Differential Signaling Profiles of MC4R Mutations with Three Different Ligands

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 GS-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 GS loss-of-function (S127L) and a GS 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 Gq/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

Evaluation of pharmacological rescue of melanocortin-4 receptor nonsense mutations by aminoglycoside

This research was funded by the Deutsche Forschungsgemeinschaft (DFG) (German Research Foundation) through SFB1423, project number 421152132, subprojects B02 to H.B. and P.K., A01 and Z03 to P.S., and C03 to P.A., and project numbers 430971019, 430970922 and KU 2673/6-1 to P.K., and by the European Union’s Horizon 2020 MSCA Program under grant agreement 956314 (ALLODD) to P.S.The melanocortin-4 receptor (MC4R) is critical for central satiety regulation, therefore presenting a potent target for pharmacological obesity treatment. Melanocortin-4 receptor mutations prevalently cause monogenetic obesity. A possibility of overcoming stop mutations is aminoglycoside-mediated translational readthrough. Promising results were achieved in COS-7 cells, but data for human cell systems are still missing, so uncertainty surrounds this potential treatment. In transfected HEK-293 cells, we tested whether translational readthrough by aminoglycoside Geneticin combined with high-affinity ligand setmelanotide, which is effective in proopiomelanocortin or leptin receptor deficiency patients, is a treatment option for affected patients. Five MC4R nonsense mutants (W16X, Y35X_D37V, E61X, W258X, Q307X) were investigated. Confocal microscopy and cell surface expression assays revealed the importance of the mutations' position within the MC4R. N-terminal mutants were marginally expressed independent of Geneticin treatment, whereas mutants with nonsense mutations in transmembrane helix 6 or helix 8 showed wild-type-like expression. For functional analysis, Gs and Gq/11 signaling were measured. N-terminal mutants (W16X, Y35X_D37V) showed no cAMP formation after challenge with alpha-MSH or setmelanotide, irrespective of Geneticin treatment. Similarly, Gs activation was almost impossible in W258X and Q307X with wild-type-like cell surface expression. Results for Gq/11 signaling were comparable. Based on our data, this approach improbably represents a therapeutic option.Publisher PDFPeer reviewe

Differential signaling profiles of MC4R mutations with three different ligands

Funding: This research was funded by SPARK BIH Validation Fund 2. P.S., P.K., H.B., P.A. and M.J.L. acknowledge funding by the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) through CRC 1423, project number 421152132, subprojects A1, A05, Z03 to P.S.; B02 to P.K. and H.B.; C03 to P.A. and M.J.L.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 GS-dependent cyclic adenosinemonophosphate (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 GS loss-of-function (S127L) and a GS 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 Gq/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.Publisher PDFPeer reviewe

Structures of active melanocortin-4 receptor−Gs-protein complexes with NDP-α-MSH and setmelanotide

The melanocortin-4 receptor (MC4R), a hypothalamic master regulator of energy homeostasis and appetite, is a class A G-protein-coupled receptor and a prime target for the pharmacological treatment of obesity. Here, we present cryo-electron microscopy structures of MC4R–Gs-protein complexes with two drugs recently approved by the FDA, the peptide agonists NDP-α-MSH and setmelanotide, with 2.9 Å and 2.6 Å resolution. Together with signaling data from structure-derived MC4R mutants, the complex structures reveal the agonist-induced origin of transmembrane helix (TM) 6-regulated receptor activation. The ligand-binding modes of NDP-α-MSH, a high-affinity linear variant of the endogenous agonist α-MSH, and setmelanotide, a cyclic anti-obesity drug with biased signaling toward Gq/11, underline the key role of TM3 in ligand-specific interactions and of calcium ion as a ligand-adaptable cofactor. The agonist-specific TM3 interplay subsequently impacts receptor–Gs-protein interfaces at intracellular loop 2, which also regulates the G-protein coupling profile of this promiscuous receptor. Finally, our structures reveal mechanistic details of MC4R activation/inhibition, and provide important insights into the regulation of the receptor signaling profile which will facilitate the development of tailored anti-obesity drugs

A new multi-system disorder caused by the Gαs mutation p.F376V

CONTEXT: The α subunit of the stimulatory G protein (Gαs) links numerous receptors to adenylyl cyclase. Gαs, encoded by GNAS, is expressed predominantly from the maternal allele in certain tissues. Thus, maternal heterozygous loss-of-function mutations cause hormonal resistance, as in pseudohypoparathyroidism type Ia, whereas somatic gain-of-function mutations cause hormone-independent endocrine stimulation, as in McCune-Albright syndrome. OBJECTIVE: We report two unrelated boys presenting with a new combination of clinical findings that suggest both gain and loss of Gαs function. DESIGN AND SETTING: Clinical features were studied and sequencing of GNAS was performed. Signaling capacities of wild-type and mutant Gαs were determined in the presence of different G protein–coupled receptors (GPCRs) under basal and agonist-stimulated conditions. RESULTS: Both unrelated patients presented with unexplained hyponatremia in infancy, followed by severe early onset gonadotrophin-independent precocious puberty and skeletal abnormalities. An identical heterozygous de novo variant (c.1136T>G; p.F376V) was found on the maternal GNAS allele in both patients; this resulted in a clinical phenotype that differed from known Gαs-related diseases and suggested gain of function at the vasopressin 2 receptor (V2R) and lutropin/choriogonadotropin receptor (LHCGR), yet increased serum PTH concentrations indicative of impaired proximal tubular PTH1 receptor (PTH1R) function. In vitro studies demonstrated that Gαs-F376V enhanced ligand-independent signaling at the PTH1R, LHCGR, and V2R and, at the same time, blunted ligand-dependent responses. Structural homology modeling suggested mutation-induced modifications at the C-terminal α5 helix of Gαs that are relevant for interaction with GPCRs and signal transduction. CONCLUSION: The Gαs p.F376V mutation causes a previously unrecognized multisystem disorder

A new multi-system disorder caused by the Gαs mutation p.F376V

Context The alpha-subunit of the stimulatory G-protein (Gαs) links numerous receptors to adenylyl cyclase. Gαs, encoded by GNAS, is expressed predominantly from the maternal allele in certain tissues. Thus, maternal heterozygous loss-of-function mutations cause hormonal resistance, as in pseudohypoparathyroidism type Ia, while somatic gain-of-function mutations cause hormone-independent endocrine stimulation, as in McCune-Albright Syndrome. Objective We here report two unrelated boys presenting with a new combination of clinical findings that suggest both gain and loss of Gαs function. Design, Setting Clinical features were studied and sequencing of GNAS was performed. Signaling capacities of wild-type and mutant-Gαs were determined in the presence of different G protein-coupled receptors (GPCRs) under basal and agonist-stimulated conditions. Results Both unrelated patients presented with unexplained hyponatremia in infancy, followed by severe early-onset gonadotrophin-independent precocious puberty and skeletal abnormalities. An identical heterozygous de novo variant (c.1136T>G; p.F376V) was found on the maternal GNAS allele, in both patients; this resulted in a clinical phenotype that differ from known Gαs-related diseases and suggested gain-of-function at the receptors for vasopressin (V2R) and lutropin (LHCGR), yet increased serum parathyroid hormone (PTH) concentrations indicative of impaired proximal tubular PTH1 receptor (PTH1R) function. In vitro studies demonstrated that Gαs-F376V enhanced ligand-independent signaling at the PTH1R, LHCGR and V2R and, at the same time, blunted ligand-dependent responses. Structural homology modeling suggested mutation-induced modifications at the C-terminal α5-helix of Gαs that are relevant for interaction with GPCRs and signal transduction. Conclusions The Gαs p.F376V mutation causes a previously unrecognized multi-system disorder

Differential Signaling Profiles of MC4R Mutations with Three Different Ligands

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 GS-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 GS loss-of-function (S127L) and a GS 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 Gq/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

In vitro Validierung von Peptid-T3-Konjugaten als Behandlungsoption für MCT8-Defizienz über einen "Trojan Horse"-ähnlichen Mechanismus

Cells of an organism have to take up all substances needed for survival and function to maintain normal performance. Several hormones are crucial for a huge variety of different functions. Some of these hormones are transported over the plasma membranes by specific hormone transporters. Inactivating mutations in the genes of these transporters can lead to severe pathological phenotypes. To overcome these defects, alternative ways of transportation need to be discovered. The aim of this dissertation was to validate a new treatment option to overcome transporter deficiency by using G protein-coupled receptor (GPCR) internalization mechanism. A conjugate of a hormone tethered to a peptide-GPCR-ligand was designed that can be internalized and the hormone can subsequently be released to reach its intracellular receptor. Two conjugates, Glucagon-like-peptide 1(GLP1)-estrogen and glucagon-triiodothyronine (T3), have already been shown to target-specifically deliver the hormone to the peptide receptor expressing tissues in vivo (Finan et al. 2012, Finan et al. 2016). The proposed mechanism, named the “Trojan Horse”-like mechanism includes the activation of a GPCR by binding the respective peptide-homrone conjugate and the internalization of this receptor-ligand complex into the cell, where the conjugate can be released, the peptide and hormone are cleaved and the hormone exert intracellular effects. Thus, the hormone transporter on the membrane has been bypassed altogether. To elucidate the mechanism in vitro is an important step to identify adverse effects. Therefore, we established a toolbox of methods that make the validation of new conjugates possible. First, the signaling cascades and subsequent internalization of the GPCR was studied, which is activated by the peptide part of the conjugate. Afterwards the activation of hormone-dependent signaling inside of the cell was investigated, which should only occur in the presence of the appropriate GPCR, ensuring the entering of the hormone exclusively through the “Trojan Horse” mechanism. With this toolbox we are able to monitor each step of the Trojan Horse mechanism for several peptide-hormone conjugate candidates. In this dissertation, two new candidates have been validated.Zellen eines Organismus müssen alle Substanzen aufnehmen, die für das Überleben und Funktion wichtig sind, um die normale Leistungsfähigkeit aufrechtzuerhalten. Verschiedene Hormone sind essentiell für eine große Anzahl verschiedener Funktionen. Manche dieser Hormone werden durch spezifische Hormontransporter über die Plasmamembran transportiert. Inaktivierungsmutationen in den Genen dieser Transporter kann zu schweren pathologischen Phänotypen führen. Um diese Defekte zu überwinden, müssen alternative Wege des Transports gefunden werden. Das Ziel dieser Dissertation war es, einen neuen Ansatz der Behandlungsoptionen einer Transporterdefizienz zu validieren. Diese nutzen den Internalisierungsmechanismus von G-Protein gekoppelten Rezeptoren (GPCRs). Ein Konjugat zwischen einem Hormon und einem Peptid-GPCR-Liganden wurde designt, welches internalisiert werden kann. Anschließend wird das Hormone in der Zelle entlassen und kann an seinen intrazellulären Rezeptor binden. Für zwei Konjugate, Glucagon-like-peptide 1(GLP1)-Östrogen und ein Glucagon-Triiodothyronin (T3), konnte bereits gezeigt werden, dass das Hormon in vivo zielgerichtet in das Gewebe transportiert wird, das den Peptidrezeptor exprimiert (Finan et al. 2012, Finan et al. 2016). Der vermutete Mechanismus, genannt der „Trojan Horse“ (Trojanisches Pferd)-ähnlicher Mechanismus umfasst die Aktivierung des GPCRs durch die Bindung des entsprechenden Peptid-Hormon-Konjugate und der Internalisierung des Rezeptor-Liganden-Komplex in die Zelle. Hier kann das Konjugate entlassen werden, das Peptid und Hormon werden voneinander abgespalten und das Hormon kann seine intrazelluläre Wirkung ausüben. Der Hormontransporter auf der Membran wurde somit komplett umgangen. Diesen Mechanismus in vitro zu untersuchen ist ein wichtiger Schritt, um Nebenwirkungen frühzeitig zu erkennen. Deshalb wurde eine Toolbox an Methoden entwickelt, die die Validierung neuer Konjugate möglich macht. Zunächst wurde die Signalisierung und Internalisierung des GPCRs untersucht, Schritte, die vom Peptidteil des Konjugates aktiviert werden. Anschließend wurde die Hormon-abhängige Signalisierung innerhalb der Zelle gemessen. Diese sollte durch das freigewordene Hormon nach dem Eintritt des Konjugates über den „Trojan Horse“-ähnlichen Mechanismus nur in Anwesenheit des Zielrezeptors aktiviert werden. Mit dieser Toolbox ist es möglich, jeden Schritt des Mechanismus für verschiedene Peptid-Hormone-Konjugatkandidaten zu verfolgen. In dieser Arbeit wurden zwei neue Kandidaten validiert.DFG, SPP 1629, Development of T3-peptide ligands to treat MCT8 deficiency via a Trojan horse like mechanis