Covalent cross-linking of prostaglandin E receptor from bovine adrenal medulla with a pertussis toxin-insensitive guanine nucleotide-binding protein.
Prostaglandin E2 (PGE2) specifically bound to 100,000 X g pellet prepared from bovine adrenal medulla, and [3H]PGE2-bound proteins were solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The dissociation of bound [3H]PGE2 from the proteins was enhanced by GTP. [3H]PGE2-specifically bound proteins were adsorbed onto a wheat germ agglutinin column and GTP treatment decreased the amount of [3H]PGE2 retained on the column. When [3H]PGE2-bound proteins were cross-linked in the membrane by dithiobis(succinimidyl propionate) and solubilized, bound [3H]PGE2 was no longer dissociated by GTP treatment, suggesting that cross-linking produced a stable and high-affinity complex of PGE receptor with a GTP-binding protein. Covalent cross-linking of the complex was attested by adsorption of dithiobis(succinimidyl propionate)-treated [3H]PGE2-bound proteins to GTP-Sepharose, and co-elution of [35S]guanosine 5'-O-(3-thiotriphosphate) binding activity and immunoreactivities of alpha o and beta subunits of a GTP-binding protein. The cross-linked [3H]PGE2-bound complex was eluted as an apparently single radioactive peak at the position of Mr = 200,000 by gel filtration. These results have demonstrated that PGE receptor is a glycoprotein with an approximate Mr of 110,000, assuming that the Mr of the GTP-binding protein is 90,000. PGE2 neither activated nor inhibited adenylate cyclase activity, and pertussis toxin (islet-activating protein) did not affect PGE2 binding and its GTP sensitivity. These results suggest that the PGE receptor may be functionally associated with a pertussis toxin-insensitive GTP-binding protein and is not coupled to the adenylate cyclase system in bovine adrenal medulla.
D037361:Adenylate Cyclase Toxin; D000313:Adrenal Medulla / Q000378:metabolism*; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D015232:Dinoprostone; D004305:Dose-Response Relationship, Drug; D019204:GTP-Binding Proteins / Q000494:pharmacology*; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate / Q000031:analogs & derivatives / Q000494:pharmacology; D046911:Macromolecular Substances; D008970:Molecular Weight; D037342:Pertussis Toxin; D011458:Prostaglandins E / Q000378:metabolism; D011982:Receptors, Prostaglandin / Q000378:metabolism*; D018078:Receptors, Prostaglandin E; D013696:Temperature; D013873:Thionucleotides / Q000494:pharmacology; D013997:Time Factors; D010566:Virulence Factors, Bordetella / Q000494:pharmacology*
D037361:Adenylate Cyclase Toxin; D046911:Macromolecular Substances; D011458:Prostaglandins E; D011982:Receptors, Prostaglandin; D018078:Receptors, Prostaglandin E; D013873:Thionucleotides; D010566:Virulence Factors, Bordetella; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006160:Guanosine Triphosphate; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; D015232:Dinoprostone