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.
Department of Pharmacology, Saitama Medical School, Saitama 390-0452, Japan. yumisait@saitama-med.ac.jp
Yumiko
Y
Saito
Mitsue
M
Tetsuka
Yue
Y
Li
Hitoshi
H
Kurose
Kei
K
Maruyama
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D021381:Protein Transport; D051381:Rats; D017481:Receptors, Somatostatin / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D014162:Transfection
C491208:MCHR1 protein, rat; D017481:Receptors, Somatostatin; D002118:Calcium