Expression of the melanocortin 5 receptor on rat lymphocytes

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 (αN-MC5-R) and the carboxyl termini (αC-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 αN-MC5-R. The purified MC5-R was detected by Western analysis using αC-MC5-R. Both anti-receptor antisera, αN-MC5-R and αC-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.