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THE ROLES OF RGD-BINDING INTEGRINS, 
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1, AND CD9 IN MEDIATING THE BINDING OF RECOMBINANT FERTILIN TO MOUSE EGGS.
Zhu, Xiaoling1, Evans, Janice1, 1
ABSTRACT- Fertilin , a sperm surface protein, is involved in the binding of sperm to eggs, and its binding partner(s) on the egg is unknown. We investigated the roles of RGD-binding integrins (51, 81, v1, v3, v5, IIb6, v8), the 61 integrin, and the tetraspan protein CD9 in mediating fertilin binding to mouse eggs. To do this, we tested the effects of RGD peptides (to perturb the function of RGD-binding integrins), GoH3 (anti-6 function-blocking monoclonal antibodiy), and KMC.8 (anti-CD9 function-blocking monoclonal antibody) on the binding of recombinant fertilin diluted in culture medium (soluble rF), and the binding of fluorescent beads coated with recombinant fertilin (bead-immobilized rF) to eggs. The GRGDTP peptide (as a fusion protein-presented peptide) moderately inhibited the binding of both soluble rF and bead-immobilized rF to eggs. In the presence of 100 mM GRGDTP, the binding of soluble rF was reduced to 45% of control level (the binding level of soluble rF to eggs in the absence of peptide). In the presence of 100 mM GRGETP peptide or TDGPGR scrambled peptide, the binding of soluble rF was reduced to 55% and 66% of control level, respectively; these levels of inhibition were significantly different (p< 0.05) from that observed with 100 mM GRGDTP peptide. Furthermore, both GRGETP and TDGPGR peptides had no effect on the binding of bead-immobilized rF to eggs. This result indicates that rF and the RGD peptide sequence might bind to the same binding site on egg plasma membrane. GoH3 had no inhibitory effect on the binding of either soluble rF or bead-immobilized rF, even though GoH3 bound to egg plasma membrane as detected by immunofluorescence staining; thus rF and GoH3 did not appear to bind to the same site on mouse egg plasma membrane. KMC.8 had no inhibitory effect on the binding of soluble rF to the eggs, but inhibited the binding of bead-immobilized rF to the eggs. This result suggests that rF presented in different formats (soluble vs immobilized) may interact with egg surface differently, as bead-immobilized rF appears to interact with a KMC.8-sensitive site and soluble rF with a KMC.8-insensitive site. Further experiments will confirm the roles of RGD-binding integrins and/or CD9 molecule on mediating fertilin binding to eggs.
KEY WORDS: fertilin, integrin, CD9, RGD