Abstract
Co-expression of guanine nucleotide-binding regulatory (G) protein-coupled receptors (GPCRs), such as the G(i/o)-coupled human 5-hydroxytryptamine receptor 1B (5-HT(1B)R), with the G(q/11)-coupled human histamine 1 receptor (H1R) results in an overall increase in agonist-independent signaling, which can be augmented by 5-HT(1B)R agonists and inhibited by a selective inverse 5-HT(1B)R agonist. Interestingly, inverse H1R agonists inhibit constitutively H1R-mediated as well as 5-HT(1B)R agonist-induced signaling in cells co-expressing both receptors. This phenomenon is not solely characteristic of 5-HT(1B)R; it is also evident with muscarinic M2 and adenosine A1 receptors and is mimicked by mastoparan-7, an activator of G(i/o) proteins, or by over-expression of Gbetagamma subunits. Likewise, expression of the G(q/11)-coupled human cytomegalovirus (HCMV)-encoded chemokine receptor US28 unmasks a functional coupling of G(i/o)-coupled CCR1 receptors that is mediated via the constitutive activity of receptor US28. Consequently, constitutively active G(q/11)-coupled receptors, such as the H1R and HCMV-encoded chemokine receptor US28, constitute a regulatory switch for signal transduction by G(i/o)-coupled receptors, which may have profound implications in understanding the role of both constitutive GPCR activity and GPCR cross-talk in physiology as well as in the observed pathophysiology upon HCMV infection.
Highlights
GPCRs,1 which can be activated by a diverse array of stimuli, represent the largest group of integral membrane proteins involved in signal transduction
Our data indicate that both activated histamine histamine 1 receptor (H1R) and HCMVencoded receptor US28 can result in the propagation of Gi/ocoupled receptor dependent signaling
Constitutive H1R Activity and Inverse Agonistic Properties of Antihistamines—Transient expression of human H1R in COS-7 cells resulted in a high affinity binding site for the H1R radioligand [3H]mepyramine (KD ϭ 1.7 Ϯ 0.2 nM, Bmax ϭ 4.6 Ϯ 0.1 pmol/mg protein, data not shown)
Summary
Materials—pNF-B-Luc was obtained from Stratagene (La Jolla, CA). ATP disodium salt, bovine serum albumin, CGS-12066A maleate, chloroquine diphosphate, cholera toxin, DEAE-dextran (chloride form), histamine dihydrochloride, mastoparan-7, mepyramine (pyrilamine maleate), pargyline hydrochloride, pertussis toxin, polyethyleneimine, tripelennamine hydrochloride, 3,3Ј,5,5Ј-tetramethylbenzidine liquid substrate system for ELISA, triprolidine hydrochloride, and Tween 20 were purchased from Sigma. Reporter Gene Assay—Cells transiently co-transfected with pNF-BLuc (125 g/11⁄7107 cells, Stratagene) and plasmid DNA encoding the various receptors (25 g/11⁄7107 cells) were seeded in 96-well blackplates (Costar) in serum-free culture medium and incubated with drugs. 5-HT1BR Binding Studies—COS-7 cells used for 5-HT1BR binding studies were harvested 48h after transfection and homogenized in ice-cold 5-HT1BR-binding buffer (50 mM Tris-HCl (pH 7.4), containing 4 mM CaCl2, 100 M ascorbic acid, and 10 M pargyline). US28 Receptor Binding Studies—The transfected COS-7 cells used for radioligand binding studies were seeded in 24-well plates; 48 h after transfection, binding was performed on whole cells for 3 h at 4 °C using [125I]CCL5 (RANTES) in binding buffer (50 mM HEPES, pH 7.4, 1 mM CaCl2, 5 mM MgCl2, and 0.5% bovine serum albumin). ELISA—48 h after transfection, receptor expression in COS-7 cells was measured using an ELISA as described previously [18]. The data from radioligand-binding and functional assays data were evaluated by a nonlinear, least squares curve-fitting procedure using Graphpad Prism® (GraphPad Software, Inc., San Diego, CA)
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