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 Cβ; 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_1areceptor 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 A₁-adenosine receptor, another G_i-coupled receptor). 3) An antiserum directed against the Mel_1areceptor 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.