Labeling of integrin alpha v beta 3 with 58Co(III). Evidence of metal ion coordination sphere involvement in ligand binding.
Integrin-mediated cell adhesion to the extracellular matrix is divalent metal ion-dependent; however, a demonstration of the interaction between native integrins and divalent metal ions is lacking. Here we provide direct evidence that the vitronectin receptor (VNR) is a metalloprotein. The unique electron shell of Co(II), an ion which we show supports ligand recognition by VNR, enables its oxidative conversion to inert Co(III). This property facilitated "affinity labeling" of VNR with 58Co(III) by oxidation of the metal ion in situ (i.e. in position). An average of 3.5 +/- 0.5 mol of cobalt were incorporated per mol of VNR. The ability of VNR to bind metal ions was independently confirmed by examining the interaction between VNR and Mn2+ under native conditions. The apparent high affinity between VNR and Mn2+ allowed us to observe the specific binding between 54Mn2+ and VNR by equilibrium gel filtration studies. Interestingly, the oxidative incorporation of Co(III) into VNR specifically blocked ligand binding, suggesting that the coordination sphere of metal ion bound to VNR is a critical determinant in integrin-ligand recognition. Furthermore, Mn2+ abolished the oxidative affinity labeling of VNR with Co(III) and consequently blocked the inactivation of VNR by in situ incorporation of Co(III). Thus, Mn2+ and Co2+ bind to the same or mutually exclusive sites on VNR. These observations provide the first demonstration that an integrin, specifically VNR, is a metalloprotein and demonstrate a functional link between the coordination sphere of the bound metal ion and ligand recognition by this receptor.
Smith
J W
JW
Department of Immunology, Research Institute of Scripps Clinic, La Jolla, California 92037.
Cheresh
D A
DA
D000345:Affinity Labels; D002850:Chromatography, Gel; D003036:Cobalt Isotopes; D004591:Electrophoresis, Polyacrylamide Gel; D008024:Ligands; D010084:Oxidation-Reduction; D011971:Receptors, Immunologic / Q000378:metabolism*; D019030:Receptors, Vitronectin
D000345:Affinity Labels; D003036:Cobalt Isotopes; D008024:Ligands; D011971:Receptors, Immunologic; D019030:Receptors, Vitronectin