Pharmacological Evaluation of Melanocortin 2 Receptor Accessory Protein 2 on Axolotl Neural Melanocortin Signaling

Abstract

The Melanocortin-3 receptor (MC3R) and Melanocortin-4 receptor (MC4R), two members of the key hypothalamic neuropeptide signaling, function as complex mediators to control the central appetitive and energy homeostasis. The melanocortin 2 receptor accessory protein 2 (MRAP2) is well-known for its modulation on the trafficking and signaling of MC3R and MC4R in mammals. In this study, we cloned and elucidated the pharmacological profiles of MRAP2 on the regulation of central melanocortin signaling in a relatively primitive poikilotherm amphibian species, the Mexican axolotl (Ambystoma mexicanum). Our results showed the higher conservation of axolotl mc3r and mc4r across species than mrap2, especially the transmembrane regions in these proteins. Phylogenetic analysis indicated that the axolotl MC3R/MC4R clustered closer to their counterparts in the clawed frog, whereas MRAP2 fell in between the reptile and amphibian clade. We also identified a clear co-expression of mc3r, mc4r, and mrap2 along with pomc and agrp in the axolotl brain tissue. In the presence of MRAP2, the pharmacological stimulation of MC3R by α-MSH or ACTH significantly decreased. MRAP2 significantly decreased the cell surface expression of MC4R in a dose dependent manner. The co-localization and formation of the functional complex of axolotl MC3R/MC4R and MRAP2 on the plasma membrane were further confirmed in vitro. Dramatic changes of the expression levels of mc3r, mrap2, pomc, and agrp in the fasting axolotl hypothalamus indicated their critical roles in the metabolic regulation of feeding behavior and energy homeostasis in the poikilotherm aquatic amphibian.

Modulation of Canine Melanocortin-3 and -4 Receptors by Melanocortin-2 Receptor Accessory Protein 1 and 2

“…Xenopus MRAP1 increased α-MSH- and ACTH-induced cAMP signaling, and chicken MRAP1 did not affect agonist-induced signaling of MC3Rs [ 38 , 39 ]. Mrap2-decreased Mc3r signaling was also reported in channel catfish, topmouth culter (Mrap2a) [ 51 ], and Mexican axolotl [ 67 ], whereas MRAP2-increased MC3R signaling was observed in chicken and Xenopus MC3Rs [ 39 ]. Zebrafish Mrap2s did not affect agonist-induced signaling of MC3R [ 31 ].…”

“…In addition, our current results are consistent with mammalian MC3Rs in that MC3R has little or no basal cAMP signaling [ 62 , 63 , 64 , 65 ]. Of interest, high constitutive activities were present in several non-mammalian MC3Rs, including teleosts [ 49 , 52 , 66 ], amphibians (Mexican axolotl) [ 67 ], and avian (chicken) [ 38 , 68 ]. The amino acids accounting for the differences in the constitutive activity between these MC3Rs are not clear.…”

“…In other species, frog MRAP1 increased and chicken MRAP1 did not alter the surface expression of MC3Rs [ 38 , 39 ]. MRAP2 decreased the surface expression of clawed frog MC3R [ 39 ], increased the surface expression of topmouth culter Mc3r [ 52 ], and had no effect on the surface expression of Mexican axolotl and chicken MC3Rs [ 38 , 67 ]. For MC4R, hMRAP1a and hMRAP2a decreased [ 30 , 74 ] or increased [ 43 ] the cell surface expression of hMC4R.…”

“…MRAP1 had no effect on the surface expression of chicken MC3R [ 38 ] and increased frog MC3R expression [ 42 ]. MRAP2 has been reported to decrease the surface expression of tilapia and Mexican axolotl MC4Rs [ 67 , 75 ], increase the membrane expression of zebrafish (Mrap2b) [ 31 ], topmouth culter (Mrap2a and Mrap2b) [ 51 ], and Xenopus MC4Rs [ 39 ], or have no effect on the surface expression of chicken and snakehead MC4Rs [ 38 , 53 ]. Collectively, MRAP1 and MRAP2 modulate the MC3R/MC4R trafficking to the plasma membrane in a species- and receptor-specific manner.…”

Single transmembrane GPCR modulating proteins: neither single nor simple

“…On one hand, due to high evolutionary selection, the absence or duplicated homologs of melanocortin members occurs in certain species, such as lacking MC3R in tilapia while two MC5R isoforms and two MRAP2 paralogs exist in zebrafish ( Sebag et al, 2013 ; Zhu et al, 2018 ). Therefore, a group of laboratories further investigated the preservation of MRAP2 in controlling MC4R signaling from the evolutionary perspective ( Zhang et al, 2017 ; Rao et al, 2019 ; Zhu et al, 2019 ; Tai et al, 2021 ; Wen et al, 2021 ; Wang et al, 2022 d). The most ancient MRAP2 protein in the sea lamprey lacks the full C-terminus ( Zhu et al, 2019 ).…”