The basic residues in the membrane-proximal C-terminal tail of the rat melanin-concentrating hormone receptor 1 are required for receptor function.
Melanin-concentrating hormone (MCH) is a hypothalamic neuropeptide that plays a key role in food intake. It acts through two G protein-coupled receptors (GPCRs), MCH1R and MCH2R, of which MCH1R is the primary regulator of food intake. We have previously reported that N-linked glycosylation of the extracellular domain of MCH1R is necessary for cell surface expression and signal transduction. We now report a role for the rat MCH1R C-terminal region. We constructed serial C-terminal truncation mutants and determined the resulting changes in protein expression, cell surface expression, ligand binding, and MCH-stimulated calcium influx. By analyzing two mutants, deltaT317 (deletion of 36 C-terminal amino acids) and deltaR321 (deletion of 32 C-terminal amino acids), we found that the region between Phe(318) and Arg(321)) was responsible for signal transduction. A more detailed analysis was performed with single or multiple residue mutations. Single mutations of Arg(319), Lys(320), or Arg(321) exhibited a decrease in the cell surface expression, whereas mutations of either Arg(319) or Lys(320), but not Arg(321), showed a significant reduction in the calcium influx. Furthermore, simultaneous mutations of Arg(319) and Lys(320) produced a pronounced decrease in the efficacy of calcium influx stimulation compared with single mutations. A computational analysis revealed a dibasic amino acid motif that is conserved among many class 1 GPCRs and may be part of the amphiphilic cytoplasmic helix 8 (an eight-cytoplasmic helix). Our results therefore provide new insights into the role of the putative helix 8 in the regulation of GPCR function.
Tetsuka
Mitsue
M
Department of Pharmacology, Saitama Medical School, Iruma-gun, Saitama 350-0492, Japan.
Saito
Yumiko
Y
Imai
Kensaku
K
Doi
Hirofumi
H
Maruyama
Kei
K
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D017124:Conserved Sequence; D005455:Fluorescent Antibody Technique; D019204:GTP-Binding Proteins / Q000502:physiology; D006801:Humans; D018613:Microscopy, Confocal; D008969:Molecular Sequence Data; D037342:Pertussis Toxin / Q000494:pharmacology; D051381:Rats; D017481:Receptors, Somatostatin / Q000737:chemistry* / Q000502:physiology*; D015398:Signal Transduction; D013329:Structure-Activity Relationship
C103263:MCHR1 protein, human; C491208:MCHR1 protein, rat; D017481:Receptors, Somatostatin; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins