Molecular analysis of signaling pathways stimulated by melanin-concentrating hormone (MCH)
| Project/Area Number |
13680859
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Neurochemistry/Neuropharmacology
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| Research Institution | Saitama Medical School |
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Principal Investigator |
SAITO Yumiko SAITAMA Medical School, Faculty of Medicine, Lecturer, 医学部, 講師 (00215568)
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| Project Period (FY) |
2001 – 2002
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| Project Status |
Completed (Fiscal Year 2002)
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| Budget Amount *help |
¥3,600,000 (Direct Cost: ¥3,600,000)
Fiscal Year 2002: ¥1,400,000 (Direct Cost: ¥1,400,000)
Fiscal Year 2001: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | food intake / peptide / receptor / calcium influx / 神経ペプチド / Gタンパク質 / イーストツーハイブリッド |
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| Research Abstract |
1.
MCH is a hypothalamic neuropeptide that regulates several physiological functions. The orphan G protein-coupled receptors MCHR1 and MCHR2 were recently found to bind MCH with high affinity. We show here that human melanoma cell line SK-MEL-37 express SLC-1 mRNA but not MCHR2 one by RT-PCR analysis and immunofluorescence studies. Using CHO and 293 cells overexpressing SLC-1 by cDNA transfection, it was shown that SLC-1 coupled to both Gα_1/Gα_0 and Gα_0 proteins. In SK-MEL-37 cells, MCH inhibited forskolin-stimulated cyclic AMP accumulation and induced mitogen-activated protein kinase (MAPK) in a pertussis toxin-(PTX)-sensitive manner. The MAPK activity leads to the production of phospholylated forms of p42/p44 MAPK. However, an increase in the intracellular free Ca^<2+> concentration was not elicited by MCH in SK-MEL-37 cells. These results show that SLC-1 is coupled only o PTX-sensitive Gα_1/Gα_0 in SK-MEL-37 cells. This study provides for the first time a skin-derived cellular model to analyze the molecular mechanism of MCH signaling pathway.
2.
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 example 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 NCHR1.
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Report
(3 results)
Research Products
(16 results)
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[Publications] Civelli, O., Nothacker, H-P., Saito, Y., Wang, Z., Lin, S., and Reinscheid, RK.: "Novel neurotransmitters as natural ligands of orphan G-protein-coupled receptors"Trends in Neurosci. 24. 230-237 (2001)
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[Publications] Saito, Y., Wang, Z., Hagino-Yamagishi, K., Civelli, O., Kawashima, S. and Maruyama, K.: "Endogenous melanin-concentrating hormone receptor SLC-1 in human melanoma SK-MEL-37 cells"Biochem. Biophys. Res. Commun.. 289. 44-50 (2001)
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[Publications] Schlumberger, SE., Saito, Y., Giller, T., Hintermann, E., Tanner, V., Jaggin, O., Civelli, O. and Eberle, AN.: "Different structural requirement for melanin-concentrating hormone (MCH) interacting with rat MCHR1 (SLC-1) and mouse B16 cell MCH-R"J. Recept. Signal Transduct.. in press.
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