Agonist-induced GTP gamma S-35 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) sub types 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 disc overed robust (over 200%) agonist-induced GTP gamma(35)S binding mediated b y the human receptor expressed in human embryonic kidney (HEK) 293 cells, a nd investigated its pharmacology. Agonists concentration-dependently increa sed GTP gamma(35)S binding in isolated membranes, which was competitively b locked by antagonists. Intrinsic efficacies of agonists from GTP gamma(35)S binding were comparable to those from IP3 measurement. Pertussis toxin tre atment largely blocked serotonin-induced GTP gamma(35)S binding, serotonin high affinity sites by 70% without altering the total binding sites, and re duced IP3 release by 40%. GTP gamma(35)S-bound G alpha subunits from seroto nin-activated membranes were mainly G(alpha)i, judging from immobilization studies with various G alpha-specific antibodies. Inhibition of forskolin-s timulated 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) sub types, which contribute to the IP3 signal, along with pertussis toxin-insen sitive G(q/11) subtypes. (C) 1999 Elsevier Science B.V. All rights reserved .