REGULATION OF INTEGRIN AFFINITY STATES THROUGH AN NPXY MOTIF IN THE BETA-SUBUNIT CYTOPLASMIC DOMAIN

The ligand binding affinities of the integrins are regulated through their cytoplasmic domains. To identify specific residues that are involved in this process, we have generated mutants in the beta(1) and beta(3) tails and coexpressed them in Chinese hamster ovary cells with constitutively active alpha subunits. These alpha subunits are chimera of extracellular and transmembrane alpha(IIb) joined to the cytoplasmic domains of alpha(5), alpha(6A), or alpha(6B), and confer an energy-dependent high affinity state when expressed in Chinese hamster ovary cells. The affinity state of these transfectants was determined by analyzing the binding of PAC1, an antibody that specifically recognizes the activated form of the reporter group, extracellular alpha(IIb)beta(3). We have identified point mutants in several areas of the beta tails, which result in a reduced ability to bind ligand. Complete abolition of PAC1 binding was obtained with mutants in an NPXY motif found in many integrin beta subunits and implicated in the internalization of other cell surface receptors. Similar effects on PAC1 binding were observed whether coexpression was with alpha chimera containing alpha(5), alpha(6A), or alpha(6B) cytoplasmic sequences. These studies identify a novel role for the NPXY motif in the regulation of integrin binding affinity.