department of Internal Medicine and Molecular Science, Graduate School of Medicine, Osaka University, Osaka, Japan.
Tomiyama
Y
Y
Pampori
N
N
Kashiwagi
H
H
Kiyoi
T
T
Kosugi
S
S
Tadokoro
S
S
Kurata
Y
Y
Shattil
S J
SJ
Matsuzawa
Y
Y
D000595:Amino Acid Sequence; D001665:Binding Sites / Q000187:drug effects; D002448:Cell Adhesion / Q000235:genetics; D002460:Cell Line; D000939:Epitopes / Q000187:drug effects; D005340:Fibrinogen / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009842:Oligopeptides / Q000494:pharmacology; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000737:chemistry; D011485:Protein Binding / Q000235:genetics; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D019030:Receptors, Vitronectin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D014443:Tyrosine
D000939:Epitopes; D008024:Ligands; D009842:Oligopeptides; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D021122:Protein Subunits; D019030:Receptors, Vitronectin; D011994:Recombinant Proteins; C082481:arginyl-glycyl-aspartyl-tryptophan; D014443:Tyrosine; D005340:Fibrinogen