Functional modulation of PTH1R activation and signaling by RAMP2.

Receptor-activity-modifying proteins (RAMPs) are ubiquitously expressed membrane proteins that associate with different G protein-coupled receptors (GPCRs), including the parathyroid hormone 1 receptor (PTH1R), a class B GPCR and an important modulator of mineral ion homeostasis and bone metabolism. However, it is unknown whether and how RAMP proteins may affect PTH1R function. Using different optical biosensors to measure the activation of PTH1R and its downstream signaling, we describe here that RAMP2 acts as a specific allosteric modulator of PTH1R, shifting PTH1R to a unique preactivated state that permits faster activation in a ligand-specific manner. Moreover, RAMP2 modulates PTH1R downstream signaling in an agonist-dependent manner, most notably increasing the PTH-mediated Gi3 signaling sensitivity. Additionally, RAMP2 increases both PTH- and PTHrP-triggered ß-arrestin2 recruitment to PTH1R. Employing homology modeling, we describe the putative structural molecular basis underlying our functional findings. These data uncover a critical role of RAMPs in the activation and signaling of a GPCR that may provide a new venue for highly specific modulation of GPCR function and advanced drug design.

Autoimmune activation and hypersensitization of the AT1 and ETA receptors contributes to vascular injury in scleroderma renal crisis.

OBJECTIVES: Scleroderma renal crisis (SRC) is a rare vascular complication of systemic sclerosis with substantial risks for end-stage renal disease and premature death. Activating autoantibodies (Abs) targeting the angiotensin II type 1 (AT1R) and the endothelin-1 type A receptor (ETAR) have been identified as predictors for SRC. Here, we sought to determine their pathogenic significance for acute renal vascular injury potentially triggering kidney failure and malignant hypertension. METHODS: IgG from patients with SRC was studied for AT1R and ETAR dependent biologic effects on isolated rat renal interlobar arteries and vascular cells including contraction, signalling and mechanisms of receptor activation. RESULTS: In myography experiments, patient IgG exerted vasoconstriction sensitive to inhibition of AT1R and ETAR. This relied on MEK-ERK signalling indicating functional relevance of anti-AT1R and anti-ETAR Abs. The contractile response to angiotensin II and endothelin-1 was amplified by patient IgG containing anti-AT1R and anti-ETAR Abs with substantial crosstalk between both receptors implicating autoimmune receptor hypersensitization. Co-immunoprecipitation experiments indicated heterodimerization between both receptor types which may enable the observed functional interrelation by direct structural interactions. CONCLUSION: We provide experimental evidence that agonistic Abs may contribute to SRC. This effect is presumably related to direct receptor stimulation and additional allosteric effects, at least in heterodimeric receptor constellations. Novel therapies targeted at autoimmune hyperactivation of AT1R and ETAR might improve outcomes in severe cases of SRC.

Intramolecular activity regulation of adhesion GPCRs in light of recent structural and evolutionary information.

The class B2 of GPCRs known as adhesion G protein-coupled receptors (aGPCRs) has come under increasing academic and nonacademic research focus over the past decade due to their physiological importance as mechano-sensors in cell-cell and cell-matrix contexts. A major advance in understanding signal transduction of aGPCRs was achieved by the identification of the so-called Stachel sequence, which acts as an intramolecular agonist at the interface between the N terminus (Nt) and the seven-transmembrane helix domain (7TMD). Distinct extracellular signals received by the Nt are integrated at the Stachel into structural changes of the 7TMD towards an active state conformation. Until recently, little information was available on how the activation process of aGPCRs is realized at the molecular level. In the past three years several structures of the 7TMD plus the Stachel in complex with G proteins have been determined, which provide new insights into the architecture and molecular function of this receptor class. Herein, we review this structural information to extract common and distinct aGPCR features with particular focus on the Stachel binding site within the 7TMD. Our analysis extends the current view of aGPCR activation and exposes similarities and differences not only between diverse aGPCR members, but also compared to other GPCR classes.

Is the Neuropeptide PEN a Ligand of GPR83?

G protein-coupled receptor 83 (GPR83) is a class A G protein-coupled receptor with predominant expression in the cerebellum and proposed function in the regulation of food intake and in anxiety-like behavior. The neuropeptide PEN has been suggested as a specific GPR83 ligand. However, conflicting reports exist about whether PEN is indeed able to bind and activate GPR83. This study was initiated to evaluate PEN as a potential ligand of GPR83. Employing several second messenger and other GPCR activation assays as well as a radioligand binding assay, and using multiple GPR83 plasmids and PEN peptides from different sources, no experimental evidence was found to support a role of PEN as a GPR83 ligand.

Recurrent convergent evolution at amino acid residue 261 in fish rhodopsin.

The evolutionary process that occurs when a species colonizes a new environment provides an opportunity to explore the mechanisms underlying genetic adaptation, which is essential knowledge for understanding evolution and the maintenance of biodiversity. Atlantic herring has an estimated total breeding stock of about 1 trillion (1012) and has colonized the brackish Baltic Sea within the last 10,000 y. Minute genetic differentiation between Atlantic and Baltic herring populations at selectively neutral loci combined with this rapid adaptation to a new environment facilitated the identification of hundreds of loci underlying ecological adaptation. A major question in the field of evolutionary biology is to what extent such an adaptive process involves selection of novel mutations with large effects or genetic changes at many loci, each with a small effect on phenotype (i.e., selection on standing genetic variation). Here we show that a missense mutation in rhodopsin (Phe261Tyr) is an adaptation to the red-shifted Baltic Sea light environment. The transition from phenylalanine to tyrosine differs only by the presence of a hydroxyl moiety in the latter, but this results in an up to 10-nm red-shifted light absorbance of the receptor. Remarkably, an examination of the rhodopsin sequences from 2,056 species of fish revealed that the same missense mutation has occurred independently and been selected for during at least 20 transitions between light environments across all fish. Our results provide a spectacular example of convergent evolution and how a single amino acid change can have a major effect on ecological adaptation.

Molecular Effects of Auto-Antibodies on Angiotensin II Type 1 Receptor Signaling and Cell Proliferation.

The angiotensin II (Ang II) type 1 receptor (AT1R) is involved in the regulation of blood pressure (through vasoconstriction) and water and ion homeostasis (mediated by interaction with the endogenous agonist). AT1R can also be activated by auto-antibodies (AT1R-Abs), which are associated with manifold diseases, such as obliterative vasculopathy, preeclampsia and systemic sclerosis. Knowledge of the molecular mechanisms related to AT1R-Abs binding and associated signaling cascade (dys-)regulation remains fragmentary. The goal of this study was, therefore, to investigate details of the effects of AT1R-Abs on G-protein signaling and subsequent cell proliferation, as well as the putative contribution of the three extracellular receptor loops (ELs) to Abs-AT1R signaling. AT1R-Abs induced nuclear factor of activated T-cells (NFAT) signaling, which reflects Gq/11 and Gi activation. The impact on cell proliferation was tested in different cell systems, as well as activation-triggered receptor internalization. Blockwise alanine substitutions were designed to potentially investigate the role of ELs in AT1R-Abs-mediated effects. First, we demonstrate that Ang II-mediated internalization of AT1R is impeded by binding of AT1R-Abs. Secondly, exclusive AT1R-Abs-induced Gq/11 activation is most significant for NFAT stimulation and mediates cell proliferation. Interestingly, our studies also reveal that ligand-independent, baseline AT1R activation of Gi signaling has, in turn, a negative effect on cell proliferation. Indeed, inhibition of Gi basal activity potentiates proliferation triggered by AT1R-Abs. Finally, although AT1R containing EL1 and EL3 blockwise alanine mutations were not expressed on the human embryonic kidney293T (HEK293T) cell surface, we at least confirmed that parts of EL2 are involved in interactions between AT1R and Abs. This current study thus provides extended insights into the molecular action of AT1R-Abs and associated mechanisms of interrelated pathogenesis.

The intramolecular agonist is obligate for activation of glycoprotein hormone receptors.

In contrast to most rhodopsin-like G protein-coupled receptors, the glycoprotein hormone receptors (GPHR) have a large extracellular N-terminus for hormone binding. The hormones do not directly activate the transmembrane domain but mediate their action via a, thus, far only partially known Tethered Agonistic LIgand (TALI). The existence of such an intramolecular agonist was initially indicated by site-directed mutation studies and activating peptides derived from the extracellular hinge region. It is still unknown precisely how TALI is involved in intramolecular signal transmission. We combined systematic mutagenesis studies at the luteinizing hormone receptor and the thyroid-stimulating hormone receptor (TSHR), stimulation with a drug-like agonist (E2) of the TSHR, and structural homology modeling to unravel the functional and structural properties defining the TALI region. Here, we report that TALI (a) is predisposed to constitutively activate GPHR, (b) can by itself rearrange GPHR into a fully active conformation, (c) stabilizes active GPHR conformation, and (d) is not involved in activation of the TSHR by E2. In the active state conformation, TALI forms specific interactions between the N-terminus and the transmembrane domain. We show that stabilization of an active state is dependent on TALI, including activation by hormones and constitutively activating mutations.

A New Mechanism in THRA Resistance: The First Disease-Associated Variant Leading to an Increased Inhibitory Function of THRA2.

The nuclear thyroid hormone receptors (THRs) are key mediators of thyroid hormone function on the cellular level via modulation of gene expression. Two different genes encode THRs (THRA and THRB), and are pleiotropically involved in development, metabolism, and growth. The THRA1 and THRA2 isoforms, which result from alternative splicing of THRA, differ in their C-terminal ligand-binding domain (LBD). Most published disease-associated THRA variants are located in the LBD of THRA1 and impede triiodothyronine (T3) binding. This keeps the nuclear receptor in an inactive state and inhibits target gene expression. Here, we investigated a new dominant THRA variant (chr17:g.38,241,010A > G, GRCh37.13 | c.518A > G, NM_199334 | p.(E173G), NP_955366), which is located between the DNA- and ligand-binding domains and affects both splicing isoforms. Patients presented partially with hypothyroid (intellectual disability, motor developmental delay, brain atrophy, and constipation) and partially with hyperthyroid symptoms (tachycardia and behavioral abnormalities) to varying degrees. Functional characterization of THRA1p.(E173G) by reporter gene assays revealed increased transcriptional activity in contrast to THRA1(WT), unexpectedly revealing the first gain-of-function mutation found in THRA1. The THRA2 isoform does not bind T3 and antagonizes THRA1 action. Introduction of p.(E173G) into THRA2 increased its inhibitory effect on THRA1, which helps to explain the hypothyroid symptoms seen in our patients. We used protein structure models to investigate possible underlying pathomechanisms of this variant with a gain-of-antagonistic function and suggest that the p.(E173G) variant may have an influence on the dimerization domain of the nuclear receptor.

Zebrafish Bioassay for Screening Therapeutic Candidates Based on Melanotrophic Activity.

In this study, we used the zebrafish animal model to establish a bioassay by which physiological efficacy differential of alpha-melanocyte-stimulating hormone (α-MSH) analogues could be measured by melanosome dispersion in zebrafish larvae. Brain-skin connection research has purported the interconnectedness between the nervous system and skin physiology. Accordingly, the neuropeptide α-MSH is a key regulator in several physiological processes, such as skin pigmentation in fish. In mammals, α-MSH has been found to regulate motivated behavior, appetite, and emotion, including stimulation of satiety and anxiety. Several clinical and animal model studies of autism spectrum disorder (ASD) have already demonstrated the effectiveness of α-MSH in restoring the social deficits of autism. Therefore, we sought to analyze the effect of synthetic and naturally-occurring α-MSH variants amongst different species. Our results showed that unique α-MSH derivatives from several fish species produced differential effects on the degree of melanophore dispersion. Using α-MSH human form as a standard, we could identify derivatives that induced greater physiological effects; particularly, the synthetic analogue melanotan-II (MT-II) exhibited a higher capacity for melanophore dispersion than human α-MSH. This was consistent with previous findings in an ASD mouse model demonstrating the effectiveness of MT-II in improving ASD behavioral symptoms. Thus, the melanophore assay may serve as a useful screening tool for therapeutic candidates for novel drug discovery.

Autoantibodies Targeting AT1- and ETA-Receptors Link Endothelial Proliferation and Coagulation via Ets-1 Transcription Factor.

Scleroderma renal crisis (SRC) is an acute life-threatening manifestation of systemic sclerosis (SSc) caused by obliterative vasculopathy and thrombotic microangiopathy. Evidence suggests a pathogenic role of immunoglobulin G (IgG) targeting G-protein coupled receptors (GPCR). We therefore dissected SRC-associated vascular obliteration and investigated the specific effects of patient-derived IgG directed against angiotensin II type 1 (AT1R) and endothelin-1 type A receptors (ETAR) on downstream signaling events and endothelial cell proliferation. SRC-IgG triggered endothelial cell proliferation via activation of the mitogen-activated protein kinase (MAPK) pathway and subsequent activation of the E26 transformation-specific-1 transcription factor (Ets-1). Either AT1R or ETAR receptor inhibitors/shRNA abrogated endothelial proliferation, confirming receptor activation and Ets-1 signaling involvement. Binding of Ets-1 to the tissue factor (TF) promoter exclusively induced TF. In addition, TF inhibition prevented endothelial cell proliferation. Thus, our data revealed a thus far unknown link between SRC-IgG-induced intracellular signaling, endothelial cell proliferation and active coagulation in the context of obliterative vasculopathy and SRC. Patients' autoantibodies and their molecular effectors represent new therapeutic targets to address severe vascular complications in SSc.

Structural Complexity and Plasticity of Signaling Regulation at the Melanocortin-4 Receptor.

The melanocortin-4 receptor (MC4R) is a class A G protein-coupled receptor (GPCR), essential for regulation of appetite and metabolism. Pathogenic inactivating MC4R mutations are the most frequent cause of monogenic obesity, a growing medical and socioeconomic problem worldwide. The MC4R mediates either ligand-independent or ligand-dependent signaling. Agonists such as α-melanocyte-stimulating hormone (α-MSH) induce anorexigenic effects, in contrast to the endogenous inverse agonist agouti-related peptide (AgRP), which causes orexigenic effects by suppressing high basal signaling activity. Agonist action triggers the binding of different subtypes of G proteins and arrestins, leading to concomitant induction of diverse intracellular signaling cascades. An increasing number of experimental studies have unraveled molecular properties and mechanisms of MC4R signal transduction related to physiological and pathophysiological aspects. In addition, the MC4R crystal structure was recently determined at 2.75 Å resolution in an inactive state bound with a peptide antagonist. Underpinned by structural homology models of MC4R complexes simulating a presumably active-state conformation compared to the structure of the inactive state, we here briefly summarize the current understanding and key players involved in the MC4R switching process between different activity states. Finally, these perspectives highlight the complexity and plasticity in MC4R signaling regulation and identify gaps in our current knowledge.

Evaluation of a rare glucose-dependent insulinotropic polypeptide receptor variant in a patient with diabetes.

AIMS: Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone that augments insulin secretion in pancreatic ß-cells via its glucose-dependent insulinotropic polypeptide receptor (GIPR). Recent genome-wide association studies identified a single nucleotide variant (SNV) in the GIPR encoding gene (GIPR), rs1800437, that is associated with obesity and insulin resistance. In the present study, we tested whether GIPR variants contribute to obesity and disturb glucose homeostasis or diabetes in specific patient populations. MATERIALS AND METHODS: Exon sequencing of GIPR was performed in 164 children with obesity and insulin resistance and in 80 children with paediatric-onset diabetes of unknown origin. The Study of Health in Pomerania (SHIP) cohort, comprising 8320 adults, was screened for the GIPR variant Arg217Leu. GIPR variants were expressed in COS-7 cells and cAMP production was measured upon stimulation with GIP. Cell surface expression was determined by ELISA. Protein homology modelling of the GIPR variants was performed to extract three-dimensional information of the receptor. RESULTS: A heterozygous missense GIPR variant Arg217Leu (rs200485112) was identified in a patient of Asian ancestry. Functional characterization of Arg217Leu revealed reduced surface expression and signalling after GIP challenge. The homology model of the GIPR structure supports the observed functional relevance of Arg217Leu. CONCLUSION: In vitro functional studies and protein homology modelling indicate a potential relevance of the GIPR variant Arg217Leu in receptor function. The heterozygous variant displayed partial co-segregation with diabetes. Based on these findings, we suggest that GIPR variants may play a role in disturbed glucose homeostasis and may be of clinical relevance in homozygous patients.

Evidence of G-protein-coupled receptor and substrate transporter heteromerization at a single molecule level.

G-protein-coupled receptors (GPCRs) can constitute complexes with non-GPCR integral membrane proteins, while such interaction has not been demonstrated at a single molecule level so far. We here investigated the potential interaction between the thyrotropin receptor (TSHR) and the monocarboxylate transporter 8 (MCT8), a member of the major facilitator superfamily (MFS), using fluorescence cross-correlation spectroscopy (FCCS). Both the proteins are expressed endogenously on the basolateral plasma membrane of the thyrocytes and are involved in stimulation of thyroid hormone production and release. Indeed, we demonstrate strong interaction between both the proteins which causes a suppressed activation of Gq/11 by TSH-stimulated TSHR. Thus, we provide not only evidence for a novel interaction between the TSHR and MCT8, but could also prove this interaction on a single molecule level. Moreover, this interaction forces biased signaling at the TSHR. These results are of general interest for both the GPCR and the MFS research fields.

GPCR-SSFE 2.0-a fragment-based molecular modeling web tool for Class A G-protein coupled receptors.

G-protein coupled receptors (GPCRs) are key players in signal transduction and therefore a large proportion of pharmaceutical drugs target these receptors. Structural data of GPCRs are sparse yet important for elucidating the molecular basis of GPCR-related diseases and for performing structure-based drug design. To ameliorate this problem, GPCR-SSFE 2.0 (http://www.ssfa-7tmr.de/ssfe2/), an intuitive web server dedicated to providing three-dimensional Class A GPCR homology models has been developed. The updated web server includes 27 inactive template structures and incorporates various new functionalities. Uniquely, it uses a fingerprint correlation scoring strategy for identifying the optimal templates, which we demonstrate captures structural features that sequence similarity alone is unable to do. Template selection is carried out separately for each helix, allowing both single-template models and fragment-based models to be built. Additionally, GPCR-SSFE 2.0 stores a comprehensive set of pre-calculated and downloadable homology models and also incorporates interactive loop modeling using the tool SL2, allowing knowledge-based input by the user to guide the selection process. For visual analysis, the NGL viewer is embedded into the result pages. Finally, blind-testing using two recently published structures shows that GPCR-SSFE 2.0 performs comparably or better than other state-of-the art GPCR modeling web servers.

The Pathogenic TSH ß-subunit Variant C105Vfs114X Causes a Modified Signaling Profile at TSHR.

1) Background: Central congenital hypothyroidism (CCH) is a rare endocrine disorder that can be caused by mutations in the ß-subunit of thyrotropin (TSHB). The TSHB mutation C105Vfs114X leads to isolated thyroid-stimulating-hormone-(TSH)-deficiency and results in a severe phenotype. The aim of this study was to gain more insight into the underlying molecular mechanism and the functional effects of this mutation based on two assumptions: a) the three-dimensional (3D) structure of TSH should be modified with the C105V substitution, and/or b) whether the C-terminal modifications lead to signaling differences. 2) Methods: wild-type (WT) and different mutants of hTSH were generated in human embryonic kidney 293 cells (HEK293 cells) and TSH preparations were used to stimulate thyrotropin receptor (TSHR) stably transfected into follicular thyroid cancer cells (FTC133-TSHR cells) and transiently transfected into HEK293 cells. Functional characterization was performed by determination of Gs, mitogen activated protein kinase (MAPK) and Gq/11 activation. 3) Results: The patient mutation C105Vfs114X and further designed TSH mutants diminished cyclic adenosine monophosphate (cAMP) signaling activity. Surprisingly, MAPK signaling for all mutants was comparable to WT, while none of the mutants induced PLC activation. 4) Conclusion: We characterized the patient mutation C105Vfs114X concerning different signaling pathways. We identified a strong decrease of cAMP signaling induction and speculate that this could, in combination with diverse signaling regarding the other pathways, accounting for the patient's severe phenotype.

Relevance of polymorphisms in MC4R and BDNF in short normal stature.

BACKGROUND: Variation in genes of the leptinergic-melanocortinergic system influence both body weight and height. Because short normal stature (SNS) is characterized by reduced body height, delayed maturation and leanness, allelic variation of genes in this pathway are hypothesized to affect this common condition. METHODS: We analyzed the coding regions of LEP, MC4R, MRAP2 and BDNF in 185 children with SNS (height < 5th percentile) to search for non-synonymous and frameshift variants. For association studies (two-sided χ2-tests) population-based data sets (ExAC, EVS and KORA) were used. Cyclic AMP accumulation, cell surface expression, central expression and MAP kinase activation were assayed in vitro to determine the functional implications of identified variants. RESULTS: We detected eleven variants predicted to be protein-altering, four in MC4R, four in BDNF, and three in MRAP2. No variants were found in LEP. In vitro analysis implied reduced function for the MC4R variant p.Met215Ile. Loss-of-function is contrary to expectations based on obesity studies, and thus does not support that this variant is relevant for SNS. The minor SNP alleles at MC4R p.Val103Ile and BDNF p.Val66Met were nominally associated with SNS. CONCLUSION: Taken together, although genes of the leptinergic-melanocortinergic system are important for normal growth, our data do not support the involvement of rare mutations in LEP, MC4R, MRAP2 or BDNF in short normal stature.

The Activation Mechanism of Glycoprotein Hormone Receptors with Implications in the Cause and Therapy of Endocrine Diseases.

Glycoprotein hormones (GPHs) are the main regulators of the pituitary-thyroid and pituitary-gonadal axes. Selective interaction between GPHs and their cognate G protein-coupled receptors ensure specificity in GPH signaling. The mechanisms of how these hormones activate glycoprotein hormone receptors (GPHRs) or how mutations and autoantibodies can alter receptor function were unclear. Based on the hypothesis that GPHRs contain an internal agonist, we systematically screened peptide libraries derived from the ectodomain for agonistic activity on the receptors. We show that a peptide (p10) derived from a conserved sequence in the C-terminal part of the extracellular N terminus can activate all GPHRs in vitro and in GPHR-expressing tissues. Inactivating mutations in this conserved region or in p10 can inhibit activation of the thyroid-stimulating hormone receptor by autoantibodies. Our data suggest an activation mechanism where, upon extracellular ligand binding, this intramolecular agonist isomerizes and induces structural changes in the 7-transmembrane helix domain, triggering G protein activation. This mechanism can explain the pathophysiology of activating autoantibodies and several mutations causing endocrine dysfunctions such as Graves disease and hypo- and hyperthyroidism. Our findings highlight an evolutionarily conserved activation mechanism of GPHRs and will further promote the development of specific ligands useful to treat Graves disease and other dysfunctions of GPHRs.

A new variant in signal peptide of the human luteinizing hormone receptor (LHCGR) affects receptor biogenesis causing leydig cell hypoplasia.

The human luteinizing hormone/chorionic gonadotropin receptor (LHCGR) plays a fundamental role in male and female reproduction. In males, loss-of-function mutations in LHCGR have been associated with distinct degrees of impairment in pre- and postnatal testosterone secretion resulting in a variable phenotypic spectrum, classified as Leydig cell hypoplasia (LCH) type 1 (complete LH resistance and disorder of sex differentiation) and type 2 (partial LH resistance with impaired masculinization and fertility). Here, we report the case of an adolescent who came to the pediatric endocrinologist at the age of 12 years old for micropenis and cryptorchidism. Testis biopsy showed profound LCH and absent germinal line elements (Sertoli-only syndrome). The sequence analysis of the LHCGR gene showed the presence of a compound heterozygosity, being one variation, c.1847C>A p.S616Y, already described in association to Hypergonadotropic Hypogonadism, and the other, c.29 C>T p.L10P, a new identified variant in the putative signal peptide (SP) of LHCGR. Functional and structural studies provide first evidence that LHCGR have a functional and cleavable SP required for receptor biogenesis. Moreover, we demonstrate the pathogenic role of the novel p.L10P allelic variant, which has to be considered a loss-of-function mutation significantly contributing, in compound heterozygosity with p.S616Y, to the LCH type 2 observed in our patient.

Construction of Structural Mimetics of the Thyrotropin Receptor Intracellular Domain.

The signaling of a G protein-coupled receptor (GPCR) is dictated by the complementary responsiveness of interacting intracellular effectors such as G proteins. Many GPCRs are known to couple to more than one G protein subtype and induce a multitude of signaling pathways, although the in vivo relevance of particular pathways is mostly unrecognized. Dissecting GPCR signaling in terms of the pathways that are activated will boost our understanding of the molecular fundamentals of hormone action. The structural determinants governing the selectivity of GPCR/G protein coupling, however, remain obscure. Here, we describe the design of soluble GPCR mimetics to study the details of the interplay between G-proteins and activators. We constructed functional mimetics of the intracellular domain of a model GPCR, the thyrotropin receptor. We based the construction on a unique scaffold, 6-Helix, an artificial protein that was derived from the elements of the trimer-of-hairpins structure of HIV gp41 and represents a bundle of six α-helices. The 6-Helix scaffold, which endowed the substituted thyrotropin receptor intracellular domain elements with spatial constraints analogous to those found in native receptors, enabled the reconstitution of a microdomain that consists of intracellular loops 2 and 3, and is capable of binding and activating Gα-(s). The 6-Helix-based mimetics could be used as a platform to study the molecular basis of GPCR/G protein recognition. Such knowledge could help investigators develop novel therapeutic strategies for GPCR-related disorders by targeting the GPCR/G protein interfaces and counteracting cellular dysfunctions via focused tuning of GPCR signaling.

What are they waiting for?-Tethered agonism in G protein-coupled receptors.

In classical pharmacology agonists bind to their respective receptors by specific interaction and induce structural changes followed by cellular responses. However, some G protein-coupled receptor (GPCRs), such as rhodopsin and protease-activated receptors (PARs), have their agonists already covalently bound and are parts of the receptor proteins, respectively. Recent studies add adhesion GPCRs and glycoprotein hormone receptors (GPHRs) to the group of GPCRs activated by integral agonists. In contrast to rhodopsin and PARs, adhesion GPCRs and GPHRs exhibit large ectodomains (ECDs) which bind a number of different proteins and other extracellular molecules. It seems that these large size ECDs are required to integrate a multitude of extracellular signals, such as protein ligand binding, cell-cell contacts and even mechanical forces, into uniform intracellular signals. Upon extracellular ligand binding, the intramolecular agonist of those receptors is exposed or isomerizes and induces structural changes in the 7-transmembrane helix domain triggering G-protein activation. The existence of activating structures integrated in receptor molecules challenges our current pharmacological definition of an agonist. We summarized and discussed the specifics of tethered agonist pharmacology which add a number of new features of the already broad signaling abilities of GPCRs and may find useful applications in designer GPCRs.