Agonist-induced GTPγ 35S binding mediated by human 5-HT 2C receptors expressed in human embryonic kidney 293 cells

Abstract

The 5-HT 2C receptor as heterologously expressed in various mammalian cells mediates inositol 1,4,5-triphosphate (IP 3) signal by activating G q/11 subtypes of G proteins, but minimally promotes agonist-induced GTPγ 35S binding in membranes due to slow GTP turnover rates of the G proteins. Here we discovered robust (over 200%) agonist-induced GTPγ 35S binding mediated by the human receptor expressed in human embryonic kidney (HEK) 293 cells, and investigated its pharmacology. Agonists concentration-dependently increased GTPγ 35S binding in isolated membranes, which was competitively blocked by antagonists. Intrinsic efficacies of agonists from GTPγ 35S binding were comparable to those from IP 3 measurement. Pertussis toxin treatment largely blocked serotonin-induced GTPγ 35S binding, serotonin high affinity sites by 70% without altering the total binding sites, and reduced IP 3 release by 40%. GTPγ 35S-bound Gα subunits from serotonin-activated membranes were mainly Gα i, judging from immobilization studies with various Gα-specific antibodies. Inhibition of forskolin-stimulated cAMP formation, however, was not observed. Apparently, the 5-HT 2C receptor-mediated GTPγ 35S binding is a unique phenotype observed in HEK293 cells, reflecting its coupling to pertussis toxin-sensitive G i subtypes, which contribute to the IP 3 signal, along with pertussis toxin-insensitive G q/11 subtypes.