Functions of the DRY motif and intracellular loop 2 of human melanocortin 3 receptor

Abstract

The melanocortin 3 receptor (MC3R) regulates several physiological functions, including feed efficiency, nutrient partitioning, fasting response, natriuresis, and immune reactions. Naturally occurring mutations in the MC3R gene have been shown to be associated with increased adiposity and lung diseases such as tuberculosis and cystic fibrosis. The DRY motif at the cytoplasmic end of transmembrane domain 3 (TM3) and the second intracellular loop 2 (ICL2) are known to be important for receptor function in several G protein-coupled receptors (GPCRs). To gain a better understanding of the functions of this domain in MC3R, we performed alanine-scanning mutagenesis on 18 residues. We showed that alanine mutation of 11 residues reduced the maximal binding and maximal cAMP production stimulated by agonists. Mutation of two residues did not change maximal binding but resulted in impaired signaling in the G s -cAMP pathway. Mutation of five residues impaired signaling in the ERK1/2 pathway. We have also shown that alanine mutants of seven residues that were defective in the cAMP pathway were not defective in the ERK1/2 pathway, demonstrating biased signaling. In summary, we demonstrated that the cytoplasmic end of TM3 and the ICL2 were critical for MC3R function. We also reported for the first time biased signaling in MC3R. Key Words" melanocortin 3 receptor " intracellular loop 2 " DRY motif " MAP kinase " biased signaling

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

“…Receptor retention or mislocalization, which has previously been described as the major defect for human disease caused by GPCR mutations ( Tao 2006 ; Tao and Conn 2014 ), was not observed in present study. Similarly, in our studies on the second and third intracellular loops, we also showed that the alanine mutants are expressed normally at the cell surface ( Huang and Tao 2014 ; Wang and Tao 2013 ).…”

“…Unbalanced cAMP and ERK1/2 signaling was reported in some human MC4R mutants ( He and Tao 2014 ; Huang and Tao 2012 ; Mo et al 2012 ) (reviewed in ( Tao 2014 )). In our recent study of the DRY motif and intracellular loop 2 of hMC3R, the presence of this biased signaling was also suggested ( Huang and Tao 2014 ). To test it further, in the current study, the ERK1/2 phosphorylation levels of nine hMC3R mutants, including eight binding- or signaling-defective mutants (P333A, I335A, Y336A, E342A, L343A, R344A, I350A, and L351A) which led to less than 20% cAMP production compared with WT hMC3R, and one constitutively active mutant (F347A) with enhanced basal cAMP activity, were investigated using western blots.…”

“…Although it is uncommon that residues important for ligand binding are located in the cytoplasmic side of receptor, observations have been reported previously in MC3R ( Huang and Tao 2014 ) as well as other GPCRs, such as gonadotropin-releasing hormone receptor ( Lu et al 2005 ), angiotensin II type 2 receptor ( Moore et al 2002 ), and V2 vasopressin receptor ( Pan et al 1994 ). These residues are not expected to directly participate in ligand binding; rather they might indirectly participate in ligand–receptor interaction through intramolecular interactions and conformational changes ( Kobilka and Deupi 2007 ; Rovati et al 2007 ).…”

“…Although it is uncommon, similar observations have been previously reported in other GPCRs, such as MC4R ( Huang and Tao 2012 ) and gonadotropin-releasing hormone receptor ( Bedecarrats et al 2003 ). This might due to the faster dissociation of the ligand from the MC3R mutants compared with WT receptor making the binding difficult to detect, but ERK1/2 could still be activated by this transient binding ( Huang and Tao 2014 ). NDP-MSH was reported to induce ERK1/2 phosphorylation in HEK293 cells transfected with WT MC3R in a very fast and potent pattern, peaking at only 5 min with an EC 50 of 3.3 ± 1.5 nM ( Chai et al 2007 ).…”

“…We generated 20 mutants and studied the cell surface expression, ligand binding and signaling properties of the mutant receptors. Since MC3R activation has also been reported to stimulate ERK1/2 phosphorylation ( Begriche et al 2012 ; Chai et al 2007 ) (one report suggested that the MC3R does not activate ERK1/2 ( Daniels et al 2003 )), and we and others recently reported biased signaling in the MC3R ( Huang and Tao 2014 ; Montero-Melendez et al 2015 ; Yang et al 2015 ), the ERK1/2 signaling pathway of the hMC3R was also investigated.…”