Ligand efficacy and potency at recombinant human MT2 melatonin receptors:: evidence for agonist activity of some mt1-antagonists

1 NIH3T3 fibroblast cells transfected with the full-length coding region of the MT(2) human melatonin receptor stably expressed the receptor that is coupled to a pertussis toxin-sensitive G protein and exhibits high affinity for melatonin (K(1) = 261 pM). 2 The order of apparent affinity for selected compounds was: 4-phenyl-2-propionamidotetralin (4P-PDOT) > 2-phenylmelatonin > 2-iodomelatonin > 2-bromomelatonin > 6-chloromelatonin greater than or equal to melatonin > luzindole > N-acetyl-tryptamine greater than or equal to N-[(2-phenyl-1 H-indol-3-yl)ethyl]cyclobutanecarboxamide (compound 6)> N-acetylserotonin. 3 4P-PDOT exhibited a very high selectivity (similar to 22,000 times) for the MT2 receptor with respect to the mti receptor subtype, as tested in comparative experiments with membrane preparations from NIH3T3 cells stably transfected with the human mt(1) receptor. 4 MT(2) melatonin receptors mediated incorporation of [(35)S]-GTP gamma S into isolated membranes via receptor catalyzed exchange of [(35)S]-GTP gamma S for GDP. The relative intrinsic activity and potency of the compounds were subsequently studied by using [(35)S]-GTP gamma S incorporation. The order of potency was equal to the order of apparent affinity. Melatonin and full agonists increased [(35)S]-GTP gamma S binding by 250% over basal (taken as 100%). Luzindole did not increase basal [(35)S]-GTP gamma S binding but competitively inhibited melatonin-stimulated [(35)S]-GTP gamma S binding, thus exhibiting antagonist action. 5 The other two mt(1) antagonists used here, 4P-PDOT and N-[2-phenyl-1 H-indol-3-yl)ethyl]cyclobutanecarboxamide, behaved as partial agonists at the MT(2) subtype, with relative intrinsic activities of 0.37 and 0.39, respectively. 6 These findings show, for the first time, important differences in the intrinsic activity of analogues between the human mt, and MTI melatonin receptor subtypes.