PT - JOURNAL ARTICLE AU - Roka, Florian AU - Brydon, Lena AU - Waldhoer, Maria AU - Strosberg, A. Donny AU - Freissmuth, Michael AU - Jockers, Ralf AU - Nanoff, Christian TI - Tight Association of the Human Mel<sub>1a</sub>-Melatonin Receptor and G<sub>i</sub>: Precoupling and Constitutive Activity AID - 10.1124/mol.56.5.1014 DP - 1999 Nov 01 TA - Molecular Pharmacology PG - 1014--1024 VI - 56 IP - 5 4099 - http://molpharm.aspetjournals.org/content/56/5/1014.short 4100 - http://molpharm.aspetjournals.org/content/56/5/1014.full SO - Mol Pharmacol1999 Nov 01; 56 AB - If stably expressed in human embryonic kidney (HEK)293 cells, the human Mel1a-melatonin receptor activates Gi-dependent, pertussis toxin-sensitive signaling pathways, i.e., inhibition of adenylyl cyclase and stimulation of phospholipase Cβ; the latter on condition that Gq is coactivated. The antagonist luzindole blocks the effects of melatonin and acts as an inverse agonist at the Mel1a receptor in both intact cells and isolated membranes. This suggests that the Mel1areceptor is endowed with constitutive activity, a finding confirmed on reconstitution of the Mel1a receptor with Gi. 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 Mel1a receptor forms a very tight complex with Gi 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 Gi is inaccessible to the toxin in cells expressing Mel1a receptors (but not the A1-adenosine receptor, another Gi-coupled receptor). 3) An antiserum directed against the Mel1areceptor coprecipitates Gi even in the absence of an agonist. We therefore conclude that the Mel1a 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.