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 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.
Hiraiwa
M
M
Department of Neurosciences, University of California, School of Medicine, San Diego, La Jolla 92093-0634, USA.
Campana
W M
WM
Martin
B M
BM
O'Brien
J S
JS
D000595:Amino Acid Sequence; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D019204:GTP-Binding Proteins / Q000378:metabolism*; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D009414:Nerve Growth Factors; D009447:Neuroblastoma / Q000378:metabolism; D010215:Papio; D010446:Peptide Fragments / Q000737:chemistry; D011956:Receptors, Cell Surface / Q000302:isolation & purification / Q000378:metabolism*; D043562:Receptors, G-Protein-Coupled; D049231:Saposins; D014407:Tumor Cells, Cultured
D006023:Glycoproteins; D009414:Nerve Growth Factors; D010446:Peptide Fragments; C483344:Psap protein, mouse; D011956:Receptors, Cell Surface; D043562:Receptors, G-Protein-Coupled; D049231:Saposins; C109336:prosaposin receptor; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019204:GTP-Binding Proteins