Identification of novel GPCR partners of the central melanocortin signaling

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.
Highlights • More GPCRs are found in the neurons than other cell types in the mouse hypothalamus, and their functions are significantly enriched in energy homeostasis and metabolism. • A total of 23 and 32 GPCRs are confirmed to be novel interacting partners of MC3R and MC4R in vitro, respectively. • The hetero-dimerization of identified GPCRs with MC3R and MC4R alters the signal transduction and surface translocation.