Necessity of conserved asparagine residues in the leucine-rich repeats of platelet glycoprotein Ib alpha for the proper conformation and function of the ligand-binding region

The polypeptides of the platelet von Willebrand factor (vWf) receptor, the GP Ib-IX-V complex, each contain tandem repeats of a sequence that assigns them to the leucine-rich repeat protein family. Here, we studied the role o f conserved Asn residues in the leucine-rich repeats of GP Ib alpha, the li gand-binding subunit of the complex. We replaced the Asn residue in the six th position of the first or sixth leucine-rich repeat (of seven) either wit h a bulky, charged Lys residue or with a Ser residue (sometimes found in th e same position of other leucine-rich repeats) and studied the effect of th e mutations on complex expression, modulator-dependent vWf binding, and int eractions with immobilized vWf under fluid shear stress. As predicted, the Lys substitutions yielded more severe phenotypes, producing proteins that e ither were rapidly degraded within the cell (mutant N158K) or failed to bin d vWf in the presence of ristocetin or roll on immobilized vWf under fluid shear stress (mutant N41K), The binding of function-blocking GP Ib alpha an tibodies to the N41K mutant was either significantly reduced (AK2 and SZ2) or abolished (AN51 and CLB-MB45). Ser mutations were tolerated much better, although both mutants demonstrated subtle defects in vWf binding. These re sults suggest a vital role for the conserved asparagine residues in the leu cine-rich repeats of GP Ib alpha for the structure and functions of this po lypeptide, The finding that mutations in the first leucine-rich repeat had a much more profound effect on vWf binding indicates that the more N-termin al repeats may be directly involved in this interaction.