hMT 1 Melatonin (MLT) receptor: amino acids within transmembrane VII (TMVII) are involved in ligand binding

An in silico model of the hMT 1 MLT receptor using the crystal structure of bovine rhodopsin as a template generated by us (unpublished) and by Ivanov et al .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‐[ 125 I]‐iodomelatonin for the hMT 1 receptor triple mutant (S110A/S114A/H195A) decreased by only 3‐fold as compared to the wild‐type (WT) [K i : 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 hMT 1 receptor model. The affinity of MLT to compete for 2‐[ 125 I]‐iodomelatonin binding to HEK cell membranes expressing the double T37V/D41A (TMI) or Y81F (TMII) hMT 1 mutant receptors was not different from the affinity for binding to the hMT 1 WT [EC 50 : 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 MT 1 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 hMT 1 melatonin receptor as demonstrated (J Biol Chem 278 :36628, 2003) for other class A GPCRs. Supported by MH 42922.