Analysis of the roles of RGD-binding integrins, alpha(4)/alpha(9) integrins, alpha(6) integrins, and CD9 in the interaction of the fertilin beta (ADAM2) disintegrin domain with the mouse egg membrane

Fertilin beta (also known as ADAM2), a mammalian sperm protein that mediates gamete cell adhesion during fertilization, is a member of the ADAM protein family whose members have disintegrin domains with homology to integrin ligands found in snake venoms. Fertilin beta utilizes an ECD sequence within its disintegrin domain to interact with the egg plasma membrane; the Asp is especially critical. Based on what is known about different integrin subfamilies and their ligands, we sought to characterize fertilin beta binding sites on mouse eggs, focusing on integrin subfamilies that recognize short peptide sequences that include an Asp residue: the alpha(5)/alpha(8)/alpha(v)/alpha(IIb) or RGD-binding subfamily (alpha(5)beta(1), alpha(8)beta(1), alpha(V)beta(1), alpha(V)beta(3), alpha(V)beta(5), alpha(V)beta(6), alpha(V)beta(8), and alpha(IIb)beta(3)) and the alpha(4)/alpha(9) subfamily (alpha(4)beta(1), alpha(9)beta(1), and alpha(4)beta(7)). We tested peptide sequences known to perturb interactions mediated by these integrins in two different assays for fertilin beta binding. Peptides with the sequence MLDG, which perturb alpha(4)/alpha(9) integrin-mediated interactions, significantly inhibit fertilin beta binding to eggs, which suggests a role for a member of this integrin subfamily as a fertilin beta receptor. RGD peptides, which perturb alpha(5)/alpha(8)/alpha(v)/alpha(IIb) integrin-mediated interactions, have partial inhibitory activity. The anti-alpha(6) antibody GoH3 has little or no inhibitory activity. An antibody to the integrin-associated tetraspanin protein CD9 inhibits the binding of a multivalent presentation of fertilin beta (immobilized on beads) but not soluble fertilin beta, which we speculate has implications for the role of CD9 in the strengthening of fertilin beta-mediated cell adhesion but not in initial ligand binding.