Melanocortin-4 Receptor in Spotted Sea Bass, Lateolabrax maculatus: Cloning, Tissue Distribution, Physiology, and Pharmacology

Abstract

Melanocortin-4 receptor (MC4R) plays important roles in regulation of multiple physiological processes including energy homeostasis, reproduction, sexual function, and other functions in mammals. Recent studies suggested that teleost MC4Rs have different physiological functions and pharmacological characteristics when compared to mammalian MC4Rs. In this study, we investigated spotted sea bass (Lateolabrax maculatus) MC4R (LmMC4R) physiology and pharmacology. Spotted sea bass mc4r consisted of a 984 bp open reading frame encoding a protein of 327 amino acids. LmMC4R was homologous to those of several teleost MC4Rs and human MC4R (hMC4R). qRT-PCR and in situ hybridization revealed that mc4r transcripts were highly expressed in the brain, followed by pituitary and liver. Brain mc4r transcripts were down-regulated in long-term and short-term fasting challenges. LmMC4R was a functional receptor with lower maximal binding and higher basal activity than hMC4R. THIQ was not able to displace 125I-NDP-MSH but could affect intracellular cAMP accumulation, suggesting that it was an allosteric ligand for LmMC4R. In vitro studies with spotted sea bass brain cells indicated that mRNA levels of neuropeptide Y and Agouti-related peptide were down-regulated by α-MSH. In summary, we cloned spotted sea bass MC4R, and showed that it had different pharmacological properties compared to hMC4R, and potentially different functions.

Regulation of Melanocortin-4 Receptor Pharmacology by Two Isoforms of Melanocortin Receptor Accessory Protein 2 in Topmouth Culter (Culter alburnus)

“…Our results showed that NDP-MSH bound to caMC4R with the highest affinity (IC 50 of 4.87 nM) and activated caMC4R with the highest potency (EC 50 was 0.44 nM) (Tables 1, 2). Lower binding capacity was observed in caMC4R (about 35% of that of hMC4R) (Figure 4 and Table 1), consistent with previous studies in spotted scat (27), grass carp (28), swamp eel (29), sea bass (31), and orange-spotted grouper (30). In culter, ACTH had higher affinity and was more efficacious than α-MSH for caMC4R, consistent with the suggestion that ACTH may be the original ligand for the MCRs (57).…”

“…In the present study, we demonstrated that cloned culter mc4r was predicted to encode a protein of 326 amino acids with similar structural characteristics as MC4Rs of other species (Figure 1A and Supplementary Figure 2), including other teleost MC4Rs (27)(28)(29)(30)(31)37). Phylogenetic analysis revealed that caMC4R clustered with teleost MC4Rs (Figure 1B).…”

“…Mutations leading to decreased constitutive activity are associated with obesity pathogenesis ( 6 , 59 , 60 ). Compared to hMC4R, teleost MC4Rs showed much higher constitutive activity in cAMP signaling ( 27 – 31 ). Our present study also showed that caMC4R significantly increased basal activities in Gs-cAMP and ERK1/2 signaling ( Figure 6 ).…”

“…The influence of caMRAP2a or caMRAP2b on the cell surface and total expression levels of caMC4R was studied using flow cytometry as described earlier ( 31 ). Cells were plated into 6-well plates and transfected with caMC4R (N-terminal c-myc tag) and caMRAP2a or caMRAP2b plasmids in four ratios (1:0, 1:1, 1:3, and 1:5).…”

“…MC4R has also been characterized in teleosts (21,(24)(25)(26)(27)(28)(29)(30)(31)(32), and shown to act as a regulator in energy balance, sexual behavior, and reproduction (33)(34)(35)(36)(37). Non-mammalian MRAPs are different from those of mammals.…”