The melanocortin receptor (MCR) family consists of five G-protein-coupled receptors (MC1R–MC5R) with diverse physiological roles. MC1R controls pigmentation, MC2R is a critical component of the hypothalamic–pituitary–adrenal axis, MC3R and MC4R have a vital role in energy homeostasis and MC5R is involved in exocrine function. The melanocortin receptor accessory protein (MRAP) and its paralogue MRAP2 are small single-pass transmembrane proteins that have been shown to regulate MCR expression and function. In the adrenal gland, MRAP is an essential accessory factor for the functional expression of the MC2R/ACTH receptor. The importance of MRAP in adrenal gland physiology is demonstrated by the clinical condition familial glucocorticoid deficiency, where inactivating MRAP mutations account for ∼20% of cases. MRAP is highly expressed in both the zona fasciculata and the undifferentiated zone. Expression in the undifferentiated zone suggests that MRAP could also be important in adrenal cell differentiation and/or maintenance. In contrast, the role of adrenal MRAP2, which is highly expressed in the foetal gland, is unclear. The expression of MRAPs outside the adrenal gland is suggestive of a wider physiological purpose, beyond MC2R-mediated adrenal steroidogenesis. In vitro, MRAPs have been shown to reduce surface expression and signalling of all the other MCRs (MC1,3,4,5R). MRAP2 is predominantly expressed in the hypothalamus, a site that also expresses a high level of MC3R and MC4R. This raises the intriguing possibility of a CNS role for the MRAPs.
T V Novoselova, D Jackson, D C Campbell, A J L Clark and L F Chan
R J Lacey, S L F Chan, H C Cable, R F L James, C W Perret, J H B Scarpello and N G Morgan
Sequences from cDNA molecules encoding α2-adrenoceptor subtype genes were subcloned into prokaryotic vectors and riboprobes generated to hybridise selectively with each of the human α2C2-, α2C4- and α2C10-adrenoceptor subtype mRNA species. The riboprobes were labelled with either 32P or digoxigenin and used to study the expression of α2-adrenoceptor subtypes in sections of human pancreas, in isolated human islets of Langerhans and in clonal HIT-T15 pancreatic β-cells. Using a ribonuclease protection assay protocol, expression of mRNA species encoding both α2C2 and α2C10 was demonstrated in preparations of isolated human islets of Langerhans. mRNA encoding α2C4 was also detected in human islet RNA, using reverse transcription coupled with the polymerase chain reaction. In situ hybridisation was then employed to examine the distribution of each α2-adrenoceptor subtype in sections of human pancreas. All three subtypes of α2-adrenoceptor mRNA were identified in sections of formalin-fixed, paraffinembedded human pancreas using riboprobes labelled with digoxigenin. Although some labelling of the three α2-adrenoceptor mRNA subtypes was seen in the islets, the labelling was most intense in the exocrine tissue of the pancreas for each receptor subtype. The specificity of the digoxigenin-labelled RNA probes was confirmed in several control tissues and by in situ hybridisation studies using sense probes in the pancreas. The integrity of the pancreas sections was confirmed by in situ hybridisation with an antisense riboprobe derived from human insulin cDNA. The results demonstrate that multiple α2-adrenoceptor subtypes are expressed in human pancreas. Both the exocrine and endocrine cells express more than one receptor subtype, although the islets stain less intensely than the bulk of the tissue suggesting that the islet cells may have lower levels of expression than the acinar tissue. The presence of α2-adrenoceptor subtype mRNA species in pancreatic β-cells was confirmed by Northern blotting of RNA extracted from the clonal β-cell line, HIT-T15. Transcripts encoding each of the three cloned α2-adrenoceptor subtypes were detected in HIT-T15 cells.
Hybridisation of sections of human pancreas with oligodeoxynucleotide probes designed to hybridise with β2-adrenoceptor mRNA revealed expression of this species in islet β-cells but not in the exocrine tissue of the pancreas.
Journal of Endocrinology (1996) 148, 531–543
T V Novoselova, R Larder, D Rimmington, C Lelliott, E H Wynn, R J Gorrigan, P H Tate, L Guasti, The Sanger Mouse Genetics Project, S O’Rahilly, A J L Clark, D W Logan, A P Coll and L F Chan
Melanocortin receptor accessory protein 2 (MRAP2) is a transmembrane accessory protein predominantly expressed in the brain. Both global and brain-specific deletion of Mrap2 in mice results in severe obesity. Loss-of-function MRAP2 mutations have also been associated with obesity in humans. Although MRAP2 has been shown to interact with MC4R, a G protein-coupled receptor with an established role in energy homeostasis, appetite regulation and lipid metabolism, the mechanisms through which loss of MRAP2 causes obesity remains uncertain. In this study, we used two independently derived lines of Mrap2 deficient mice (Mrap2 tm1a/tm1a) to further study the role of Mrap2 in the regulation of energy balance and peripheral lipid metabolism. Mrap2 tm1a/tm1a mice have a significant increase in body weight, with increased fat and lean mass, but without detectable changes in food intake or energy expenditure. Transcriptomic analysis showed significantly decreased expression of Sim1, Trh, Oxt and Crh within the hypothalamic paraventricular nucleus of Mrap2 tm1a/tm1a mice. Circulating levels of both high-density lipoprotein and low-density lipoprotein were significantly increased in Mrap2 deficient mice. Taken together, these data corroborate the role of MRAP2 in metabolic regulation and indicate that, at least in part, this may be due to defective central melanocortin signalling.