Identification of the prostacyclin receptor by use of [15-3H1]19-(3-azidophenyl)-20-norisocarbacyclin, an irreversible specific photoaffinity probe.
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.
Ito
S
S
Department of Cell Biology, Osaka Bioscience Institute, Suita, Japan.
Hashimoto
H
H
Negishi
M
M
Suzuki
M
M
Koyano
H
H
Noyori
R
R
Ichikawa
A
A
D000345:Affinity Labels; D000818:Animals; D001386:Azides; D001792:Blood Platelets / Q000737:chemistry; D002462:Cell Membrane / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D011464:Epoprostenol / Q000031:analogs & derivatives*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000737:chemistry; D012515:Mast-Cell Sarcoma; D051379:Mice; D010777:Photochemistry; D011869:Radioligand Assay; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin / Q000032:analysis*; D013379:Substrate Specificity; D013552:Swine; D014407:Tumor Cells, Cultured
D000345:Affinity Labels; D001386:Azides; D044006:Receptors, Epoprostenol; D011982:Receptors, Prostaglandin; C077220:19-(3-azidophenyl)-20-norisocarbacyclin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D011464:Epoprostenol