Site-directed mutagenesis was used to study two residues, valine 208 and histidine 211, in transmembrane domain 5 of the ovine Mel(1a beta) melatonin receptor. A series of 4 mutants were constructed (V208A, V208L, H211F, H211L), and each engineered to contain a FLAG-epitope. Immunocytochemistry demonstrated that all the mutants were expressed in COS-7 cells at levels comparable to the FLAG-epitope tagged wild-type Mel(1a beta) receptor (similar to 120 fmol/mg protein). Ligand binding revealed however that all mutants had reduced affinities for 2-[I-125]-iodomelatonin (Kd wild-type 139 pM, Kd mutants 320 to 989 pM). Competition studies, with a series of melatonin analogues, identified a probable interaction between histidine 211 and the 5-methoxy group of melatonin. The wild-type receptor and both valine 208 mutants displayed a dose-dependent melatonin mediated inhibition of cyclic AMP levels in HEK293 cells, with IC50 values in the same rank-order as their melatonin binding affinities. Both H211F and H211L, however, did not display any melatonin mediated effects and may suggest that histidine 211 is critical for melatonin mediated receptor activation. (C) 1997 Academic Press.
|Number of pages||6|
|Journal||Biochemical and Biophysical Research Communications|
|Publication status||Published - 20 Oct 1997|
- serine residues
- pars tuberalis