Human melanocortin-3 receptor: structure-function relationship of DPLIY motif and helix 8 and biased signaling

Abstract

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 functions. 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 eleven residues were important for ligand binding and one was indispensable for downstream cAMP generation. F347A was identified to be constitutively active in cAMP signaling while all the other mutants had normal basal activities. We also 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. Not only mutant receptors can be biased, different ligands of the same receptor have been shown to induce distinct receptor conformations that lead to the activation of diverse signaling pathways, resulting in biased physiological responses. The orexigenic agouti-related peptide (AgRP), which was initially identified as an endogenous antagonist for both neural MCRs, has been suggested to be a biased agonist of MC4R independently of its antagonizing effects. However, little is known about the underlying mechanism or whether the biased agonism of AgRP also exists in MC3R. Herein, we investigated the potential of AgRP to regulate the activation of intracellular kinases through both neural MCRs. We showed that AgRP acted as a biased agonist in MC3R, decreasing the basal cAMP activity of constitutively active mutant (F347A) but stimulating ERK1/2 activation in both wide type and F347A hMC3Rs. AgRP-stimulated ERK1/2 phosphorylation through MC3R was abolished by protein kinase A (PKA) inhibitor H-89, whereas AgRP-initiated ERK1/2 activation through MC4R was inhibited by phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. Both NDP-MSH and AgRP treatment induced significant AKT phosphorylation in GT1-7 cells but not in either MC3R- or MC4R-transfected HEK293T cells. The pAMPK levels in both GT1-7 cells and HEK293T cells transfected with neural MCRs were significantly decreased upon stimulation with NDP-MSH but not AgRP. In summary, we showed that the DPLIY motif and Helix 8 are important for MC3R activation and signal transduction. We also provided novel data for the biased agonism of AgRP in neural MCRs. Our result established a theoretical basis for the structure-function relationship of MC3R and AgRP-initiated multiple intracellular signaling pathways, leading to a better understanding of neural MCR pharmacology.