Agonist-induced GTPγ35S binding mediated by human 5-HT2C receptors expressed in human embryonic kidney 293 cells

The 5-HT2C receptor as heterologously expressed in various mammalian cells mediates inositol 1,4,5-triphosphate (IP3) signal by activating G(q/11) subtypes of G proteins, but minimally promotes agonist-induced GTPy S binding in membranes due to slow GTP turnover rates of the G proteins. Here we discovered robust (over 200%) agonist-induced GTP gamma(35)S binding mediated by the human receptor expressed in human embryonic kidney (HEK) 293 cells, and investigated its pharmacology. Agonists concentration-dependently increased GTP gamma(35)S binding in isolated membranes, which was competitively blocked by antagonists. Intrinsic efficacies of agonists from GTP gamma(35)S binding were comparable to those from IP3 measurement. Pertussis toxin treatment largely blocked serotonin-induced GTP gamma(35)S binding, serotonin high affinity sites by 70% without altering the total binding sites, and reduced IP3 release by 40%. GTP gamma(35)S-bound G alpha subunits from serotonin-activated membranes were mainly G(alpha)i, judging from immobilization studies with various G alpha-specific antibodies. Inhibition of forskolin-stimulated cAMP formation, however, was not observed. Apparently, the 5-HT2C receptor-mediated GTP gamma(35) binding is a unique phenotype observed in HEK293 cells, reflecting its coupling to pertussis toxin-sensitive G(i) subtypes, which contribute to the IP3 signal, along with pertussis toxin-insensitive G(q/11) subtypes. (C) 1999 Elsevier Science B.V. All rights reserved.