TY - JOUR A1 - Paisdzior, Sarah A1 - Dimitriou, Ioanna Maria A1 - Schöpe, Paul Curtis A1 - Annibale, Paolo A1 - Scheerer, Patrick A1 - Krude, Heiko A1 - Lohse, Martin J. A1 - Biebermann, Heike A1 - Kühnen, Peter T1 - Differential signaling profiles of MC4R mutations with three different ligands JF - International Journal of Molecular Sciences N2 - 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. KW - Melanocortin 4 receptor (MC4R) KW - Melanocyte stimulating hormones MSH KW - G protein coupled receptor (GPCR) KW - biased signaling Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-285108 SN - 1422-0067 VL - 21 IS - 4 ER - TY - JOUR A1 - Wölfel, Angela A1 - Sättele, Mathias A1 - Zechmeister, Christina A1 - Nikolaev, Viacheslov O. A1 - Lohse, Martin J. A1 - Boege, Fritz A1 - Jahns, Roland A1 - Boivin-Jahns, Valérie T1 - Unmasking features of the auto-epitope essential for β\(_1\)-adrenoceptor activation by autoantibodies in chronic heart failure JF - ESC Heart Failure N2 - Aims Chronic heart failure (CHF) can be caused by autoantibodies stimulating the heart via binding to first and/or second extracellular loops of cardiac β1-adrenoceptors. Allosteric receptor activation depends on conformational features of the autoantibody binding site. Elucidating these features will pave the way for the development of specific diagnostics and therapeutics. Our aim was (i) to fine-map the conformational epitope within the second extracellular loop of the human β\(_1\)-adrenoceptor (β1ECII) that is targeted by stimulating β\(_1\)-receptor (auto)antibodies and (ii) to generate competitive cyclopeptide inhibitors of allosteric receptor activation, which faithfully conserve the conformational auto-epitope. Methods and results Non-conserved amino acids within the β\(_1\)EC\(_{II}\) loop (compared with the amino acids constituting the ECII loop of the β\(_2\)-adrenoceptor) were one by one replaced with alanine; potential intra-loop disulfide bridges were probed by cysteine–serine exchanges. Effects on antibody binding and allosteric receptor activation were assessed (i) by (auto)antibody neutralization using cyclopeptides mimicking β1ECII ± the above replacements, and (ii) by (auto)antibody stimulation of human β\(_1\)-adrenoceptors bearing corresponding point mutations. With the use of stimulating β\(_1\)-receptor (auto)antibodies raised in mice, rats, or rabbits and isolated from exemplary dilated cardiomyopathy patients, our series of experiments unmasked two features of the β\(_1\)EC\(_{II}\) loop essential for (auto)antibody binding and allosteric receptor activation: (i) the NDPK\(^{211–214}\) motif and (ii) the intra-loop disulfide bond C\(^{209}\)↔C\(^{215}\). Of note, aberrant intra-loop disulfide bond C\(^{209}\)↔C\(^{216}\) almost fully disrupted the functional auto-epitope in cyclopeptides. Conclusions The conformational auto-epitope targeted by cardio-pathogenic β\(_1\)-receptor autoantibodies is faithfully conserved in cyclopeptide homologues of the β\(_1\)EC\(_{II}\) loop bearing the NDPK\(^{211–214}\) motif and the C\(^{209}\)↔C\(^{215}\) bridge while lacking cysteine C216. Such molecules provide promising tools for novel diagnostic and therapeutic approaches in β\(_1\)-autoantibodypositive CHF. KW - antibody/autoantibody KW - β1-adrenoceptor/β1-adrenergic receptor KW - chronic heart failure KW - conformational auto-epitope KW - cyclic peptides/cyclopeptides KW - cyclopeptide therapy Y1 - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:bvb:20-opus-235974 VL - 7 IS - 4 ER -