Prosaposin Receptor: Evidence for a G-Protein-Associated Receptor

Prosaposin, the precursor of sphingolipid activator protein (saposins A-D), has been identified as a neurotrophic factor capable of inducing neural differentiation and preventing cell death. The putative prosaposin receptor was partially purified from baboon brain membranes by affinity chromatography using a saposin C-column. The purified preparation gave a single major protein band with an apparent molecular weight of 54 kDa on SDS-PAGE. Affinity cross-linking of 11 kDa125I-saposin C demonstrated the presence of a 66 kDa product, indicative of an apparent molecular weight of 55 kDa for the receptor. A GTPγS-binding assay using cell membranes from SHSY5Y neural cells demonstrated agonist stimulated binding of [35S]GTPγS upon treatment with prosaptide TX14(A) a peptide from the neurotrophic region; maximal binding was obtained at 2 nM. TX14(A) stimulated binding was abolished by prior treatment of SHSY5Y cells with pertussis toxin and by a scrambled and an all D-amino acid-derivative of the 14-mer. A 14-mer mutant prosaptide (6N→6D) competed with TX14(A) with a Ki of 0.7 nM. Immunoblot analysis using an antibody against the G0αsubunit demonstrated that the purified receptor preparation contained a 40 kDa reactive band consistent with association of G0αand the receptor. These findings indicate that the signaling induced by prosaposin and TX14(A) is generated by binding to a G0-protein associated receptor.