Abstract/Summary

Over the last 40 years, numerous studies have reported the role of the N-terminal of proopiomelanocortin (N-POMC) in adrenal steroidogenesis and growth; however, little is known about the cell surface receptor that it activates. Identifying this receptor will help to understand the pathophysiology of certain adrenal disorders, such as familial glucocorticoid deficiency (FDG) and adrenal carcinomas. Recent data from the Bicknell lab identified the orphan G protein-coupled receptor 19 (GPR19) as a possible N-POMC receptor in adrenal cells. Preliminary data showed that overexpression of GPR19 leads to the accumulation of the protein inside the cell, so we proposed that it might require the co-expression of the melanocortin 2 receptor accessory protein (MRAP) to be translocated to the plasma membrane. In this study, we generated three stably transformed cell lines in HEK-293: the first overexpresses GPR19, the second expresses MRAP, and the third overexpresses GPR19 and MRAP. The gene expression of GPR19 and MRAP was confirmed by RT-PCR, whilst the protein expression was confirmed by immunocytochemistry (ICC) and immunoblotting. ICC showed that in the cell line that expresses MRAP the fluorescent staining of GPR19 was greater on the plasma membrane in comparison to the cell lines that do not express it. ICC images of cells expressing GPR19/MRAP revealed that both are localised on the cell membrane and inside the cells. Co-IP results showed that GPR19 and MRAP precipitate together, and an N-POMC1-28 antibody could precipitate the GPR19 cross-linked to N-POMC1-28. Moreover, cells treated with N-POMC1-28, either unlabelled or labelled with Alexa-Fluor 488 or biotin, showed a significant increase in signal intensity in the cell lines that overexpress GPR19 and/or MRAP in comparison to the wild-type. In competitive binding assays, the N-POMC fragments 1-49 and 1-77 were shown to compete with N-POMC1-28. In addition, N-POMC1-28 activates GPR19, and that leads to the reduction of cAMP levels in the cells, the increase of ERK1/2 phosphorylation and the activation of the downstream transcription factors, which consequently stimulate cell proliferation. These results support the hypothesis that GPR19 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.