Arginine residue 155 in the second intracellular loop plays a critical role in rat melanin-concentrating hormone receptor 1 activation.

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.

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.

Functional role of N-linked glycosylation on the rat melanin-concentrating hormone receptor 1.

Melanin-concentrating hormone (MCH) is known to act through two G-protein-coupled receptors MCHR1 and MCHR2. MCHR1 has three potential sites (Asn13, Asn16 and Asn23) 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 Asn23 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.

Properties of rat melanin-concentrating hormone receptor 1 internalization.

Melanin-concentrating hormone (MCH) is a neuropeptide that plays an important role in several physiological processes. It activates two G protein-coupled receptors (GPCRs), MCH1R and MCH2R, of which MCH1R seems to be a key regulator of food intake. By using HEK293T cells stably transfected with Flag-tagged rat MCH1R, we investigated the mechanism underlying the MCH-induced internalization pathway, which is important for the desensitization or regulation of the receptor response. Quantitative analysis by flow cytometry indicated that the rate of MCH1R internalization progressed in a rapid and time-dependent manner during the first 30 min, and was partly inhibited by pretreatment with the selective protein kinase C (PKC) inhibitor Go6850. Overexpression of dominant-negative beta-arrestin-2 (284-409) or dynamin I-K44A significantly prevented MCH-induced internalization of MCH1R, while overexpression of dominant-negative beta-arrestin-1-V53D had no effect. A triple-substituted mutant at Thr317, Ser325 and Thr342 to Ala residue in the C-terminus significantly prevented MCH-induced receptor internalization. Similar extents of internalization prevention were noted with the deletion mutants DeltaThr342 and DeltaGlu346, lacking 11 and 7 residues in the C-terminal tail, respectively. Our data suggest that MCH1R undergoes rapid MCH-induced internalization through a PKC-, beta-arrestin-2- and dynamin I-dependent pathway and that a portion of the C-terminal tail plays an important role in the internalization process.

Distinct roles of the DRY motif in rat melanin-concentrating hormone receptor 1 in signaling control.

Rhodopsin family (class A) G protein-coupled receptors possess common key residues or motifs that appear to be important for receptor function. To clarify the roles of the highly conserved amino acid triplet Asp(3.49)-Arg(3.50)-Tyr(3.51) (DRY motif), we examined how single-substitution mutations of the amino acids in the motif influenced specific features of rat melanin-concentrating hormone receptor 1 (MCH1R) activity. Substitution of either Asp140(3.49) or Tyr142(3.51) to Ala resulted in nonfunctional receptors, despite the retention of apparent potencies for agonist binding. These loss-of-function phenotypes may be caused by the lack of stimulation for GDP-GTP exchange observed in GTPgammaS-binding assays. On the other hand, substitution of Arg141(3.50) to Ala caused a 4-fold reduction in the agonist binding affinity and, concomitantly, a rightward shift of the dose-dependency curve for calcium mobilization and inhibition of cyclic AMP production. Although many experimental studies have suggested that the DRY motif is involved in maintaining the receptor in its ground state, none of the DRY motif substitutions to Ala in MCH1R led to constitutive activation, in terms of the basal signaling level for ERK1/2 activation or GTPgammaS binding. These data suggest that the major contribution of the DRY motif in MCH1R is to govern receptor conformation and G protein coupling/recognition.