The interaction of MC3R and MC4R with MRAP2, ACTH, α-MSH and AgRP in chickens

Abstract

The interaction of melanocortin-4 (MC4R) and melanocortin-3 (MC3R) receptors with proopiomelanocortin (POMC)-derived peptides (e.g. α-MSH), agouti-related protein (AgRP) and melanocortin-2 receptor accessory protein 2 (MRAP2) is suggested to play critical roles in energy balance of vertebrates. However, evidence on their interaction in birds remains scarce. Our study aims to reveal their interaction in chickens and the results showed that (1) chicken (c-)MC3R and cMC4R expressed in Chinese hamster ovary (CHO) cells can be activated by α-MSH and ACTH equipotently, monitored by a pGL3-CRE-luciferase reporter system; (2) cMC3R and cMC4R, when co-expressed with cMRAP2 (or cMRAP, a cMRAP2 homolog), show increased sensitivity to ACTH treatment and thus likely act as ACTH-preferring receptors, and the interaction between cMC3R/cMC4R and cMRAP2 was demonstrated by co-immunoprecipitation assay; (3) both cMC3R and cMC4R display constitutive activity when expressed in CHO cells, as monitored by dual-luciferase reporter assay, and cMRAP2 (and cMRAP) can modulate their constitutive activity; (4) AgRP inhibits the constitutive activity of cMC3R/cMC4R, and it also antagonizes ACTH/α-MSH action on cMC4R/cMC3R, indicating that AgRP functions as the inverse agonist and antagonist for both receptors. These findings, together with the co-expression of ,, , and in chicken hypothalamus detected by quantitative real-time PCR, suggest that within the hypothalamus, α-MSH/ACTH, AgRP and MRAP2 may interact at the MC4R(/MC3R) interface to control energy balance. Furthermore, our data provide novel proof for the involvement of MRAP2 (and MRAP) in fine-tuning the constitutive activity and ligand sensitivity and selectivity of both MC3R and MC4R in vertebrates.

Pathological cyclic strain promotes proliferation of vascular smooth muscle cells via the ACTH/ERK/STAT3 pathway

“…Fridmanis ' s study reported that ACTH stimulation increases the expression of MC2R, but this phenomenon has always been considered to occur via ACTH activation of the cell membrane surface MC2R, which activates MC2R production through a series of signal pathways to increase its own expression level . Many studies have shown that the lack of MRAP‐trapped MC2R becomes the endoplasmic reticulum and impairs membrane localization, which may participate in the activation process of MC2R . In our study, MRAP expression was observed in VSMCs, as was an increase in MRAP dimerization and glycosylation levels after ACTH stimulation, but no localization changes in MRAP and MC2R were observed.…”

“…25 Many studies have shown that the lack of MRAP-trapped MC2R becomes the endoplasmic reticulum and impairs membrane localization, which may participate in the activation process of MC2R. 24 In our study, MRAP expression was observed in VSMCs, as was an increase in MRAP dimerization and glycosylation levels after ACTH stimulation, but no localization changes in MRAP and MC2R were observed. The above questions require further study.…”

“…In VSMCs, most MRAP and MC2R were concentrated in the cytoplasm. Related studies have shown that in the pituitary, MC2R is generally located at the cell membrane . The relationship between MC2R localization and function needs further investigation.…”

Pharmacological modulation of two melanocortin-5 receptors by MRAP2 proteins in zebrafish

“…In chickens, MC3R and MC4R can be activated by α-MSH or ACTH. But AgRP can antagonize α-MSH or ACTH actions on MC3R/MC4R and lower the constitutive activity of MC3R and MC4R (Zhang et al 2017). In mice, knockout of MC5R exhibit a severe dysfunction of exocrine secretion (Chen et al 1997).…”

“…MRAP2b can decrease the constitutive activity of MC4R during fasting which suggests that the deletion of MRAP2b may induce growth changes or obesity. As in zebrafish, MRAP2 can enhance the sensitivity of MC4R for ACTH and block the constitutive activity of MC4R in chickens (Zhang et al 2017). Unlike MC4R, the physiological functions between MC5Rs and MRAP2 proteins are largely unknown in zebrafish.…”