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

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Abstract

The 5-HT2C receptor as heterologously expressed in various mammalian cells mediates inositol 1,4,5-triphosphate (IP3) signal by activating Gq/11 subtypes of G proteins, but minimally promotes agonist-induced GTPγ35S binding in membranes due to slow GTP turnover rates of the G proteins. Here we discovered robust (over 200%) agonist-induced GTPγ35S binding mediated by the human receptor expressed in human embryonic kidney (HEK) 293 cells, and investigated its pharmacology. Agonists concentration-dependently increased GTPγ35S binding in isolated membranes, which was competitively blocked by antagonists. Intrinsic efficacies of agonists from GTPγ35S binding were comparable to those from IP3 measurement. Pertussis toxin treatment largely blocked serotonin-induced GTPγ35S binding, serotonin high affinity sites by 70% without altering the total binding sites, and reduced IP3 release by 40%. GTPγ35S-bound Gα subunits from serotonin-activated membranes were mainly Gαi, judging from immobilization studies with various Gα-specific antibodies. Inhibition of forskolin-stimulated cAMP formation, however, was not observed. Apparently, the 5-HT2C receptor-mediated GTPγ35S binding is a unique phenotype observed in HEK293 cells, reflecting its coupling to pertussis toxin-sensitive Gi subtypes, which contribute to the IP3 signal, along with pertussis toxin-insensitive Gq/11 subtypes.

Introduction

The 5-HT2C receptor, a G-protein coupled receptor with seven transmembrane segments, is highly expressed in the brain Conn et al., 1979, Julius et al., 1988, Stam et al., 1994 and has been implicated for a number of diseases such as anxiety, obesity, depression, schizophrenia and affective disorders Canton et al., 1990, Sanders-Bush and Breeding, 1991, Moreau et al., 1993, Dourish, 1995, Tecott et al., 1995, Cowen et al., 1996, Kennett et al., 1996, Epstein et al., 1997. Recently, the receptor has received much attention as a potential target for anxiolytics and anti-obesity agents Dourish, 1995, Cowen et al., 1996, Kennett et al., 1996, and its recombinant clones have been often used for pharmacological studies. The human 5-HT2C receptor, since the cloning of its cDNA Julius et al., 1988, Salzman et al., 1991, Stam et al., 1994, Xie et al., 1996, has been heterologously expressed in various cells, including NIH3T3 fibroblast, Chinese hamster ovary, African green monkey SV40 transformed, human embryonic kidney (HEK)293, SH-SY5Y cells, insect Sf9 cells and Xenopus oocytes Barker et al., 1994, Chen et al., 1994, Akiyoshi et al., 1995, Tohda et al., 1995, Hartman and Northup, 1996, Kennett et al., 1996, Newton and Elliott, 1997. Pharmacological characterization of the cloned receptor have shown that its activation elevates intracellular inositol 1,4,5-triphosphate (IP3), via Gq/11 subtypes of G proteins and phospholipase C pathway, and subsequently the Ca2+ level Conn et al., 1979, Julius et al., 1988, Stam et al., 1994, Kaufman et al., 1995. Direct coupling of the receptor with Gq (squid retinal) has been shown in reconstituted receptors from the Sf9 cell expression system (Hartman and Northup, 1996), and also its coupling with Go and Gi1 in the Xenopus oocytes expression system, using antisense probes (Chen et al., 1994).

For G protein-coupled receptors, an early and common signaling step is the agonist-induced binding of GTP to Gα subunits, and this step, using slowly hydrolyzable GTPγ35S, has been frequently used for investigations of receptor-ligand interactions. Interestingly, agonist-induced GTPγ35S binding has been reported to be low or non-existent for the 5-HT2C receptor expressed in NIH3T3 cells (Burns et al., 1997), and this has been attributed to low intrinsic GTP-turnover rate for the receptors coupled to the Gq family of G proteins. Our preliminary study with the human 5-HT2C receptor expressed in HEK293 cells revealed, however, robust enhancements of GTPγ35S binding by serotonin and several agonists, and led us to investigate its pharmacology.

Section snippets

Materials and methods

The cDNA for the human 5-HT2C receptor was cloned into the PCRscript vector via a blunt end ligation. The directionality of the insert was determined with polymerase chain reaction (PCR) using primers annealing to the vector and the insert. Then the cDNA insert was transferred to a mammalian expression vector, PCI-Neo™ from Promega, and the recombinant vector was used to transfect HEK293 cells using Ca2+ phosphate precipitation techniques. Stably transfected cells were selected in the presence

Ligand binding

We examined binding of standard serotonergic ligands to the human 5-HT2C receptor in HEK293 cell membranes (Fig. 1 and Table 1). The binding data for [3H]mesulergine at various concentrations fit well to the binding model for a single class of sites, and yielded a dissociation constant (Kd) of 3.0±0.2 nM and maximal binding (Bmax) of 62±2 pmol/mg protein (Fig. 1A). It should be noted that membranes from untransfected HEK293 cells show no detectable specific binding for [3H]mesulergine or [3

Discussion

In this study we have shown that serotonin-activation of the human 5-HT2C receptor, as heterologously expressed in HEK293 cells, enhanced GTPγ35S binding via pertussis toxin-sensitive Gi, and IP3 release via both pertussis toxin-sensitive Gi and -insensitive G proteins (probably Gq/11). Inhibition of adenylyl cyclases was not observed, despite activation of Gi which are best known as the primary G proteins to inhibit adenylyl cyclases. Binding studies showed that about 3% of the total binding

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