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hMT1 Melatonin (MLT) receptor: amino acids within transmembrane VII (TMVII) are involved in ligand binding

An in silico model of the hMT1 MLT receptor using the crystal structure of bovine rhodopsin as a template generated by us (unpublished) and by Ivanov et al. (Doklady Biochem Biophys 394:49, 2004) predicted that S110, S114 and H195 in TMIII and TMV, are critical for MLT binding. However, the affinity of MLT to compete with 2‐[125I]‐iodomelatonin for the hMT1 receptor triple mutant (S110A/S114A/H195A) decreased by only 3‐fold as compared to the wild‐type (WT) [Ki: 2.29 ± 0.84 nM (n=3) vs 0.67 ± 0.12 nM (n= 3), respectively]. We identified an alternate cluster of conserved hydrophilic amino acids within TMI (T37, D41), TMII (Y81) and TMVII (Y281, Y285, S288, C289) of the hMT1 receptor model. The affinity of MLT to compete for 2‐[125I]‐iodomelatonin binding to HEK cell membranes expressing the double T37V/D41A (TMI) or Y81F (TMII) hMT1 mutant receptors was not different from the affinity for binding to the hMT1 WT [EC50: high = 0.23 ± 0.20 pM (n=3); low = 0.60 ± 0.08 nM (n=3)]. However, the affinity for MLT to compete for radioligand binding to the MT1 Y281F/Y285F mutant decreased by 10 fold at both the high and low affinity sites compared to the WT receptor (p < 0.01). Expression of the S288A/C289A mutant was decreased by 99.7 % of the WT (1286 ± 174 fmol/mg protein, n=3). We suggest that point mutations on TMVII within the cluster of conserved hydrophilic amino acids significantly affected ligand binding to the hMT1 melatonin receptor as demonstrated (J Biol Chem 278:36628, 2003) for other class A GPCRs.