Functional activation of Gαq via serotonin2A (5-HT2A) and muscarinic acetylcholine M1 receptors assessed by guanosine-5′-O-(3-[35S]thio) triphosphate ([35S]GTPγS) binding/immunoprecipitation in rat brain membranes

Functional coupling between serotonin(2A) (5-HT2A) receptors and G alpha(q) proteins in native brain membranes has been sparsely reported thus far. In the present study, the guanosine-5'-O-(3-[S-35]thio) triphosphate ([S-35]GTP gamma S) binding assay combined with immunoprecipitation using magnetic beads (Dynabeads Protein A) coated with anti-G alpha(q), antibody was developed. Under experimental conditions optimised for assay constituents (GDP, MgCl2, and NaCl), for contents of membrane protein, anti-G alpha(q) antibody, and Dynabeads Protein A, and for the incubation period, 5-HT stimulated specific [S-35]GTP gamma S binding to G alpha(q), in rat cerebral cortical membranes in a concentration-dependent and saturable manner, with a signal/noise ratio that was sufficiently high for further detailed pharmacological characterisation. This characterisation revealed an involvement of 5-HT2A receptors. Activation of G alpha(q) proteins was also detectable by the addition of carbachol via muscarinic acetylcholine M-1 receptors, (-)-epinephrine, and dopamine, but not by L-glutamate or (+/-)-baclofen. When 5-HT2A receptors and M-1 receptors were stimulated simultaneously, there were non-additive effects, indicating that the two receptors were coupled to the same components of G alpha(q) proteins in the rat cerebral cortex. This method will serve as an efficacious strategy for neurobiological investigations aimed at elucidating the physiological and pathological implications of signal transduction systems mediated via G alpha(q) proteins coupled with 5-HT2A receptors and muscarinic acetylcholine M-1 receptors. (C) 2014 Elsevier B.V. All rights reserved.