Differential regulation of cAMP‐mediated gene transcription and ligand selectivity by MC3R and MC4R melanocortin receptors

Abstract

Melanocortins are known to be involved in the regulation of feeding behavior. These hormones mediate their effects through G-protein-coupled receptors by stimulating adenylate cyclase. In this study we describe the functional response of melanocortin 4 receptor (MC4R) and melanocortin 3 receptor (MC3R) in HEK 293T cells, by using a luciferase reporter gene under the transcriptional control of a cAMP-responsive element (CRE) as a monitor of intracellular cAMP levels and cAMP-regulated gene expression. We were able to show that MC4R and MC3R expressed in the human cell line HEK 293T stimulate transcription induced by stimulation with different analogs of a-melanocyte-stimulating hormone (a-MSH) at different levels. In our assay of CRE-mediated gene transcription activity, a-MSH-ND was the most efficient a-MSH analog for MC4R whereas NDP-MSH was the most efficient for MC3R. Changing the His6 residue of a-MSH-ND to Gln or Lys markedly decreased CRE-mediated luciferase activity for MC3R compared with MC4R. On analysis by modeling the receptor±ligand complex by NMR, [Gln6]a-MSH-ND and [Lys6]a-MSH-ND showed different conformational interactions between MC3R and MC4R. Furthermore, the maximum coupling efficiency of MC4R and MC3R to G proteins was different; MC4R showed only 30±50% of the maximum activity induced by MC3R. In total, our results suggest that a differential receptor±ligand interaction is involved and that the relative interactions of MC3R and MC4R with G protein are possibly quantitatively and qualitatively different.Keywords: cAMP; G protein; melanocortin; molecular modeling; obesity.Melanocortins are peptide hormones derived through a series of proteolytic cleavages from the precursor peptide pro-opiomelanocortin [1]. They are known to have a broad spectrum of physiological actions including regulation of melanocyte pigmentation [2], thermoregulation [3], regulation of obesity [4], control of the cardiovascular system [5] and learning and memory [6], as well as immunomodulatory effects [7]. These hormones mediate their effects through G-protein-coupled receptors by stimulating adenylate cyclase [8]. To date, five melanocortin receptor subtypes with different patterns of tissue expression in brain and the peripheral body have been cloned and characterized [8±12].It has been reported that activation of melanocortin 4 receptor (MC4R) by a-melanocyte-stimulating hormone (a-MSH) increases energy expenditure and decreases food intake; genetic disruption of MC4R also caused obesity in mice [13]. Thus, a-MSH derived from the arcuate nucleus pro-opiomelanocortin neurons, the primary source of ligands for MC4R, appears to play an inhibitory role in feeding and energy storage. Therefore, MC4R-selective agonists have been considered as potential candidates for the treatment of abnormal eating behavior including obesity and anorexia. MC3R is also abundantly expressed in the brain and peripheral tissues such as the placenta, gut tissues and the human heart [14]. Recent experiments in MC3R-null mice indicate that inacti...

Peripheral Effect of α-Melanocyte-stimulating Hormone on Fatty Acid Oxidation in Skeletal Muscle

“…Unexpectedly, [Glu 6 ]␣-MSH-ND, which has ample potency for activation of both MC3R and MC4R, revealed very weak potency for the activation of FAO in C2C12 cells (only at 10 Ϫ5 M, p Ͻ 0.05) (Fig. 3A) (38). These data suggest that neither MC3R nor MC4R is functioning in ␣-MSH induction of FAO in skeletal muscle.…”

Agouti-Related Peptide and MC3/4 Receptor Agonists Both Inhibit Excitatory Hypothalamic Ventromedial Nucleus Neurons

“…MC3R is thought to play a complementary role to MC4R by regulating feeding efficiency and partitioning of nutri- ents into fat (Chen et al, 2000a). It has also been suggested that MC3/4R show different receptor-ligand interactions (Lee et al, 2001) and may couple to differential downstream signaling systems (Konda et al, 1994). To study whether MC3/4R activation shows different effects in vGluT2 VMH neurons, selective MC4R and MC3R agonists were used.…”

“…12 A-C). Because AgRP did not act via MC3/4 receptors, which are coupled to G s -and/or G q -proteins (Konda et al, 1994;Lee et al, 2001;Srinivasan et al, 2003), we investigated the possibility that AgRP might act through the G i/o -protein pathway. The G i/o -protein antagonists PTX and NF023 were used Freissmuth et al, 1996).…”

“…Both MC4R and MC3R agonists showed consistent inhibitory actions in all cells tested. MC3R and MC4R show different receptor-ligand interaction (Lee et al, 2001) and may couple to different downstream signaling systems (Konda et al, 1994). Figure 11.…”

Lack of cAMP-response Element-binding Protein 1 in the Hypothalamus Causes Obesity

“…The MC4R is known to signal through G s ␣, which in turn activates adenylyl cyclase, leading to increased intracellular levels of cAMP (1,3,18). Elevated cAMP levels induce phosphorylation and activation of the transcription factor cAMP-response element-binding protein (CREB1) through increased activity of protein kinase A, or PKA (19 -22).…”