Kokkotou E, Mastaitis J W, Qu D, Hoersch D, Slieker L, Bonter K, Tritos N A, Maratos-Flier E
Research Division, Joslin Diabetes Center and Harvard Medical School, 1 Joslin Place, Boston, MA 02215, USA.
Neuropeptides. 2000 Jun-Aug;34(3-4):240-7. doi: 10.1054/npep.2000.0821.
Melanin concentrating hormone (MCH), a hypothalamic neuropeptide, is an important regulator of energy homeostasis in mammals. Characterization of an MCH specific receptor has been hampered by the lack of a suitable radioligand. The [Phe(13), Tyr(19)]-MCH analog has been shown by different investigators to bind specifically to cell lines of epithelial or pigment cell origin. Recently, using functional assays, the MCH receptor has been characterized as a seven transmembrane G-coupled protein initially identified as SLC-1. In the present study, we used tyrosine iodinated [Phe(13), Tyr(19)]-MCH analog, which stimulates food intake in a manner similar to that of MCH, as well as native MCH to conduct binding studies. Specific binding could not be demonstrated in intact cells of several cell lines, including A431 and B16. Specific binding associated with membranes localized to the microsomal, not the plasma membrane, fraction. Message for SLC-1 was absent in these cell lines, as assessed by Northern blot analysis. We conclude that cells previously reported to express the MCH receptor do not express SLC-1 and that both iodinated MCH and the [Phe(13), Tyr(19)]-MCH have a large component of non-specific binding. These ligands may be useful for binding studies in transfected cells with high levels of SLC-1 expression. However they do not appear to be suitable for screening for the MCH receptor as most cells demonstrate significant low affinity non-specific binding.
黑色素浓缩激素（MCH）是一种下丘脑神经肽，是哺乳动物能量平衡的重要调节因子。由于缺乏合适的放射性配体，MCH特异性受体的特性研究受到了阻碍。不同的研究人员已证明，[苯丙氨酸（13），酪氨酸（19）]-MCH类似物能特异性结合上皮或色素细胞来源的细胞系。最近，通过功能测定，MCH受体已被鉴定为一种最初被确定为SLC-1的七跨膜G偶联蛋白。在本研究中，我们使用酪氨酸碘化的[苯丙氨酸（13），酪氨酸（19）]-MCH类似物（其刺激食物摄入的方式与MCH类似）以及天然MCH进行结合研究。在包括A431和B16在内的几种细胞系的完整细胞中未证明有特异性结合。与膜相关的特异性结合定位于微粒体部分，而非质膜部分。通过Northern印迹分析评估，这些细胞系中不存在SLC-1的信息。我们得出结论，先前报道表达MCH受体的细胞不表达SLC-1，并且碘化MCH和[苯丙氨酸（13），酪氨酸（19）]-MCH都有很大一部分非特异性结合。这些配体可能对转染了高表达SLC-1的细胞的结合研究有用。然而，由于大多数细胞表现出显著的低亲和力非特异性结合，它们似乎不适合用于筛选MCH受体。
