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Abstract Background The melanocortin receptor accessory proteins (MRAP1 and MRAP2) are well‐known endocrine regulators for the trafficking and signalling of all five melanocortin receptors (MC1R–MC5R). The observation of MRAP2 on regulating several non‐melanocortin G protein‐coupled receptors (GPCRs) has been sporadically reported, whereas other endogenous GPCR partners of the MRAP protein family are largely unknown. Methods Here, we performed single‐cell transcriptome analysis and drew a fine GPCR blueprint and MRAPs‐associated network of two major endocrine organs, the hypothalamus and adrenal gland at single‐cell resolution. We also integrated multiple bulk RNA‐seq profiles and single‐cell datasets of human and mouse tissues, and narrowed down a list of 48 GPCRs with strong endogenous co‐expression correlation with MRAPs. Results 36 and 46 metabolic‐related GPCRs were consequently identified as novel interacting partners of MRAP1 or MRAP2, respectively. MRAPs exhibited protein–protein interactions and varying pharmacological properties on the surface translocation, constitutive activities and ligand‐stimulated downstream signalling of these GPCRs. Knockdown of MRAP2 expression by hypothalamic administration of adeno‐associated virus (AAV) packed shRNA stimulated body weight gain in mouse model. Co‐injection of corticotropinreleasing factor (CRF), the agonist of corticotropin releasing hormone receptor 1 (CRHR1), suppressed feeding behaviour in a MRAP2‐dependent manner. Conclusions Collectively, our study has comprehensively elucidated the complex GPCR networks in two major endocrine organs and redefined the MRAP protein family as broad‐spectrum GPCR modulators. MRAP proteins not only serve as a vital endocrine pivot on the regulation of global GPCR activities in vivo that could explain the composite physiological phenotypes of the MRAP2 null murine model but also provide us with new insights of the phenotyping investigation of GPCR–MRAP functional complexes.

Objective: Homo- or heterodimerization of G protein–coupled receptors (GPCRs) generally alters the normal functioning of these receptors and mediates their responses to a variety of physiological stimuli in vivo. It is well known that melanocortin-3 receptor (MC3R) and melanocortin-4 receptor (MC4R) are key regulators of appetite and energy homeostasis in the central nervous system (CNS). However, the GPCR partners of MC3R and MC4R are not well understood. Our objective is to analyze single cell RNA-seq datasets of the hypothalamus to explore and identify novel GPCR partners of MC3R and MC4R and examine the pharmacological effect on the downstream signal transduction and membrane translocation of melanocortin receptors. Methods: We conducted an integrative analysis of multiple single cell RNA-seq datasets to reveal the expression pattern and correlation of GPCR families in the mouse hypothalamus. The emerging GPCRs with important metabolic functions were selected for cloning and co-immunoprecipitation validation. The positive GPCR partners were then tested for the pharmacological activation, competitive binding assay and surface translocation ELISA experiments. Results: Based on the expression pattern of GPCRs and their function enrichment results, we narrowed down the range of potential GPCR interaction with MC3R and MC4R for further confirmation. Co-immunoprecipitation assay verified 23 and 32 novel GPCR partners that interacted with MC3R and MC4R in vitro. The presence of these GPCR partners exhibited different effects in the physiological regulation and signal transduction of MC3R and MC4R. Conclusions: This work represented the first large-scale screen for the functional GPCR complex of central melanocortin receptors and defined a composite metabolic regulatory GPCR network of the hypothalamic nucleuses.