Activation of MAPK by inverse agonists in six naturally occurring constitutively active mutant human melanocortin-4 receptors

Abstract

The melanocortin-4 receptor (MC4R) is a G protein-coupled receptor that plays an essential role in regulating energy homeostasis. Defects in MC4R are the most common monogenic form of obesity, with about 170 distinct mutations identified in human. In addition to the conventional Gs-stimulated adenylyl cyclase pathway, it has been recently demonstrated that MC4R also activates mitogen-activated protein kinases, extracellular signal-regulated kinases 1 and 2 (ERK1/2). Herein, we investigated the potential of four MC4R ligands that are inverse agonists at the Gs-cAMP signaling pathway, including agouti-related peptide (AgRP), MCL0020, Ipsen 5i and ML00253764, to regulate ERK1/2 activation (pERK1/2) in wild type and six naturally occurring constitutively active mutant (CAM) MC4Rs. We showed that these four inverse agonists acted as agonists for the ERK1/2 signaling cascade in wild type and CAM MC4Rs. Three mutants (P230L, L250Q and F280L) had significantly increased pERK1/2 level upon stimulation with all four inverse agonists, with maximal induction ranging from 1.6 to 4.2-fold. D146N had significantly increased pERK1/2 level upon stimulation with AgRP, MCL0020 or ML00253764, but not Ipsen 5i. The pERK1/2 levels of H76R and S127L were significantly increased only upon stimulation with AgRP or MCL0020. In summary, our studies demonstrated for the first time that MC4R inverse agonists at the Gs-cAMP pathway could serve as agonists in the MAPK pathway. These results suggested that there were multiple activation states of MC4R with ligand-specific and/or mutant-specific conformations capable of differentially coupling the MC4R to distinct signaling pathways.

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

“…To the best of our knowledge, this is the first report describing biased signaling in MC3R. Biased signaling induced either by mutation or by biased ligand has been reported in the MC4R (Patten et al 2007, Büch et al 2009, Huang & Tao 2012, Mo et al 2012, Mo & Tao 2013; reviewed by Breit et al (2011) andTao (2014)) and several other GPCRs (reviewed by Violin & Lefkowitz (2007) and Lefkowitz (2013)). It has been suggested that biased ligands interact with different residues of the receptor and, therefore, induce different conformational changes in b 1 -AR (Warne et al 2012) and serotonin receptors (Wacker et al 2013).…”

“…In addition to biased ligands, biased signaling may also be induced by mutations in the GPCRs (Rajagopal et al 2010). We and others have previously identified both biased ligands and receptors in MC4R (Patten et al 2007, Büch et al 2009, Huang & Tao 2012, Mo et al 2012, Mo & Tao 2013; reviewed by Breit et al (2011) and Tao (2014)). However, until now, no biased signaling of MC3R has been reported.…”

Biased Agonism as a Novel Strategy To Harness the Proresolving Properties of Melanocortin Receptors without Eliciting Melanogenic Effects

“…AGRP can actually initiate G i/o proteininduced signaling (44), as well as internalization of MC 3 and MC 4 , a receptor turn-off mechanism attributed to agonistic activity (45). More recently, AGRP has been identified as a biased agonist at MC 4 causing, indeed, activation of the ERK1/2 pathway (46).…”

Identification and pharmacological analyses of eight naturally occurring caprine melanocortin-1 receptor mutations in three different goat breeds

“…In the mouse MC1R, a mutant receptor constitutively active in the cAMP pathway is not active in both basal and a-MSH induced ERK1/2 signaling, also representing a case of biased signaling (Benned-Jensen et al, 2011). Of note, biased signaling has been reported for MC4R and MC3R mutants (Breit et al, 2011;He and Tao, 2014;Mo and Tao, 2013;Mo et al, 2012;Yang et al, 2015), including biased constitutive signaling (Tao, 2014).…”