Identification of the prostacyclin receptor by use of [15-3H1]19-(3-azidophenyl)-20-norisocarbacyclin, an irreversible specific photoaffinity probe

• Published in: The Journal of biological chemistry 1992-10, Vol.267 (28), p.20326-20330
• Main Authors: Ito, S, Hashimoto, H, Negishi, M, Suzuki, M, Koyano, H, Noyori, R, Ichikawa, A
• Format: Article
• Language: English
• Subjects: Affinity Labels; Animals; Azides; Blood Platelets - chemistry; Cell Membrane - chemistry; Electrophoresis, Polyacrylamide Gel; Epoprostenol - analogs & derivatives; Guanosine 5'-O-(3-Thiotriphosphate) - chemistry; Mast-Cell Sarcoma; Mice; Photochemistry; Radioligand Assay; Receptors, Epoprostenol; Receptors, Prostaglandin - analysis; Substrate Specificity; Swine; Tumor Cells, Cultured
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(19)88705-7

The prostaglandin I (PGI2) receptor of mouse mastocytoma P-815 cells was characterized by photo-affinity labeling with the stable PGI2 analogue [15-3H1]-19-(3-azidophenyl)-20-norisocarbacyclin ([3H] APNIC) used as a potential photoaffinity probe for the receptor. [3H]APNIC bound to the mastocytoma membrane with high affinity and in a saturable manner. Scatchard plot analysis indicated a single binding site with a Kd of 4.7 nM and a Bmax of 0.58 pmol/mg protein. The binding of [3H]APNIC was dose dependently inhibited by APNIC and iloprost, another stable PGI2 agonist, and to a much lesser extent by PGE2. The binding of the radioligand showed sensitivity to the guanine nucleotide guanosine 5'-O-(3-thio-triphosphate) (GTP gamma S). Photolysis of [3H]APNIC-prelabeled membranes resulted in incorporation of radiolabel into a protein of approximately 43 kDa. Photolabeling was inhibited by PGI2 agonists and other prostaglandins with specificity for the PGI2 receptor and was modulated by GTP gamma S. A protein of approximately 45 kDa was also labeled by [3H]APNIC in the membrane of porcine platelets, membranes that are known to be abundant in PGI2 receptors. These results demonstrate that [3H]APNIC specifically labels a protein that may represent the PGI2 receptor and that this radioprobe will be a useful reagent for further characterization and purification of the PGI2 receptor.