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Expression of the melanocortin 5 receptor on rat lymphocytes
S Akbulut1, C A Byersdorfer, C P Larsen
1Department of Medical Microbiology and Immunology, University of Minnesota-Duluth, Duluth, Minnesota, 55812, USA.
Abstract:
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.
Insights
Researchers identified melanocortin-5 receptor (MC5-R) mRNA and protein in rat lymphocytes. This supports the hypothesis that MC5-R acts as the ACTH receptor on these immune cells.
Area of Science:
- Immunology
- Endocrinology
- Molecular Biology
Background:
- The melanocortin-5 receptor (MC5-R) is involved in various physiological processes.
- Its presence and function on immune cells, particularly lymphocytes, are not fully understood.
- Investigating MC5-R expression on lymphocytes could elucidate novel signaling pathways in immune responses.
Purpose of the Study:
- To characterize the expression of melanocortin-5 receptor (MC5-R) mRNA and protein in isolated rat lymphocytes.
- To confirm the identity and localization of MC5-R within lymphocyte populations.
- To provide evidence supporting the role of MC5-R as the ACTH receptor on lymphocytes.
Main Methods:
- Reverse transcription-polymerase chain reaction (RT-PCR) to detect MC5-R mRNA in spleen and thymus tissues.
- Western blot analysis using specific polyclonal antisera against MC5-R epitopes to detect the protein in various tissues and isolated lymphocytes.
- Protein purification from spleen and thymic lymphocytes followed by characterization.
- Treatment with endoglycosidase F to analyze protein glycosylation.
Main Results:
- MC5-R mRNA was detected in rat spleen and thymus tissues.
- A prominent MC5-R protein band (p77) was identified in spleen, thymus, lachrymal glands, adipose, and adrenal tissues, but not in the pancreas.
- The p77 protein was confirmed to be MC5-R through antibody specificity and peptide inhibition assays.
- Purified MC5-R from lymphocytes was detected by specific antisera, and deglycosylation yielded a smaller protein (p35), indicating N-linked glycosylation.
Conclusions:
- The study confirms the expression of MC5-R mRNA and protein in rat lymphocytes.
- The identified MC5-R protein (p77) is glycosylated, with a core protein size of approximately 37 kDa.
- These findings support the hypothesis that MC5-R functions as the ACTH receptor on rat lymphocytes, suggesting a role in immune cell regulation.