Shafir I, Schmidt-Sole J, Shai E, Salomon Y
Department of Hormone Research, Weizmann Institute of Science, Rehovot, Israel.
Melanoma Res. 1993 Jun;3(3):157-68. doi: 10.1097/00008390-199306000-00003.
Several properties of the MSH receptor in solid melanotic and amelanotic mouse M2R tumour isografts were studied in C57BL mice. Using cell membrane fractions prepared from such tumours and the superpotent [Nle4,D-Phe7]alpha MSH analogue, the affinity and receptor contents of the two tumour variants were found to be similar. When occupied by MSH, the receptor-MSH complex (R.MSH) was readily soluble in cholate. In the solubilized form, R.MSH was extremely stable and dissociated to an extent of only 30% within 12 days at 4 degrees C. While this high stability can be maintained in the pH range of 7.0-8.5, the solubilized R.MSH complex becomes increasingly unstable below pH 7.0 and totally dissociates at a pH < 6.0. In the membrane-bound form, the R.MSH complex shows a parallel pH stability profile which is shifted down by approximately two pH units. In addition to low pH, the R.MSH complex becomes unstable and totally dissociates in the presence of 10 mM EGTA, suggesting that the calcium-sensitive function of the receptor is still associated with the receptor in the detergent-soluble state. The R.MSH complexes in the soluble and membrane-bound forms are also totally resistant to proteolytic digestion by V8 protease, but were slowly digested by trypsin. Treatment of R.MSH with 1-ethyl-3-(3-dimethylamino-propyl)carbodiimide hydrochloride or bis (sulphosuccinimidyl) suberate led to covalent crosslinking of MSH to the receptor molecule. The electrophoretic mobility on SDS-PAGE of the 43/46 kD doublet of the receptor-MSH conjugate (RMSH) was identical to the photoaffinity labelled MSH receptor product described earlier in cultured M2R cells. However, the efficiency of production of the crosslinked product was approximately 30%, much higher than that achieved previously by photoaffinity labelling. Using rabbit polyclonal anti-alpha MSH antibodies, the RMSH conjugate was identifiable on Western immunoblots. These results provide a basis for further development of procedures for purification of the MSH receptor molecule and studying its protein structure.
在C57BL小鼠中研究了实体黑素瘤和无黑素小鼠M2R肿瘤同基因移植瘤中促黑素（MSH）受体的几种特性。使用从此类肿瘤制备的细胞膜组分和超强效的[Nle4,D-Phe7]α-MSH类似物，发现这两种肿瘤变体的亲和力和受体含量相似。当被MSH占据时，受体-MSH复合物（R.MSH）很容易溶于胆酸盐。以可溶形式存在时，R.MSH极其稳定，在4℃下12天内仅解离30%。虽然这种高稳定性可在pH 7.0 - 8.5范围内维持，但可溶的R.MSH复合物在pH 7.0以下变得越来越不稳定，在pH < 6.0时完全解离。以膜结合形式存在时，R.MSH复合物显示出平行的pH稳定性曲线，该曲线向下移动约两个pH单位。除了低pH外，R.MSH复合物在10 mM乙二醇双四乙酸（EGTA）存在下变得不稳定并完全解离，这表明受体的钙敏感功能在去污剂可溶状态下仍与受体相关。可溶形式和膜结合形式的R.MSH复合物也完全抵抗V8蛋白酶的蛋白水解消化，但会被胰蛋白酶缓慢消化。用盐酸1-乙基-3-(3-二甲基氨基丙基)碳二亚胺或双(磺基琥珀酰亚胺基)辛二酸酯处理R.MSH导致MSH与受体分子共价交联。受体-MSH偶联物（RMSH）的43/46 kD双峰在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）上的电泳迁移率与先前在培养的M2R细胞中描述的光亲和标记的MSH受体产物相同。然而，交联产物的产生效率约为30%，远高于先前通过光亲和标记获得的效率。使用兔多克隆抗α-MSH抗体，RMSH偶联物可在蛋白质免疫印迹法（Western免疫印迹）上鉴定出来。这些结果为进一步开发MSH受体分子的纯化程序及其蛋白质结构研究提供了基础。
