Tight Association of the Human Mel_1a-Melatonin Receptor and G_i: Precoupling and Constitutive Activity

Abstract

If stably expressed in human embryonic kidney (HEK)293 cells, the human Mel(1a)-melatonin receptor activates G(i)-dependent, pertussis toxin-sensitive signaling pathways, i.e., inhibition of adenylyl cyclase and stimulation of phospholipase Cbeta; the latter on condition that G(q) is coactivated. The antagonist luzindole blocks the effects of melatonin and acts as an inverse agonist at the Mel(1a) receptor in both intact cells and isolated membranes. This suggests that the Mel(1a) receptor is endowed with constitutive activity, a finding confirmed on reconstitution of the Mel(1a) receptor with G(i). Because the receptor density is in the physiological range, constitutive activity is not an artifact arising from overexpression of the receptor. In addition, the following findings indicate that the Mel(1a) receptor forms a very tight complex with G(i) which can be observed both in the presence and absence of an agonist. 1) In intact cells and in membranes, high-affinity agonist binding is resistant to the destabilizing effect of guanine nucleotides. 2) The ability to bind an agonist with high affinity is preserved even after exposure of the cells to pertussis toxin, because a fraction of G(i) is inaccessible to the toxin in cells expressing Mel(1a) receptors (but not the A(1)-adenosine receptor, another G(i)-coupled receptor). 3) An antiserum directed against the Mel(1a) receptor coprecipitates G(i) even in the absence of an agonist. We therefore conclude that the Mel(1a) receptor is tightly precoupled and that its constitutive activity may play a role in pacing the biological clock, an action known to involve the melatonin receptors in the suprachiasmatic nucleus.

The PDZ Protein Mupp1 Promotes Gi Coupling and Signaling of the Mt1 Melatonin Receptor

“…G i has been shown to be precoupled to MT 1 in its inactive form and to remain stably associated upon agonist stimulation despite the presence of high GTP concentrations in intact cells (29,36). Destabilization of the MT 1 -G i complex in the absence of MUPP1 has different effects on the signaling capacities of MT 1 .…”

“…Previous studies have shown that high affinity agonist binding to MT 1 depends on the coupling of the receptor to G i proteins (36). Accordingly, decreased high affinity agonist binding would be expected when MUPP1 is displaced from MT 1 .…”

Monitoring of Ligand-independent Dimerization and Ligand-induced Conformational Changes of Melatonin Receptors in Living Cells by Bioluminescence Resonance Energy Transfer

“…Crude Membrane Preparation, Solubilization, and Immunoprecipitation-Crude membranes were prepared, solubilized with 1% digitonin, a detergent known to maintain MTR in a native conformation, and immunoprecipitated as described recently (13,14) with 10 g/ml of the FLAG-specific M2 antibody or 1 g/ml of the polyclonal anti-green fluorescence protein antibody (CLONTECH, Palo Alto, CA). Precipitates were analyzed by assessing luciferase activity in a luminometer using coelenterazine h as substrate (Molecular Probes, Eugene, OR).…”

“…Radioligand Binding Experiments-Whole cell radioligand binding assays were performed as described (13 S binding assays with membranes prepared from HEK 293 cells stably expressing the MT2R as described recently (14).…”

Binding of Calmodulin to the D2-Dopamine Receptor Reduces Receptor Signaling by Arresting the G Protein Activation Switch

“…The latter only weakly stimulates GTP␥S binding because of its tight association with G proteins (15). EGTA-washed cell membranes (ϳ10 g) were suspended in an assay volume of 30 l of buffer containing 25 mM HepesNaOH, pH 7.5, 1.5 mM MgCl 2 , 100 mM NaCl, 1 mM EDTA, and 0.01 mM GDP.…”