The melanocortin-4 receptor (MC4R) is a hypothalamic regulator of energy homeostasis and appetite. Hence, this G-protein coupled receptor (GPCR) is a prime pharmaceutical target for obesity treatment. During this thesis, the central experimental prerequisites for in vitro GPCR studies were established: (1) cell culture techniques for expressing receptors and G-proteins, (2) a fluorescent-based in cell ligand-binding assay for assessing suitable ligands, and (3) a receptor-G-protein complex formation and purification protocol. These biochemical experiments lead to the first cryo-electron microscopy structures of a GPCR-G-protein complex in Germany, the MC4R-Gs complexes with two novel FDA- approved drugs, the peptide agonists NDP-α-MSH and setmelanotide, with 2.9 Å and 2.6 Å resolution, respectively. Both complexes demonstrate the importance of calcium as a cofactor for agonist binding, precisely adjusted depending on the different ligands. Furthermore, the structures together with signaling data reveal both the agonist-induced origin of the transmembrane helix (TM) 6-dependent receptor activation and the mediator role of TM3 in fine-tuned activation, driven by interactions with the respective agonist and calcium. This interplay proceeds towards the receptor Gs-protein interface, particularly at intracellular loop three, impacting MC4R's G-protein coupling specificity. These structures uncover mechanistic details of MC4R activation, inhibition, and G-protein coupling specificity, facilitating the development of optimized drugs.
