Endocrine Abstracts

Background: Numerous studies have reported the role of the N-terminal of pro-opiomelanocortin (N-POMC1-76) and its smaller fragments; 1-28 and 1-49, in adrenal steroidogenesis and mitogenesis. A full understanding of this area will help to understand the pathophysiology of certain adrenal tumours but exactly how these peptides elicit this effect is unclear. We have recently identified an orphan G protein-coupled receptor (GPCR) as a possible N-POMC receptor. Preliminary data showed that overexpression of this receptor leads to its accumulation inside the cell so we hypothesised that the accessory protein MRAP (melanocortin 2 receptor accessory protein) might be needed to translocate the receptor to the plasma membrane.

Objective: To investigate the binding affinity and specificity of this GPCR to N-POMC and if the co-expression of MRAP is required for functionality.

Methods: GPCR was overexpressed in HEK-293 cells with or without co-expression of MRAP. Ligand specificity was determined by non-radioactive ligand binding assay and association of GPCR and MRAP was determined by immunocytochemistry (ICC) and co-immunoprecipitation (co-IPs).

Results: ICC showed that co-expression of MRAP increases cell surface expression of the GPCR and co-IPs showed that the two proteins associate with each other. Non-radioactive ligand binding assay using biotinylated N-POMC1-28 as a ligand showed significant binding to cells expressing both MRAP and the GPCR while competitive binding assays showed other N-POMC fragments 1-49 and 1-77 could compete with N-POMC1-28 although N-POMC1-77 was found to be around 10-fold lower affinity than either N-POMC1-28 or 1-49.

Conclusions: These results support the hypothesis that the identified GPCR is the receptor for N-POMC and MRAP is required for full functionality. The identification of a receptor for N-POMC is significant and might be a potential target in adrenocortical carcinomas.