Molecular and Cellular Endocrinology 320 (2010) 145–152 Contents lists available at ScienceDirect Molecular and Cellular Endocrinology journal homepage: www.elsevier.com/locate/mce Role of melanocortin receptor accessory proteins in the function of zebrafish melanocortin receptor type 2 Maria Josep Agulleiro a,1 , Simon Roy b,1 , Elisa Sánchez a , Sara Puchol a , Nicole Gallo-Payet b,2 , José Miguel Cerdá-Reverter a,∗,2 a Department of Fish Physiology and Biotechnology, Instituto de Acuicultura de Torre de la Sal, Consejo Superior de Investigaciones Científicas, Ribera de Cabanes, Castellón, Spain b Service of Endocrinology, Department of Medicine, University of Sherbrooke, Sherbrooke, Québec, Canada article info Article history: Received 13 November 2009 Received in revised form 8 January 2010 Accepted 23 January 2010 Keywords: ACTH MRAP MC2R HPA-axis Stress Fish abstract In this paper, we identify three different MRAPs in zebrafish, zfMRAP1, zfMRAP2a and zfMRAP2b, and demonstrate that zfMC2R is not functional in the absence of MRAP expression. ZfMRAP1 expression was restricted to adipose tissue and the anterior kidney whereas MRAP2a and MRAP2b were expressed in all the tissues tested. Quantification of surface receptor and immunofluorescence studies indicated that the receptor is unable to translocate to membrane in the absence of MRAP isoforms. MRAP1 and MRAP2b are localized in the plasma membrane in the absence of zfMC2R expression but MRAP2b is retained in perinuclear position. MRAP1 and MRAP2a displayed an equivalent translocation capacity to the mem- brane of zfMC2R but only zfMRAP1 expression led to intracellular cAMP increases after ACTH stimulation. ZfMRAP2b had no effect on zfMC2R activity but both zfMRAP2 isoforms enhanced the zfMRAP1-assisted cAMP intracellular increase, suggesting an interaction between zfMRAP1 and zfMRAP2s when regulating zfMC2R activity. © 2010 Elsevier Ireland Ltd. All rights reserved. 1. Introduction Melanocortin 2 receptor (MC2R) is the smallest member of a G protein-coupled receptor subfamily, which includes five recep- tors (MC1R–MC5R). These receptors are involved in the regulation of diverse physiological functions, including skin pigmentation, stress response, metabolism, energy balance and exocrine secretion (Cone, 2006). MC2R is adrenocorticotropic hormone (ACTH)- specific, whereas the other four MCRs distinctively recognize MSHs (Schiöth et al., 2005). ACTH is posttranscriptionally processed from proopiomelanocortin (POMC) after tissue-specific cleavage in the corticotrophs of the anterior pituitary (Castro and Morrison, 1997), and secreted in response to hypothalamic corticotropin-releasing factor (CRF). ACTH binding to MC2R increases intracellular cAMP, thus stimulating steroidogenesis and protein synthesis in the adrenal gland (Sewer and Waterman, 2003). The expression of the MC2R mRNA is mainly restricted to the adrenal cortex and adi- pose tissue (Mountjoy et al., 1992; Boston and Cone, 1996) and ∗ Corresponding author at: Department of Fish Reproductive Physiology, Instituto de Acuicultura de Torre de la Sal, 12595 Torre de la Sal, Ribera de Cabanes, Castellón, Spain. Tel.: +34 964319500; fax: +34 964319509. E-mail addresses: cerdarev@iats.csic.es, cerdarev@iats.csic.es (J.M. Cerdá-Reverter). 1 Both authors have contributed equally to this study. 2 Co-senior authors. is a key point in the peripheral response to stress (Dallman et al., 2004). Unlike in the case of other melanocortin receptors, the study of MC2R activation has been constrained because of the lack of a readily transfectable heterologous expression system. Suitable expression data have been obtained only in adrenocortical-derived cell lines (Y6 or OS3), in which endogenous MCR expression is absent (Schimmer et al., 1995). The expression experiments of MC2R in a range of non-adrenal cells suggested a common expla- nation for failed functional expression in that the receptor cannot reach plasma membrane and is retarded in the endoplasmic reticu- lum. This explanation involved the presence of an accessory factor that works as an MC2R-specific transport system in the adrenocor- tical cells (Noon et al., 2002). One gene candidate that encodes for a small single-transmembrane domain protein with high expression in the adrenal cortex was identified in humans, showing famil- ial glucocorticoid deficiency (FGD) and no MC2R mutations. This syndrome is characterized by resistance to ACTH, i.e. high plasma ACTH levels but lacking adrenal glucocorticoids. The protein is now known as melanocortin 2 receptor accessory protein (MRAP), even though alternative splicing of the last two exons gives rise to two isoforms that differ in the C-terminal region (MRAP and MRAP) (Metherell et al., 2005). Knockdown of endogenous mouse MRAP in Y1 adrenocortical cells, which express a functional endogenous MC2R, leads to insensitivity to ACTH, demonstrating that MRAP is essential for producing an ACTH responsive MC2R (Cooray et al., 2008). MRAP interacts with the MC2R to facilitate correct folding, 0303-7207/$ – see front matter © 2010 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2010.01.032