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

Non-functional trace amine-associated receptor 1 variants in patients with mental disorders

Background: The G protein-coupled receptor (GPCR) trace amine-associated receptor 1 (TAAR1) is expressed across brain areas involved in emotions, reward and cognition, and modulates monoaminergic and glutamatergic neurotransmissions. TAAR1 is stimulated with nanomolar affinity by 3-iodothyronamine (T1AM), an endogenous messenger considered a novel branch of thyroid hormone signaling. The human gene for TAAR1 maps to locus 6q23, within a region associated with major mental disorders. Materials and Methods: We screened a cohort of patients with major mental disorders (n = 104) and a group of healthy controls (n = 130) for TAAR1 variants. HEK293 cells were transiently transfected with: i) wild-type TAAR1 and ii) mutated TAAR1, either in homozygous or heterozygous state. Cell surface expression and Gs/adenylyl cyclase activation upon administration of β-phenylethylamine (PEA), T1AM, and RO5166017, were assessed. Results: We detected 13 missense variants in TAAR1 coding region, with a significant enrichment in patients as compared to healthy controls (11 vs. 1, 1 variant in both groups, p < 0.01). In silico analysis identified four dysfunctional variants, all in patients. Three of these-R23C, Y131C, and C263R-were functionally characterized. In cells co-transfected with wild-type and mutated TAAR1, we observed a significant reduction of cell surface expression. In heterozygosity, the three TAAR1 variants substantially dampened Gs signaling in response to PEA, and, more robustly, to T1AM. Co-stimulation with PEA and RO5166017 did not yield any improvement in Gs signaling. R23C, Y131C, and C263R are rare in the general population and map in functionally important highly conserved positions across TAAR1 orthologous and paralogous genes. Conclusions: Our findings suggest that disruptions of TAAR1 activity may be relevant to the pathophysiology of mental disorders, thereby providing a promising target for novel psychopharmacological interventions

The Trace Amine-Associated Receptor 1 Agonist 3-Iodothyronamine Induces Biased Signaling at the Serotonin 1b Receptor

Trace amine-associated receptors (TAARs) belong to the class A G-protein-coupled receptors (GPCR) and are evolutionary related to aminergic receptors. TAARs have been identified to mediate effects of trace amines. TAAR1 signaling is mainly mediated via activation of the Gs/adenylyl cyclase pathway. In addition to classical trace amines, TAAR1 can also be activated by the thyroid hormone derivative 3-iodothyronamine (3-T1AM). Pharmacological doses of 3-T1AM induced metabolic and anapyrexic effects, which might be centrally mediated in the hypothalamus in rodents. However, the observed anapyrexic effect of 3-T1AM persists in Taar1 knock-out mice which raises the question whether further GPCRs are potential targets for 3-T1AM and mediate the observed physiological effect. Anapyrexia has been observed to be related to action on aminergic receptors such as the serotonin receptor 1b (5-HT1b). This receptor primarily activates the Gi/o mediated pathway and PLC signaling through the Gβγ of Gi/o. Since the expression profiles of TAAR1 and 5-HT1b overlap, we questioned whether 3-T1AM may activate 5-HT1b. Finally, we also evaluated heteromerization between these two GPCRs and tested signaling under co-expressed conditions. In this study, we showed, that 3-T1AM can induce Gi/o signaling through 5-HT1b in a concentration of 10 μM. Strikingly, at 5-HT1b the ligand 3-T1AM only activates the Gi/o mediated reduction of cAMP accumulation, but not PLC activation. Co-stimulation of 5-HT1b by both ligands did not lead to additive or synergistic signaling effects. In addition, we confirmed the capacity for heteromerization between TAAR1 and 5-HT1b. Under co-expression of TAAR1 and HTR1b, 3-T1AM action is only mediated via TAAR1 and activation of 5-HT1b is abrogated. In conclusion, we found evidence for 5-HT1b as a new receptor target for 3-T1AM, albeit with a different signaling effect than the endogenous ligand. Altogether, this indicates a complex interrelation of signaling effects between the investigated GPCRs and respective ligands