Pharmacological characterization of the human melatonin Mel1a receptor following stable transfection into NIH3T3 cells

Mouse fibroblasts (NIH3T3) transfected with the full‐length coding region of the Mel1a melatonin receptor stably expressed the receptor, coupled to a pertussis toxin‐sensitive G‐protein(s) and exhibiting high affinity and adequate pharmacological profile. The receptor protein had the tendency of a strong coupling to the G‐protein and therefore low‐affinity state was induced by uncoupling the receptor from its G‐protein in presence of high concentrations of NaCl (500–700 mM) and/or GTPγS (100 μM). Thereafter, the affinity of a series of melatonin analogues was determined to both, high‐ and low‐affinity receptor states, thus providing a basis for the prediction of their efficacy, according to the ternary complex model. The cells were subsequently used to study the agonist‐induced G‐protein activation, determined by calculating the rate of GDP‐GTP exchange measured in presence of 35S‐labelled GTPγS. The natural ligand melatonin induced a significant increase in the GDP‐GTP exchange rate, the presence of GDP and NaCl being necessary to observe this effect. The full agonists 2‐phenylmelatonin, 2‐bromomelatonin and 6‐chloromelatonin equally induced an increase of the GDP‐GTP exchange. 5‐Hydroxy‐N‐acetyltryptamine activated the GTP‐GDP exchange to a much lesser extent (53%) than melatonin, thus behaving as a partial agonist. As predicted by the model, the melatonin antagonist (N‐[(2‐phenyl‐1H‐indol‐3‐yl)ethyl]cyclobutanecarboxamide) was without effect on basal G protein activation. Coincubation of this compound with melatonin induced a dose‐dependent rightward shift in the melatonin concentration‐effect curve, thus exhibiting the behaviour of a competitive and surmountable antagonist. Using the equation proposed by Venter (1997) we were able to determine that there were no ‘spare’ receptors in the system. Therefore, the approach proposed in the present work can be successfully used for the determination of ‘drug action’ at the level of the human Mel1a melatonin receptor and evaluation of the efficacy of new selective melatonin analogues.