Expression of the melanocortin 5 receptor on rat lymphocytes

The expression of melanocortin-5 receptor (MC5-R) mRNA and protein was char acterized from isolated rat lymphocytes. The presence of MC5-R mRNA in sple en and thymus tissues was demonstrated by RT-PCR. The RT-PCR product was se quenced to confirm the identification of MC5-R. Tissues from lachrymal glan ds, adipose, adrenals, thymus, pancreas, and isolated splenic lymphocytes w ere detergent solubilized. The elude proteins were resolved by SDS-PAGE, tr ansblotted to a nitrocellulose membrane, and probed for MC5-R using anti-re ceptor rabbit antisera. Two different types of polyclonal rabbit antisera w ere raised against synthetic peptides representing epitopes found at the am ino (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 pancr eas. Preimmune sera did not detect p77 by Western analysis and the addition of peptide antigen neutralized the detection of p77 by the specific antise ra. The receptor protein was purified from spleen and thymic lymphocytes us ing protein A agarose that precipitated material complexed to alphaN-MC5-R. The purified MC5-R was detected by Western analysis using alphaC-MC5-R. Bo th 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 pr otein band between 34-38,000 (p35); the inferred size is 37,000 based on th e cDNA sequence. The data suggest that Asn-linked carbohydrate groups accou nt for much of the p77 mass of the MC5-R. The data also demonstrate the exp ression of MC5-R protein on rat lymphocytes, thus, supporting the hypothesi s that MC5-R is the ACTH receptor on lymphocytes. (C) 2001 Academic Press.