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

Abstract

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.

• Authors: S Ito, H Hashimoto, M Negishi, M Suzuki, H Koyano, R Noyori, A Ichikawa
• Journal: The Journal of biological chemistry (J Biol Chem) Vol. 267 Issue 28 Pg. 20326-30 (Oct 05 1992) ISSN: 0021-9258 [Print] United States
• PMID: 1400351 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Affinity Labels
• Azides
• Receptors, Epoprostenol
• Receptors, Prostaglandin
• 19-(3-azidophenyl)-20-norisocarbacyclin
• Guanosine 5'-O-(3-Thiotriphosphate)
• Epoprostenol

Topics

• 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