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.

Melanin-concentrating hormone (MCH) is known to act through two G-protein-coupled receptors MCHR1 and MCHR2. MCHR1 has three potential sites (Asn 13 , Asn 16 and Asn 23 ) for N-linked glycosylation in its extracellular amino-terminus which may modulate its reactivity. Site-directed mutagenesis of the rat MCHR1 cDNA at single or multiple combinations of the three potential glycosylation sites was used to examine the role of the putative carbohydrate chains on receptor activity. It was found that all three potential N-linked glycosylation sites in MCHR1 were glycosylated, and that N-linked glycosylation of Asn 23 was necessary for full activity. Furthermore, disruption of all three glycosylation sites impaired proper expression at the cell surface and receptor activity. These data outline the importance of the N-linked glycosylation of the MCHR1.

Melanin-concentrating hormone (MCH) receptor 1 (MCH1R) is a class A G protein-coupled receptor. The MCH system has been linked to a variety of physiological functions, including the regulation of feeding and energy metabolism. We recently reported the importance of a dibasic motif in the membrane-proximal C-terminal region for MCH1R function. Here we reveal that an Arg residue in intracellular loop 2 of MCH1R plays a critical role in receptor function. We analyzed the roles of two distinct motifs, BBXXB and BXBB (in which B is a basic residue and X is a nonbasic residue), located in the three intracellular loops of MCH1R. Triple-substitution mutants of intracellular loops 1 and 3 could still activate calcium mobilization, albeit with lower efficacy or potency. However, mutations in intracellular loop 2 led to a complete loss of induction of signal transduction without changing the high affinity constant (Kd) value. By analyzing a series of single-substitution mutants, a point mutation of Arg155 in intracellular loop 2 was found to be responsible for the signaling pathway elicited by MCH. In addition, substitution at positions corresponding to Arg155 in human MCH receptor 2 and rat somatostatin receptor 2 also markedly abolished their ligand-induced signaling capacities, indicating that this Arg is a recognition determinant in several G protein-coupled receptors.

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