Genetic defects underlying the melanocortin-4 receptor (MC4R) signaling pathway lead to severe obesity. Three severely obese LEPR-deficient individuals were administered the MC4R agonist setmelanotide, resulting in substantial and durable reductions in hyperphagia and body weight over an observation period of 45-61 weeks. Compared to formerly developed and tested MC4R agonists, setmelanotide has the unique capability of activating nuclear factor of activated T cell (NFAT) signaling and restoring function of this signaling pathway for selected MC4R variants. Our data demonstrate the potency of setmelanotide in treatment of individuals with diverse MC4R-related pathway deficiencies.

PEN is an abundant peptide in the brain that has been implicated in the regulation of feeding. We identified a receptor for PEN in mouse hypothalamus and Neuro2A cells. PEN bound to and activated GPR83, a G protein (heterotrimeric guanine nucleotide–binding protein)–coupled receptor (GPCR). Reduction of GPR83 expression in mouse brain and Neuro2A cells reduced PEN binding and signaling, consistent with GPR83 functioning as the major receptor for PEN. In some brain regions, GPR83 colocalized with GPR171, a GPCR that binds the neuropeptide bigLEN, another neuropeptide that is involved in feeding and is generated from the same precursor protein as is PEN. Coexpression of these two receptors in cell lines altered the signaling properties of each receptor, suggesting a functional interaction. Our data established PEN as a neuropeptide that binds GPR83 and suggested that these two ligand-receptor systems—PEN-GPR83 and bigLEN-GPR171—may be functionally coupled in the regulation of feeding.

Seven-transmembrane-spanning receptors (7TMRs) are prominent drug targets. However, small-molecule ligands for 7-transmembrane-spanning receptors for which the natural ligands are large, heterodimeric glycoprotein hormones, like thyroid-stimulating hormone (TSH; thyrotropin), have only recently been reported, and none are approved for human use. We have used quantitative high-throughput screening to identify a small-molecule TSH receptor (TSHR) agonist that was modified to produce a second agonist with increased potency. We show that these agonists are highly selective for human TSHR versus other glycoprotein hormone receptors and interact with the receptor's serpentine domain. A binding pocket within the transmembrane domain was defined by docking into a TSHR homology model and was supported by site-directed mutagenesis. In primary cultures of human thyrocytes, both TSH and the agonists increase mRNA levels for thyroglobulin, thyroperoxidase, sodium iodide symporter, and deiodinase type 2, and deiodinase type 2 enzyme activity. Moreover, oral administration of the agonist stimulated thyroid function in mice, resulting in increased serum thyroxine and thyroidal radioiodide uptake. Thus, we discovered a small molecule that activates human TSHR in vitro, is orally active in mice, and could be a lead for development of drugs to use in place of recombinant human TSH in patients with thyroid cancer.7TMR ͉ G protein-coupled receptor ͉ low-molecular-weight ligands ͉ radioiodide uptake ͉ TSH receptor S mall-molecule agonists and antagonists for 7-transmembrane-spanning receptors (7TMRs; G protein-coupled receptors) make up approximately 40% of the drugs in clinical use (1). The natural ligands for the 7TMRs for which drugs have been discovered are themselves primarily small molecules, such as norepinephrine, adenosine, and acetylcholine. Smallmolecule ligands for the subclass of 7TMRs for which the natural ligands are 30-kDa heterodimeric glycoprotein hormones have recently begun to be described, but none have been approved for use in humans. For example, small-molecule agonists (2-5) and an antagonist (6) for the luteinizing hormone/chorionic gonadotropin receptor (LHCGR), and small-molecule agonists (7-9) and antagonists (10-12) for the FSH receptor (FSHR) have been reported. The thyrotropin (thyroid-stimulating hormone, TSH) receptor (TSHR) is the third member of the glycoprotein hormone receptor subfamily. We recently reported smallmolecule agonists (13) and an antagonist (14) for human TSHR; however, the agonists were of low affinity and were shown to activate TSHRs only in a model cell system in which human TSHRs were ectopically over-expressed.The physiologic role of TSHRs in the hypothalamic-pituitarythyroid axis is well known. TSH, which is produced in the thyrotrophs of the anterior pituitary gland, is secreted into the circulation in response to TSH-releasing hormone stimulation and in turn stimulates the function of the thyroid follicular cells (i.e., thyrocytes) leading, in particular, to increases in size ...

The G protein-coupled receptor 83 (Gpr83) is widely expressed in brain regions regulating energy metabolism. Here we report that hypothalamic expression of Gpr83 is regulated in response to nutrient availability and is decreased in obese mice compared with lean mice. In the arcuate nucleus, Gpr83 colocalizes with the ghrelin receptor (Ghsr1a) and the agouti-related protein. In vitro analyses show heterodimerization of Gpr83 with Ghsr1a diminishes activation of Ghsr1a by acyl-ghrelin. The orexigenic and adipogenic effect of ghrelin is accordingly potentiated in Gpr83-deficient mice. Interestingly, Gpr83 knock-out mice have normal body weight and glucose tolerance when fed a regular chow diet, but are protected from obesity and glucose intolerance when challenged with a high-fat diet, despite hyperphagia and increased hypothalamic expression of agouti-related protein, Npy, Hcrt and Ghsr1a. Together, our data suggest that Gpr83 modulates ghrelin action but also indicate that Gpr83 regulates systemic metabolism through other ghrelin-independent pathways.

Monocarboxylate transporter 8 (MCT8, SLC16A2) is a thyroid hormone (TH) transmembrane transport protein mutated in Allan-Herndon-Dudley syndrome, a severe X-linked psychomotor retardation. The neurological and endocrine phenotypes of patients deficient in MCT8 function underscore the physiological significance of carrier-mediated TH transmembrane transport. MCT8 belongs to the major facilitator superfamily of 12 transmembrane-spanning proteins and mediates energy-independent bidirectional transport of iodothyronines across the plasma membrane. Structural information is lacking for all TH transmembrane transporters. To gain insight into structure-function relations in TH transport, we chose human MCT8 as a paradigm. We systematically performed conventional and liquid chromatography-tandem mass spectrometrybased uptake measurements into MCT8-transfected cells using a large number of compounds structurally related to iodothyronines. We found that human MCT8 is specific for L-iodothyronines and requires at least one iodine atom per aromatic ring. Neither thyronamines, decarboxylated metabolites of iodothyronines, nor triiodothyroacetic acid and tetraiodothyroacetic acid, TH derivatives lacking both chiral center and amino group, are substrates for MCT8. The polyphenolic flavonoids naringenin and F21388, potent competitors for TH binding at transthyretin, did not inhibit T 3 transport, suggesting that MCT8 can discriminate its ligand better than transthyretin. Bioinformatic studies and a first molecular homology model of MCT8 suggested amino acids potentially involved in substrate interaction. Indeed, alanine mutation of either Arg 445 (helix 8) or Asp 498(helix 10) abrogated T 3 transport activity of MCT8, supporting their predicted role in substrate recognition. The MCT8 model allows us to rationalize potential interactions of amino acids including those mutated in patients with Allan-HerndonDudley syndrome.

The TSH receptor (TSHR) together with the homologous lutropin/choriogonadotropin receptor and the follitropin receptor are glycoprotein hormone receptors (GPHRs). They constitute a subfamily of the rhodopsin-like G protein-coupled receptors with seven transmembrane helices. GPHRs and their corresponding hormones are pivotal proteins with respect to a variety of physiological functions. The identification and characterization of intra- and intermolecular signaling determinants as well as signaling mechanisms are prerequisites to gaining molecular insights into functions and (pathogenic) dysfunctions of GPHRs. Knowledge about activation mechanisms is fragmentary, and the specific aspects have still not been understood in their entirety. Therefore, here we critically review the data available for these receptors and bring together structural and functional findings with a focus on the important large extracellular portion of the TSHR. One main focus is the particular function of structural determinants in the initial steps of the activation such as: 1) hormone binding at the extracellular site; 2) hormone interaction at a second binding site in the hinge region; 3) signal regulation via sequence motifs in the hinge region; and 4) synergistic signal amplification by cooperative effects of the extracellular loops toward the transmembrane region. Comparison and consolidation of data from the homologous glycoprotein hormone receptors TSHR, follitropin receptor, and lutropin/choriogonadotropin receptor provide an overview of extracellular mechanisms of signal initiation, conduction, and regulation at the TSHR and homologous receptors. Finally, we address the issue of structural implications and suggest a refined scenario for the initial signaling process on GPHRs.

Many cognate low molecular weight (LMW) agonists bind to seven transmembrane-spanning receptors within their transmembrane helices (TMHs). The thienopyrimidine org41841 was identified previously as an agonist for the luteinizing hormone/chorionic gonadotropin receptor (LHCGR) and suggested to bind within its TMHs because it did not compete for LH binding to the LHCGR ectodomain. Because of its high homology with LHCGR, we predicted that thyroid-stimulating hormone receptor (TSHR) might be activated by org41841 also. We show that org41841 is a partial agonist for TSHR but with lower potency than for LHCGR. Analysis of three-dimensional molecular models of TSHR and LHCGR predicted a binding pocket for org41841 in common clefts between TMHs 3, 4, 5, 6, and 7 and extracellular loop 2 in both receptors. Evidence for this binding pocket was obtained in signaling studies with chimeric receptors that exhibited improved responses to org41841. Furthermore, a key receptor-ligand interaction between the highly conserved negatively charged E3.37 and the amino group of org41841 predicted by docking of the ligand into the three-dimensional TSHR model was experimentally confirmed. These findings provide the first evidence that, in contrast to the ectodomain binding of cognate ligands, a LMW agonist can bind to and activate glycoprotein hormone receptors via interaction with their transmembrane domain.

Background: The melanocortin-3 (MC3R) and ghrelin (GHSR) receptors are important key components in hypothalamic weight regulation. Results: MC3R and GHSR di/oligomerize and have an opposite impact on each other's function. Conclusion:The high basal activity of GHSR is a determinant of heterodimer function, and MC3R may constrain GHSR function. Significance: Receptor di/oligomerization and its functional relevance contribute to the complex network of hypothalamic weight regulation.