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Updated: Aug 10, 2026

Measuring G-protein-coupled Receptor Signaling via Radio-labeled GTP Binding
Published on: June 9, 2017
Prosaposin receptor: evidence for a G-protein-associated receptor
M Hiraiwa1, W M Campana, B M Martin
1Department of Neurosciences, University of California, School of Medicine, San Diego, La Jolla 92093-0634, USA.
Abstract:
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 kDa 125I-saposin C demonstrated the presence of a 66 kDa product, indicative of an apparent molecular weight of 55 kDa for the receptor. A GTP gamma S-binding assay using cell membranes from SHSY5Y neural cells demonstrated agonist stimulated binding of [35S]-GTP gamma S 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 G0 alpha subunit demonstrated that the purified receptor preparation contained a 40 kDa reactive band consistent with association of G0 alpha and the receptor. These findings indicate that the signaling induced by prosaposin and TX14(A) is generated by binding to a G0-protein associated receptor.
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