The 5HT2C Receptor Couples to Multiple Ga Subunits

Although 5-HT2C receptors are "classically" considered to couple to Gaq, only a few studies have directly demonstrated this. Thus, stimulation of [35 S]GTPg S binding was observed in membranes of Sf9 insect cells expressing high levels of 5-HT2C receptors reconstituted with exogenous Gaq proteins (Hartman and Northup 1996), and cell-permeable peptides mimicking the C-terminal component of Gaq were shown to block activation of native 5-HT2C receptors in the choroid plexus (Chang et al. 2000). Several studies that used pertussis toxin (PTX), which uncouples G-protein-coupled receptors from Gai/o protein subtypes, have demonstrated that in addition to Gaq, 5-HT2C receptors also interact with PTX-sensitive G proteins controlling endogenous Cl- membrane currents in Xenopus laevis oocytes (Quick et al. 1994), DNA synthesis and proliferation in NIH-3 T3 cells (Westphal and Sanders-Bush 1996), and adenylyl cyclase activity in the AV12 cell line (Lucaites et al. 1996). Direct functional coupling of 5-HT2C receptors expressed in Xenopus laevis oocytes to Gao and Gai1 that resulted in phospho-lipase C activation was shown by an antisense strategy (Quick et al. 1994; Chen et al. 1994). Similarly, 5-HT2C receptors overexpressed in human embryonic kidney (HEK) 293 cells mediated [35 S]GTPy S binding to Gai proteins, and PTX-sensitive Gai subtypes contributed to stimulation of phospholipase C, along with PTX-insensitive G subtypes (Alberts et al. 1999). Finally, Cussac et al. demonstrated that the partially edited 5-HT2C-VSV receptor isoform stably expressed in Chinese hamster ovary (CHO) cells couples to both G and Gai3, which are recruited in an agonist-dependent and receptor reserve-dependent manner, and suggested that the differential influence of agonists on G-protein coupling to the 5-HT2C receptor variants may be relevant to their functional profiles in vivo (Cussac et al. 2002).

However, these studies were conducted in artificial systems in which the receptors may have promiscuous interactions with various heterotrimeric G proteins. At the same time, studies in primary cultures of choroid plexus epithelial (CPE) cells demonstrated that PLCP signaling of endogenously expressed 5-HT2C receptors is mediated by G heterotrimeric proteins, specifically by the Gaq subunit, and that Gpy subunits released from Gq/11 heterotrimers did not contribute to the activation of PI hydrolysis signal in natural systems in which the stoichiometry of signaling molecules is undisturbed. In this study, peptide mimics of G and the G interac-

J r r aq aq tion domain of PLCP1 prevented 5-HT2C receptor-mediated accumulation of inositol phosphates, whereas peptides derived from Py-binding sites of PLCP2 did not (Chang et al. 2000). Other studies in CPE cells have shown that the endogenous 5-HT2C receptor also is able to activate phospholipase D via Ga13 heterotrimers, and that both Ga13 subunit and free Py subunits are responsible for this activation (McGrew et al. 2002). In addition, 5-HT2C receptor has been shown to activate the small GTPase RhoA to induce stress fiber formation (Gohla et al. 1999).

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