Scientists changed two building blocks (valine 208 and histidine 211) in a sheep melatonin receptor to see how they affect melatonin binding and signaling. All the altered receptors still showed up in cells, but they bound melatonin less tightly, and the ones with the histidine change stopped responding to melatonin altogether.

Site-directed mutagenesis was used to study two residues, valine 208 and histidine 211, in transmembrane domain 5 of the ovine Mel1a 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 Mel1a beta receptor (approximately 120 fmol/mg protein). Ligand binding revealed however that all mutants had reduced affinities for 2-[125I]-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.