International Journal of Molecular Sciences Article Differential Signaling Profiles of MC4R Mutations with Three Different Ligands Sarah Paisdzior 1 , Ioanna Maria Dimitriou 1 , Paul Curtis Schöpe 2 , Paolo Annibale 2 , Patrick Scheerer 3 , Heiko Krude 1 , Martin J. Lohse 2 , Heike Biebermann 1, † and Peter Kühnen 1, †, * 1 Institute of Experimental Pediatric Endocrinology, Charité–Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, D-10117 Berlin, Germany; sarah.paisdzior@gmx.de (S.P.); ioanna-maria.dimitriou@charite.de (I.M.D.); heiko.krude@charite.de (H.K.); heike.biebermann@charite.de (H.B.) 2 Max Delbrück Center, Robert-Rössle-Straße 10, 13092 Berlin, Germany; Institute of Pharmacology and Toxicology, University of Würzburg, D-97078 Würzburg, Germany; paul.schoepe@charite.de (P.C.S.); paolo.annibale@mdc-berlin.de (P.A.); Martin.Lohse@mdc-berlin.de (M.J.L.) 3 Group Protein X-ray Crystallography and Signal Transduction, Institute of Medical Physics and Biophysics, Charité- Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, D-10117 Berlin, Germany; patrick.scheerer@charite.de * Correspondence: peter.kuehnen@charite.de † These authors contributed equally to this work. Received: 19 December 2019; Accepted: 6 February 2020; Published: 12 February 2020   Abstract: The melanocortin 4 receptor (MC4R) is a key player in hypothalamic weight regulation and energy expenditure as part of the leptin–melanocortin pathway. Mutations in this G protein coupled receptor (GPCR) are the most common cause for monogenetic obesity, which appears to be mediated by changes in the anorectic action of MC4R via G S -dependent cyclic adenosine-monophosphate (cAMP) signaling as well as other signaling pathways. To study potential bias in the effects of MC4R mutations between the different signaling pathways, we investigated three major MC4R mutations: a G S loss-of-function (S127L) and a G S gain-of-function mutant (H158R), as well as the most common European single nucleotide polymorphism (V103I). We tested signaling of all four major G protein families plus extracellular regulated kinase (ERK) phosphorylation and β-arrestin2 recruitment, using the two endogenous agonists, α- and β-melanocyte stimulating hormone (MSH), along with a synthetic peptide agonist (NDP-α-MSH). The S127L mutation led to a full loss-of-function in all investigated pathways, whereas V103I and H158R were clearly biased towards the G q/11 pathway when challenged with the endogenous ligands. These results show that MC4R mutations can cause vastly different changes in the various MC4R signaling pathways and highlight the importance of a comprehensive characterization of receptor mutations. Keywords: Melanocortin 4 receptor (MC4R); Melanocyte stimulating hormones MSH; G protein coupled receptor (GPCR); biased signaling 1. Introduction A complex hypothalamic network of orexigenic and anorexigenic signals regulates body weight and energy expenditure. One of the key players in this network is a G protein coupled receptor (GPCR), the melanocortin 4 receptor (MC4R), which is mainly expressed in the paraventricular nucleus (PVN) of the hypothalamus. The MC4R receives input from the leptin–melanocortin pathway and thereby integrates peripheral and central metabolic signals. Activation of the MC4R by the proopiomelanocortin (POMC)-derived melanocyte stimulation hormones alpha and beta (α-MSH, β-MSH) counteracts the Int. J. Mol. Sci. 2020, 21, 1224; doi:10.3390/ijms21041224 www.mdpi.com/journal/ijms