Photoaffinity Labeling of Adenylate Cyclase‐Linked Serotonin Receptors in Aplysia Neurons

Abstract

Serotonin stimulated adenylate cyclase in Aplysia neurons with a Kact of 0.7 .mu.M. Under the same conditions, 1-[2-(4-aminophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine stimulated adenylate cyclase with a Kact of 20 .mu.M. The azido derivative of this compound, 1-[2-(4-azidophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine, or of serotonin, (4-amino, 3-nitrophenylazido-serotonin), also stimulated the cyclase in the dark, but with lower efficiency (Kact > 10-4 M). Irradiation of the membranes in the presence of 100 .mu.M 1-[2-(4-azidophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine abolished 75% of the cyclase activity stimulated by 5 .mu.M serotonin. Under the same conditions, 100 .mu.M 4-amino, 3-nitrophenylazidoserotonin did not inhibit serotonin-stimulated adenylate cyclase activity. When [3H]1-[2-(4-azidophenyl)ethyl]4-(3-trifluoromethylphenyl)piperazine (20 .mu.M) was irradiated with membranes for 5 min at 4.degree.C, a dozen peptides were labeled, as revealed by a fluorogram of sodium dodecyl sulfate-polyacrylamide gels. Among them, the labeling of five polypeptides (molecular weights of 45,000, 55,000, 63,000, 80,000, and 94,000) was protected by the presence of 0.2 mM serotonin during photolysis. These peptides may be related to serotonin receptors.