Akbulut S, Byersdorfer C A, Larsen C P, Zimmer S L, Humphreys T D, Clarke B L
Department of Medical Microbiology and Immunology, University of Minnesota-Duluth, Duluth, Minnesota, 55812, USA.
Biochem Biophys Res Commun. 2001 Mar;281(5):1086-92. doi: 10.1006/bbrc.2001.4481.
The expression of melanocortin-5 receptor (MC5-R) mRNA and protein was characterized from isolated rat lymphocytes. The presence of MC5-R mRNA in spleen and thymus tissues was demonstrated by RT-PCR. The RT-PCR product was sequenced to confirm the identification of MC5-R. Tissues from lachrymal glands, adipose, adrenals, thymus, pancreas, and isolated splenic lymphocytes were detergent solubilized. The crude proteins were resolved by SDS-PAGE, transblotted to a nitrocellulose membrane, and probed for MC5-R using anti-receptor rabbit antisera. Two different types of polyclonal rabbit antisera were raised against synthetic peptides representing epitopes found at the amino (alphaN-MC5-R) and the carboxyl termini (alphaC-MC5-R) on the MC5-R. A prominent band at 77,000 (p77) was detected in all tissues except the pancreas. Preimmune sera did not detect p77 by Western analysis and the addition of peptide antigen neutralized the detection of p77 by the specific antisera. The receptor protein was purified from spleen and thymic lymphocytes using protein A agarose that precipitated material complexed to alphaN-MC5-R. The purified MC5-R was detected by Western analysis using alphaC-MC5-R. Both anti-receptor antisera, alphaN-MC5-R and alphaC-MC5-R, detected the p77. The p77 was treated with protein endoglycosidase F to produce a smaller protein band between 34-38,000 (p35); the inferred size is 37,000 based on the cDNA sequence. The data suggest that Asn-linked carbohydrate groups account for much of the p77 mass of the MC5-R. The data also demonstrate the expression of MC5-R protein on rat lymphocytes, thus, supporting the hypothesis that MC5-R is the ACTH receptor on lymphocytes.
从分离出的大鼠淋巴细胞中对促黑素皮质素-5受体（MC5-R）的mRNA和蛋白质表达进行了表征。通过逆转录聚合酶链反应（RT-PCR）证明了脾脏和胸腺组织中存在MC5-R mRNA。对RT-PCR产物进行测序以确认MC5-R的鉴定。将来自泪腺、脂肪、肾上腺、胸腺、胰腺和分离出的脾淋巴细胞的组织用去污剂溶解。粗蛋白通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离，转印到硝酸纤维素膜上，并用抗受体兔抗血清检测MC5-R。针对代表MC5-R氨基端（αN-MC5-R）和羧基端（αC-MC5-R）表位的合成肽制备了两种不同类型的多克隆兔抗血清。除胰腺外，在所有组织中均检测到一条77000（p77）的明显条带。免疫前血清通过蛋白质免疫印迹分析未检测到p77，添加肽抗原可中和特异性抗血清对p77的检测。使用与αN-MC5-R复合的蛋白A琼脂糖从脾脏和胸腺淋巴细胞中纯化受体蛋白。使用αC-MC5-R通过蛋白质免疫印迹分析检测纯化的MC5-R。两种抗受体抗血清αN-MC5-R和αC-MC5-R均检测到了p77。用蛋白内切糖苷酶F处理p77，产生一条34000 - 38000（p35）之间较小的蛋白条带；根据cDNA序列推断大小为37000。数据表明，N-连接的碳水化合物基团占MC5-R的p77质量的大部分。数据还证明了MC5-R蛋白在大鼠淋巴细胞上的表达，因此，支持了MC5-R是淋巴细胞上促肾上腺皮质激素（ACTH）受体的假说。
