THE ROLES SF VALINE-208 AND HISTIDINE-211 IN LIGAND-BINDING AND RECEPTOR FUNCTION OF THE OVINE MEL(1A-BETA) MELATONIN RECEPTOR

Site-directed mutagenesis was used to study two residues, valine 208 a nd histidine 211, in transmembrane domain 5 of the ovine Mel(1a beta) melatonin receptor. A series of 4 mutants were constructed (V208A, V20 8L, H211F, H211L), and each engineered to contain a FLAG-epitope. Immu nocytochemistry demonstrated that all the mutants were expressed in CO S-7 cells at levels comparable to the FLAG-epitope tagged wild-type Me l(1a beta) receptor (similar to 120 fmol/mg protein). Ligand binding r evealed however that all mutants had reduced affinities for 2-[I-125]- iodomelatonin (Kd wild-type 139 pM, Kd mutants 320 to 989 pM). Competi tion studies, with a series of melatonin analogues, identified a proba ble interaction between histidine 211 and the 5-methoxy group of melat onin. The wild-type receptor and both valine 208 mutants displayed a d ose-dependent melatonin mediated inhibition of cyclic AMP levels in HE K293 cells, with IC50 values in the same rank-order as their melatonin binding affinities. Both H211F and H211L, however, did not display an y melatonin mediated effects and may suggest that histidine 211 is cri tical for melatonin mediated receptor activation. (C) 1997 Academic Pr ess.