Molecular analysis of signaling pathways stimulated by melanin-concentrating hormone (MCH)

黑色素浓缩激素 (MCH) 刺激信号通路的分子分析

• 批准号: 13680859
• 负责人: SAITO Yumiko
• 金额: $ 2.3万
• 依托单位: Saitama Medical School
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2001
• 资助国家: 日本
• 起止时间: 2001 至 2002
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-13680859/
• 关键词: 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

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.

1. MCH是一种调节多种生理功能的下丘脑神经肽。最近发现孤儿G蛋白偶联受体MCHR 1和MCHR 2以高亲和力结合MCH。我们在这里显示，人黑色素瘤细胞系SK-MEL-37表达SLC-1 mRNA，但不表达MCHR 2的一个通过RT-PCR分析和免疫荧光研究。用cDNA转染的方法转染过表达SLC-1的CHO细胞和293细胞，发现SLC-1与Gα_1/Gα_0和Gα_0蛋白偶联。在SK-MEL-37细胞中，MCH抑制毛喉素刺激的环AMP积累，并以百日咳毒素（PTX）敏感的方式诱导丝裂原活化蛋白激酶（MAPK）。MAPK活性导致产生磷酸化形式的p42/p44 MAPK。然而，在SK-MEL-37细胞中，MCH并没有引起细胞内游离Ca^<2+>浓度的增加。这些结果表明SLC-1在SK-MEL-37细胞中仅与PTX敏感的Gα_1/Gα_0偶联。本研究首次提供了一种皮肤源性细胞模型来分析MCH信号通路的分子机制。2. MCHR 1胞外氨基端存在三个潜在的N-糖基化位点（Asn^<13>、Asn^<16>和Asn^<23>），这三个位点可能调节MCHR 1的反应性。在三个潜在糖基化位点的单个或多个组合处对大鼠MCHR 1 cDNA进行定点诱变，以举例说明推定的糖链对受体活性的作用。发现MCHRl中的所有三个潜在的N-连接糖基化位点都被糖基化，并且Asnl的N-连接糖基化<23>是完全活性所必需的。此外，所有三个糖基化位点的破坏损害了细胞表面的正确表达和受体活性。这些数据概述了NCHR 1的N-连接糖基化的重要性。

项目成果

Saito, Y. et al.: "Expression of the melanin-concentrating hormone (MCH) recepter mRNA in the rat brain"J. Comp. Neurol.. 435. 26-40 (2001)

Saito, Y. 等人：“大鼠脑中黑色素浓缩激素 (MCH) 受体 mRNA 的表达”J.

Saito, Y et al.: "Endogenous melanin-concentrating hormone receptor SLC-1 in human melanoma SK-MEL-37 cells"Biochem. Biophys. Res. Commun.. 289. 44-50 (2001)

Saito, Y 等人：“人黑色素瘤 SK-MEL-37 细胞中的内源性黑色素浓缩激素受体 SLC-1”Biochem。

Clark, S. et al.: "The urotensinII receptor is expressed in the cholinergic mesopontine tegmentum of the rat"Brain Research. 923. 120-127 (2001)

Clark, S. 等人：“尾加压素 II 受体在大鼠的胆碱能中脑桥被盖中表达”《大脑研究》。

斎藤 祐見子: "Gタンパク質キメラを用いたオーファン受容体リガンドの探索"神経精神薬理学. 21. 77-82 (2001)

Yumiko Saito：“使用 G 蛋白嵌合体寻找孤儿受体配体”《神经精神药理学》21. 77-82 (2001)。

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)

Civelli, O.、Nothacker, H-P.、Saito, Y.、Wang, Z.、Lin, S. 和 Reinscheid, RK.：“新型神经递质作为孤儿 G 蛋白偶联受体的天然配体”神经科学趋势。