Premium
hMT 1 Melatonin (MLT) receptor: amino acids within transmembrane VII (TMVII) are involved in ligand binding
Author(s) -
Pazin Marina Viktoria,
Kai Li,
Bertsch Martina,
Focia Pamela J.,
Dubocovich Margarita L
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a250-b
Subject(s) - receptor , amino acid , ligand (biochemistry) , melatonin receptor , mutant , chemistry , radioligand , rhodopsin , wild type , transmembrane domain , binding site , microbiology and biotechnology , biochemistry , stereochemistry , biophysics , biology , gene , retinal
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silico model of the hMT 1 MLT receptor using the crystal structure of bovine rhodopsin as a template generated by us (unpublished) and by Ivanov et al .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.