Activation of Adenylyl Cyclase by Endogenous G_s-Coupled Receptors in Human Embryonic Kidney 293 Cells Is Attenuated by 5-HT₇Receptor Expression

Abstract

Human 5-hydroxytryptamine 7 (5-HT 7 ) receptors display characteristics shared with receptors believed to form a tight physical coupling with G protein in the absence of ligand. Some receptors apparently preassociated with G i/o and G q/11 are reported to inhibit the signaling of other similarly coupled G protein-coupled receptors by limiting their access to activate a common G protein pool. Therefore, we determined whether 5-HT 7 receptor expression was sufficient to limit signaling of endogenously expressed G s -coupled receptors in human embryonic kidney (HEK) 293 cells. Using the ecdysone-inducible expression system, which allows for the titration of increasing receptor density in the same clonal cell line, we compared the effects of 5-HT 4(b) and 5-HT 7(a,b,d) receptor expression on adenylyl cyclase (AC) stimulation by the endogenous G s -coupled ␤-adrenergic (␤AR) and prostanoid EP (EPR) receptors. ␤AR-and EPR-stimulated AC activity was attenuated by 5-HT 7 receptor expression in both membrane preparations and intact HEK293 cells. ␤AR-and EPR-stimulated AC activity was unaffected by expression of the G s -coupled 5-HT 4 receptor. The mechanism of this heterologous desensitization seems independent of protein kinase A activation, nor does it occur at the level of G protein activation because 1) ␤AR-and EPR-stimulated AC activity was not restored to control values when G␣ s was overexpressed; and 2) ␤ 1 AR and ␤ 2 AR activation of G␣ s was unaffected by the expression of 5-HT 7 receptors. In addition, overexpression of AC isoforms was unable to rescue ␤AR-and EPR-stimulated AC activity. Therefore, 5-HT 7 receptors probably limit access and/or impede activation of AC by ␤AR and EP receptors. Although the 5-HT 7 receptor may preassociate with G protein and/or AC, the mechanism of this heterologous desensitization remains elusive.Serotonin (5-hydroxytryptamine, 5-HT) mediates its diverse physiological effects through at least 14 different receptor subtypes, of which 13 belong to the G protein-coupled receptor family (Hoyer et al., 1994). Among the human 5-HT receptors, three different subtypes, 5-HT 4 , 5-HT 6 , and 5-HT 7 , are coupled to G s and at least the 5-HT 4 and 5-HT 7 receptors are expressed as several different functional splice variants (Gerald et al., 1995;Heidmann et al., 1997). The functional significance of 5-HT 7 splice variants, which differ only in the carboxyl terminus (Heidmann et al., 1997), remains unknown (Krobert et al., 2001Krobert and Levy, 2002), whereas among the 5-HT 4 splice variants, constitutive activation of AC is dependent on the different carboxyl termini (Bockaert et al., 2004). We have shown previously that the 5-HT 4(b) and 5-HT 7(a) signaling properties differ fundamentally. The potency of 5-HT to stimulate AC increased with increasing receptor density in clones expressing 5-HT 4(b) but not 5-HT 7(a) receptors, even though 5-HT-stimulated AC activity in clones expressing 5-HT 7(a) receptors had reached asymptotic levels (Bruheim et al., 2003). This indicates that...

Antihistaminergics and inverse agonism: Potential therapeutic applications

“…However, it is worth noting that regardless of its theoretical origins, there are empirical observations supporting the CTCM. Some mutants of Į 1B -adrenergic receptors are able to spontaneously couple to G-protein inactivating it (Chen et al, 2000), and this has also been observed for non-mutant serotonin 5HT 7 and histamine H 2 receptors (Andressen et al, 2006;Tubio et al, 2010).…”

Label-free optical biosensor for probing integrative role of adenylyl cyclase in G protein-coupled receptor signaling

“…GPCRs can be desensitized in two distinct ways: 1) homologous desensitization wherein the activated GPCR is down-regulated and 2) heterologous desensitization wherein the activation of second messengerdependent protein kinases or of a different receptor causes down-regulation of a GPCR (21,22). Because forskolin directly activates AC, which is a downstream target of G s -coupled receptors, we were interested in the heterologous desensitization of G s signaling using a twostep biosensor desensitization assay, in which the cells are repeatedly stimulated with two stimuli and the two stimulations are separated by about 1 hr (23).…”

Receptor-G Protein Interaction Studied by Bioluminescence Resonance Energy Transfer: Lessons from Protease-Activated Receptor 1

“…This is true for D2 receptor and Gαi (Senogles et al, 1987), β2-adrenergic receptor and Gαs (Lachance et al, 1999), δ-opioid receptor, and Gαi (Law and Reisine, 1997), somatostatin receptor and Gαi and Gαo (Law et al, 1993), AT2 receptor and Gαi (Zhang and Pratt, 1996), melatonin MT1 receptor, and Gαi (Roka et al, 1999). Pharmacological studies have also been useful to demonstrate a tight association between a GPCR and its G protein as shown for the serotonin 5-HT7 preassociated with Gαs even in the absence of agonist (Andressen et al, 2006). As discussed later, such a preassembly is not observed with all GPCR-G protein couples, indicating that this cannot be considered as a general phenomena involved in receptor-G protein coupling, then illustrating the importance and the specificity of such association when it can be observed.…”

“…Moreover, GPCR-G protein preassembly may allow a better control of the selectivity of the signaling cascades since one would argue that a preassembled receptor has a limited availability in space (same tissue or cell) and in time (simultaneously) to interact and couple to different G proteins as we nicely demonstrated for PAR1 and Gαi1 and Gα12 (Ayoub et al, 2010). The GPCR-G protein preassembly may also control the efficiency of signaling by sequestering or limiting access to a common G protein pool as this has been shown for CXCR7 attenuating β-adrenergic-mediated Gαs/adenylate cyclase activation (Andressen et al, 2006). …”

“…This is consistent with the strong and sustained developing effects of thrombin and PAR1 on cell morphology and proliferation where stabilizing PAR1-Gα12 interaction and maintaining their long term activation may be crucial for the control of cell proliferation, differentiation, migration, or oncogenesis (Dhanasekaran and Dermott, 1996; Riobo and Manning, 2005). However, one would exclude that the stable Gα12 association with PAR1 may inhibits subsequent receptor activation and via a sequestration process this limits the pool of Gα12 available, thereby reduces the action of Gα12-dependent signaling promoted by other GPCRs as observed with the preassembly of 5-HT7 with Gαs inhibiting this G protein to be activated by the β-adrenergic receptor (Andressen et al, 2006). …”