Functional characterization of nine novel naturally occurring human melanocortin-3 receptor mutations

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The melanocortin-4 receptor (MC4R) is a critical regulator of energy homeostasis and has emerged as a premier target for obesity treatment. Numerous mutations in transmembrane domain 6 (TM6) of MC4R resulting in functional alterations have been identified in obese patients. Several mutagenesis studies also provided some data suggesting the importance of this domain in receptor function. To gain a better understanding of the structure-function relationship of the receptor, we performed alanine-scanning mutagenesis in TM6 to determine the functions of side chains. Of the 31 residues, two were important for cell surface expression, five were indispensable for a-melanocyte-stimulating hormone (a-MSH) and b-MSH binding, and six were important for signaling in the Gs-cAMP-PKA pathway. H264A, targeted normally to the plasma membrane, was undetectable by competitive binding assay and severely defective in basal and stimulated cAMP production and ERK1/2 phosphorylation. Nine mutants had decreased basal cAMP signaling. Seven mutants were constitutively active in cAMP signaling and their basal activities could be inhibited by two MC4R inverse agonists, Ipsen 5i and ML00253764. Five mutants were also constitutively active in the MAPK pathway with enhanced basal ERK1/2 phosphorylation. In summary, our study provided comprehensive data on the structure-function relationship of the TM6 of MC4R. We identified residues that are important for cell surface expression, ligand binding, cAMP generation, and residues for maintaining the WT receptor in active conformation. We also reported constitutive activation of the MAPK pathway and biased signaling. These data will be useful for rationally designing MC4R agonists and antagonists for treatment of eating disorders.

Section: Discussionmentioning

confidence: 90%

Pleiotropic functions of the transmembrane domain 6 of human melanocortin-4 receptor

Huang¹,

Tao²

2012

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“…After blocking with PBS-IH containing 5% BSA for 1h, the cells were then incubated with the primary antibody anti-HA.11 (Covance, Princeton, NJ, USA), which was diluted 1:100 in PBS-IH containing 5% BSA, for another 1 h. The cells were washed once with 0.5% BSA in PBS-IH and then incubated with the secondary antibody Alexa Fluor 488-labeled goat anti-mouse IgG (Invitrogen) for 1 h. The cell surface expression of WT and mutant hMC3Rs was analyzed by Accuri flow cytometer (Accuri Cytometers, Ann Arbor, MI, USA). The expression levels of the mutant hMC3Rs were calculated as the percentage of WT hMC3R expression using the formula: [mutant - pcDNA3.1] / [WT - pcDNA3.1] ×100%, where the pcDNA3.1 were used as control for background staining ( Wang et al 2008 ; Yang and Tao 2012 ).…”

Section: Methodsmentioning

confidence: 99%

“…The MC4R plays an undisputed role in human obesity pathogenesis since mutations in MC4R have been characterized as the most common monogenic form of obesity in human ( Farooqi et al 2003 ; Hinney et al 2013; Tao 2009 ). However, the role of MC3R in human obesity pathogenesis is more controversial (reviewed in ( Tao 2010b )), although some MC3R mutations (such as I183N and I335S) have been recognized as possible genetic contributors for morbid obesity ( Lee et al 2007 ; Lee et al 2002 ; Mencarelli et al 2008 ; Rached et al 2004 ; Tao 2007 ; Tao and Segaloff 2004 ; Yang et al 2015 ; Yang and Tao 2012 ).…”

Section: Introductionmentioning

confidence: 99%

Functions of DPLIY motif and helix 8 of human melanocortin-3 receptor

Zhao

Zhili²,

Tao

2015

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The melanocortin-3 receptor (MC3R) is a member of family A G protein-coupled receptors (GPCRs). The MC3R remains the most enigmatic of the melanocortin receptors with regard to its physiological functions, especially the role in energy homeostasis. The N/DPxxY motif and the eighth helix (helix 8) in the carboxyl terminus of GPCRs have been identified to be important for receptor expression, ligand binding, signal transduction and internalization. To gain a better understanding of the structure-function relationship of MC3R, we performed systematic study of all the 20 residues in this domain using alanine-scanning mutagenesis. We showed that although all mutants were expressed normally on cell surface, eleven residues were important for ligand binding and one was indispensable for downstream cAMP generation. F347A showed constitutive activity in cAMP signaling while all the other mutants had normal basal activities. We studied the signaling capacity of nine mutants in the ERK1/2 signaling pathway. All of these mutants showed normal basal ERK1/2 phosphorylation levels. The pERK1/2 levels of six binding- or signaling-defective mutants were enhanced upon agonist stimulation. The unbalanced cAMP and pERK1/2 signaling pathways suggested the existence of biased signaling in MC3R mutants. In summary, we showed that the DPLIY motif and Helix 8 was important for MC3R activation and signal transduction. Our data led to a better understanding of the structure-function relationship of MC3R.

“…Decreased total receptor expression has been reported previously for some missense GPCR mutations including the MC4R (Fan & Tao 2009, Wang & Tao 2011, Yang & Tao 2012, Zhang et al 2012, even for some synonymous mutations (Duan et al 2003, Bartoszewski et al 2010. We therefore further studied whether the decreased total receptor expression was due to accelerated protein degradation (Yang et al 2011).…”

Section: Discussionmentioning

confidence: 99%

Functions of transmembrane domain 3 of human melanocortin-4 receptor

Mo¹,

Yang²,

Tao³

2012

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The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor critical for maintaining energy homeostasis. Transmembrane domain 3 (TM3) of MC4R contains residues that were suggested to be essential in ligand binding and signaling. Several MC4R mutations in TM3 are associated with human obesity. To gain a better understanding of the functions of TM3, we analyzed the functions of 26 residues in TM3 using alanine-scanning mutagenesis. We showed that all mutants had normal cell-surface expression. Four mutants were defective in ligand binding and signaling and six mutants had normal ligand binding but impaired cAMP production. L140A had increased basal cAMP level. To further characterize the function of L140, we generated 17 additional L140 mutants. Fifteen L140 mutants had significantly decreased cell-surface expression, with L140R and L140V expressed normally. Ten L140 mutants had increased basal cAMP activities. Four L140 mutants were defective in ligand-stimulated cAMP generation. Interestingly, with the ERK1/2 pathway, we showed that nine constitutively active mutants had similar levels of basal pERK1/2 as that of WT, and two signaling defective mutants had similar levels of pERK1/2 as that of WT upon agonist stimulation, different from their cAMP signaling properties, suggesting biased signaling in these mutant receptors. In summary, we identified 13 residues in TM3 that were essential for ligand binding and/or signaling. Moreover, L140 was critical for locking MC4R in inactive conformation and several mutants showed biased signaling in cAMP and ERK1/2 signaling pathways.