IDENTIFICATION OF THE HEMOPHILIC BINDING-SITE OF THE RECEPTOR PROTEIN-TYROSINE-PHOSPHATASE PTP-MU

The receptor-type protein tyrosine phosphatase PTP mu comprises an ext racellular segment containing a MAM domain, an immunoglobulin domain a nd four fibronectin type III repeats, a transmembrane segment, and two intracellular PTP domains. We have previously shown that PTP mu binds homophilically, i.e. PTP mu on the surface of one cell binds to PTP m u on an apposing cell, and that the extracellular segment alone is suf ficient for hemophilic binding. In this study we report that in MvLu c ells PTP mu is proteolytically processed into two noncovalently associ ated fragments, one comprising most of the extracellular segment (simi lar to 100 kDa) and the other containing predominantly the transmembra ne and intracellular portions (similar to 100 kDa). We have also ident ified the hemophilic binding site within the extracellular segment. We have generated, expressed, and purified various fragments of the extr acellular segment of PTP mu and have used fluorescent beads (Covaspher es) coated with these fragments in three binding assays: (i) measureme nt of bead aggregation, (ii) binding of beads to surfaces of dishes co ated with purified PTP mu or (iii) binding to MvLu cells. Only beads c oated with recombinant fragments that contained the immunoglobulin dom ain underwent aggregation or bound to surfaces displaying PTP mu, sugg esting that neither the MAM domain nor the fibronectin type III repeat s bound homophilically in these assays. The fragment containing the Ig domain alone bound as well as any other Ig domain-containing fragment , suggesting that the Ig domain is both necessary and sufficient for h emophilic binding under these conditions.