Specific G Protein-coupled Receptors Research Articles

Exploring the Activation Mechanism of the GPR183 Receptor.

The G protein-coupled receptors (GPCRs) play a pivotal role in numerous biological processes as crucial cell membrane receptors. However, the dynamic mechanisms underlying the activation of GPR183, a specific GPCR, remain largely elusive. To address this, we employed computational simulation techniques to elucidate the activation process and key events associated with GPR183, including conformational changes from inactive to active state, binding interactions with the Gi protein complex, and GDP release. Our findings demonstrate that the association between GPR183 and the Gi protein involves the formation of receptor-specific conformations, the gradual proximity of the Gi protein to the binding pocket, and fine adjustments of the protein conformation, ultimately leading to a stable GPR183-Gi complex characterized by a high energy barrier. The presence of Gi protein partially promotes GPR183 activation, which is consistent with the observation of GPCR constitutive activity test experiments, thus illustrating the reliability of our calculations. Moreover, our study suggests the existence of a stable partially activated state preceding complete activation, providing novel avenues for future investigations. In addition, the relevance of GPR183 for various diseases, such as colitis, the response of eosinophils to Mycobacterium tuberculosis infection, antiviral properties, and pulmonary inflammation, has been emphasized, underscoring its therapeutic potential. Consequently, understanding the activation process of GPR183 through molecular dynamic simulations offers valuable kinetic insights that can aid in the development of targeted therapies.

A Study on the Robustness and Stability of Explainable Deep Learning in an Imbalanced Setting: The Exploration of the Conformational Space of G Protein-Coupled Receptors.

G-protein coupled receptors (GPCRs) are transmembrane proteins that transmit signals from the extracellular environment to the inside of the cells. Their ability to adopt various conformational states, which influence their function, makes them crucial in pharmacoproteomic studies. While many drugs target specific GPCR states to exert their effects-thereby regulating the protein's activity-unraveling the activation pathway remains challenging due to the multitude of intermediate transformations occurring throughout this process, and intrinsically influencing the dynamics of the receptors. In this context, computational modeling, particularly molecular dynamics (MD) simulations, may offer valuable insights into the dynamics and energetics of GPCR transformations, especially when combined with machine learning (ML) methods and techniques for achieving model interpretability for knowledge generation. The current study builds upon previous work in which the layer relevance propagation (LRP) technique was employed to interpret the predictions in a multi-class classification problem concerning the conformational states of the β2-adrenergic (β2AR) receptor from MD simulations. Here, we address the challenges posed by class imbalance and extend previous analyses by evaluating the robustness and stability of deep learning (DL)-based predictions under different imbalance mitigation techniques. By meticulously evaluating explainability and imbalance strategies, we aim to produce reliable and robust insights.

The tick Ixodes scapularis has five different GPCRs specifically activated by ACP (adipokinetic hormone/corazonin-related peptide)

Insects have about 50 neuropeptide genes and about 70 genes, coding for neuropeptide G protein-coupled receptors (GPCRs). An important, but small family of evolutionarily related insect neuropeptides consists of adipokinetic hormone (AKH), corazonin, and AKH/corazonin-related peptide (ACP). Normally, insects have one specific GPCR for each of these neuropeptides. The tick Ixodes scapularis is not an insect, but belongs to the subphylum Chelicerata, which comprises ticks, scorpions, mites, spiders, and horseshoe crabs. Many of the neuropeptides and neuropeptide GPCRs occurring in insects, also occur in chelicerates, illustrating that insects and chelicerates are evolutionarily closely related. The tick I. scapularis is an ectoparasite and health risk for humans, because it infects its human host with dangerous pathogens during a blood meal. Understanding the biology of ticks will help researchers to prevent tick-borne diseases. By annotating the I. scapularis genome sequence, we previously found that ticks contain as many as five genes, coding for presumed ACP receptors. In the current paper, we cloned these receptors and expressed each of them in Chinese Hamster Ovary (CHO) cells. Each expressed receptor was activated by nanomolar concentrations of ACP, demonstrating that all five receptors were functional ACP receptors. Phylogenetic tree analyses showed that the cloned tick ACP receptors were mostly related to insect ACP receptors and, next, to insect AKH receptors, suggesting that ACP receptor genes and AKH receptor genes originated by gene duplications from a common ancestor. Similar duplications have probably occurred for the ligand genes, during a process of ligand/receptor co-evolution. Interestingly, chelicerates, in contrast to all other arthropods, do not have AKH or AKH receptor genes. Therefore, the ancestor of chelicerates might have lost AKH and AKH receptor genes and functionally replaced them by ACP and ACP receptor genes. For the small family of AKH, ACP, and corazonin receptors and their ligands, gene losses and gene gains occur frequently between the various ecdysozoan clades. Tardigrades, for example, which are well known for their survival in extreme environments, have as many as ten corazonin receptor genes and six corazonin peptide genes, while insects only have one of each, or none.

Adipocyte G Protein-Coupled Receptors as Potential Targets for Novel Antidiabetic Drugs.

The functional state of adipocytes plays a central role in regulating numerous important metabolic functions, including energy and glucose homeostasis. While white adipocytes store excess calories as fat (triglycerides) and release free fatty acids as a fuel source in times of need, brown and beige adipocytes (so-called thermogenic adipocytes) convert chemical energy stored in substrates (e.g., fatty acids or glucose) into heat, thus promoting energy expenditure. Like all other cell types, adipocytes express many G protein-coupled receptors (GPCRs) that are linked to four major functional classes of heterotrimeric G proteins (Gs, Gi/o, Gq/11, and G12/13). During the past few years, novel experimental approaches, including the use of chemogenetic strategies, have led to a series of important new findings regarding the metabolic consequences of activating or inhibiting distinct GPCR/G protein signaling pathways in white, brown, and beige adipocytes. This novel information should guide the development of novel drugs capable of modulating the activity of specific adipocyte GPCR signaling pathways for the treatment of obesity, type 2 diabetes, and related metabolic disorders.

Identification and characterisation of PRXamide peptides in the western flower thrips, Frankliniella occidentalis.

Insect CAPA-PVK (periviscerokinin) and pyrokinin (PK) neuropeptides belong to the PRX family peptides and are produced from capa and pyrokinin genes. We identified and characterised the two genes from the western flower thrips, Frankliniella occidentalis. The capa gene transcribes three splice variants, capa-a, -b, and -c, encoding two CAPA-PVKs (EVQGLFPFPRVamide; QGLIPFPRVamide) and two PKs (ASWMPSSSPRLamide; DSASFTPRLamide). The pyrokinin mRNA encodes three PKs: DLVTQVLQPGQTGMWFGPRLamide, SEGNLVNFTPRLamide, and ESGEQPEDLEGSMGGAATSRQLRTDSEPTWGFSPRLamide, the most extended pheromone biosynthesis activating neuropeptide (PBAN) ortholog in insects. Multiple potential endoproteolytic cleavage sites were presented in the prepropeptides from the pyrokinin gene, creating ambiguity to predict mature peptides. To solve this difficulty, we used three G protein-coupled receptors (GPCRs) for CAPA-PVK, tryptophan PK (trpPK), and PK peptides, and evaluated the binding affinities of the peptides. The binding activities revealed each subfamily of peptides exclusively bind to their corresponding receptors, and were significant for determining the CAPA-PVK and PK peptides. Our biological method using specific GPCRs would be a valuable tool for determining mature peptides, particularly with multiple and ambiguous cleavage sites in those prepropeptides. Both capa and pyrokinin mRNAs were strongly expressed in the head/thorax, but minimally expressed in the abdomen. The two genes also were clearly expressed during most of the life stages. Whole-mounting immunocytochemistry revealed that neurons contained PRXamide peptides throughout the whole-body: four to six neurosecretory cells in the head, and three and seven pairs of immunostained cells in the thorax and abdomen, respectively. Notably, the unusual PRXamide profiles of Thysanoptera are different from the other insect groups.

Conformational flexibility of β-arrestins - How these scaffolding proteins guide and transform the functionality of GPCRs.

G protein-coupled receptors (GPCRs) constitute the largest family of transmembrane proteins and play a crucial role in regulating diverse cellular functions. They transmit their signaling via binding to intracellular signal transducers and effectors, such as G proteins, GPCR kinases, and β-arrestins. To influence specific GPCR signaling behaviors, β-arrestins recruit effectors to form larger signaling complexes. Intriguingly, they facilitate divergent functions for the binding to different receptors. Recent studies relying on advanced structural approaches, novel biosensors and interactome analyses bring us closer to understanding how this specificity is achieved. In this article, we share our hypothesis of how active GPCRs induce specific conformational rearrangements within β-arrestins to reveal distinct binding interfaces, enabling the recruitment of a subset of effectors to foster specialized signaling complexes. Furthermore, we discuss methods of how to comprehensively assess β-arrestin conformational states and present the current state of research regarding the functionality of these multifaceted scaffolding proteins.

Vesicle-associated membrane protein 2 is a cargo-selective v-SNARE for a subset of GPCRs.

Vesicle fusion at the plasma membrane is critical for releasing hormones and neurotransmitters and for delivering the cognate G protein-coupled receptors (GPCRs) to the cell surface. The SNARE fusion machinery that releases neurotransmitters has been well characterized. In contrast, the fusion machinery that delivers GPCRs is still unknown. Here, using high-speed multichannel imaging to simultaneously visualize receptors and v-SNAREs in real time in individual fusion events, we identify VAMP2 as a selective v-SNARE for GPCR delivery. VAMP2 was preferentially enriched in vesicles that mediate the surface delivery of μ opioid receptor (MOR), but not other cargos, and was required selectively for MOR recycling. Interestingly, VAMP2 did not show preferential localization on MOR-containing endosomes, suggesting that v-SNAREs are copackaged with specific cargo into separate vesicles from the same endosomes. Together, our results identify VAMP2 as a cargo-selective v-SNARE and suggest that surface delivery of specific GPCRs is mediated by distinct fusion events driven by distinct SNARE complexes.

Prospects for targeting ACKR1 in cancer and other diseases.

The chemokine network is comprised of a family of signal proteins that encode messages for cells displaying chemokine G-protein coupled receptors (GPCRs). The diversity of effects on cellular functions, particularly directed migration of different cell types to sites of inflammation, is enabled by different combinations of chemokines activating signal transduction cascades on cells displaying a combination of receptors. These signals can contribute to autoimmune disease or be hijacked in cancer to stimulate cancer progression and metastatic migration. Thus far, three chemokine receptor-targeting drugs have been approved for clinical use: Maraviroc for HIV, Plerixafor for hematopoietic stem cell mobilization, and Mogalizumab for cutaneous T-cell lymphoma. Numerous compounds have been developed to inhibit specific chemokine GPCRs, but the complexity of the chemokine network has precluded more widespread clinical implementation, particularly as anti-neoplastic and anti-metastatic agents. Drugs that block a single signaling axis may be rendered ineffective or cause adverse reactions because each chemokine and receptor often have multiple context-specific functions. The chemokine network is tightly regulated at multiple levels, including by atypical chemokine receptors (ACKRs) that control chemokine gradients independently of G-proteins. ACKRs have numerous functions linked to chemokine immobilization, movement through and within cells, and recruitment of alternate effectors like β-arrestins. Atypical chemokine receptor 1 (ACKR1), previously known as the Duffy antigen receptor for chemokines (DARC), is a key regulator that binds chemokines involved in inflammatory responses and cancer proliferation, angiogenesis, and metastasis. Understanding more about ACKR1 in different diseases and populations may contribute to the development of therapeutic strategies targeting the chemokine network.

Allosteric modulation of G protein-coupled receptor signaling.

G protein-coupled receptors (GPCRs), the largest family of transmembrane proteins, regulate a wide array of physiological processes in response to extracellular signals. Although these receptors have proven to be the most successful class of drug targets, their complicated signal transduction pathways (including different effector G proteins and β-arrestins) and mediation by orthosteric ligands often cause difficulties for drug development, such as on- or off-target effects. Interestingly, identification of ligands that engage allosteric binding sites, which are different from classic orthosteric sites, can promote pathway-specific effects in cooperation with orthosteric ligands. Such pharmacological properties of allosteric modulators offer new strategies to design safer GPCR-targeted therapeutics for various diseases. Here, we explore recent structural studies of GPCRs bound to allosteric modulators. Our inspection of all GPCR families reveals recognition mechanisms of allosteric regulation. More importantly, this review highlights the diversity of allosteric sites and presents how allosteric modulators control specific GPCR pathways to provide opportunities for the development of new valuable agents.

Molecular determinants of distinctive opioid receptor subtype affinities.

Despite being targeted by over 30% of the drugs available on the market, G-Protein Coupled Receptors (GPCR) have proven to be notoriously difficult to characterize on an atomistic level. While it is known that helix 6 (TM6) movement is necessary for signal transduction, structural details of activation remain elusive and each receptor is generally studied individually, making comparisons between homologous proteins challenging. Given their homology, any newly discovered drug targeting a specific GPCR may be promiscuous, adding work- and cost-intensive dimensions to the drug discovery process. Some of the GPCRs that seem the most appealing as drug targets are opioid receptors. As opioids are potent pain relievers with widespread use and addictive properties, understanding how drugs bind particular opioid receptor subtypes would allow the design of non-addictive derivatives and thus help alleviate the ongoing opioid crisis - in the United States only, it is estimated that 4 million people use opioids recreationally or are dependent on them, with about half overdose-related deaths being related to synthetic opioids. In this work, we have used enhanced-sampling molecular dynamics simulations to explore the conformational dynamics of different subtypes of opioid receptors and uncovered the molecular determinants of their affinity to various endogenous peptides and synthetic opioids. Using enhanced sampling techniques, we have obtained free energy surfaces (FES) of activation of the μ-, κ-, and δ-subtypes. By comparing the metastable states obtained from the FES, we have pinpointed key structural differences relevant to the activation process. These findings will allow the development of simple and accurate structure-activity relationship, which in turn will guide the design of new, less addictive opioids.

Off-target pharmacological profiling of synthetic cannabinoid receptor agonists including AMB-FUBINACA, CUMYL-PINACA, PB-22, and XLR-11.

Synthetic cannabinoid receptor agonists (SCRAs) are a diverse class of new psychoactive substances that have been associated with multiple instances and types of toxicity. Some SCRAs appear to carry a greater toxicological burden than others, or compared to the prototypical cannabis-derived agonist Δ9-tetrahydrocannabinol (Δ9-THC), despite a common primary mechanism of action via cannabinoid 1 (CB1) receptors. "Off-target" (i.e., non-CB1 receptor) effects could underpin this differential toxicity, although there are limited data around the activity of SCRAs at such targets. A selection of 7 SCRAs (AMB-FUBINACA, XLR11, PB-22, AKB-48, AB-CHMINICA, CUMYL-PINACA, and 4F-MDMB-BUTINACA), representing several distinct chemotypes and toxicological profiles, underwent a 30 μM single-point screen against 241 G protein-coupled receptor (GPCR) targets in antagonist and agonist mode using a cellular β-arrestin recruitment assay. Strong screening "hits" at specific GPCRs were followed up in detail using concentration-response assays with AMB-FUBINACA, a SCRA with a particularly notable history of toxicological liability. The single-point screen yielded few hits in agonist mode for any compound aside from CB1 and CB2 receptors, but many hits in antagonist mode, including a range of chemokine receptors, the oxytocin receptor, and histamine receptors. Concentration-response experiments showed that AMB-FUBINACA inhibited most off-targets only at the highest 30 μM concentration, with inhibition of only a small subset of targets, including H1 histamine and α2B adrenergic receptors, at lower concentrations (≥1 μM). AMB-FUBINACA also produced concentration-dependent CB1 receptor signaling disruption at concentrations higher than 1 μM, but did not produce overt cytotoxicity beyond CP55,940 or Δ9-THC in CB1 expressing cells. These results suggest that while some "off-targets" could possibly contribute to the SCRA toxidrome, particularly at high concentrations, CB1-mediated cellular dysfunction provides support for hypotheses concerning on-target, rather than off-target, toxicity. Further investigation of non-GPCR off-targets is warranted.

Combinatorial depletions of G-protein coupled receptor kinases in immune cells identify pleiotropic and cell type-specific functions

G-protein coupled receptor kinases (GRKs) participate in the regulation of chemokine receptors by mediating receptor desensitization. They can be recruited to agonist-activated G-protein coupled receptors (GPCRs) and phosphorylate their intracellular parts, which eventually blocks signal propagation and often induces receptor internalization. However, there is growing evidence that GRKs can also control cellular functions beyond GPCR regulation. Immune cells commonly express two to four members of the GRK family (GRK2, GRK3, GRK5, GRK6) simultaneously, but we have very limited knowledge about their interplay in primary immune cells. In particular, we are missing comprehensive studies comparing the role of this GRK interplay for (a) multiple GPCRs within one leukocyte type, and (b) one specific GPCR between several immune cell subsets. To address this issue, we generated mouse models of single, combinatorial and complete GRK knockouts in four primary immune cell types (neutrophils, T cells, B cells and dendritic cells) and systematically addressed the functional consequences on GPCR-controlled cell migration and tissue localization. Our study shows that combinatorial depletions of GRKs have pleiotropic and cell-type specific effects in leukocytes, many of which could not be predicted. Neutrophils lacking all four GRK family members show increased chemotactic migration responses to a wide range of GPCR ligands, whereas combinatorial GRK depletions in other immune cell types lead to pro- and anti-migratory responses. Combined depletion of GRK2 and GRK6 in T cells and B cells shows distinct functional outcomes for (a) one GPCR type in different cell types, and (b) different GPCRs in one cell type. These GPCR-type and cell-type specific effects reflect in altered lymphocyte chemotaxis in vitro and localization in vivo. Lastly, we provide evidence that complete GRK deficiency impairs dendritic cell homeostasis, which unexpectedly results from defective dendritic cell differentiation and maturation in vitro and in vivo. Together, our findings demonstrate the complexity of GRK functions in immune cells, which go beyond GPCR desensitization in specific leukocyte types. Furthermore, they highlight the need for studying GRK functions in primary immune cells to address their specific roles in each leukocyte subset.

Activation of G protein-coupled receptors by ketone bodies: Clinical implication of the ketogenic diet in metabolic disorders.

Ketogenesis takes place in hepatocyte mitochondria where acetyl-CoA derived from fatty acid catabolism is converted to ketone bodies (KB), namely β-hydroxybutyrate (β-OHB), acetoacetate and acetone. KB represent important alternative energy sources under metabolic stress conditions. Ketogenic diets (KDs) are low-carbohydrate, fat-rich eating strategies which have been widely proposed as valid nutritional interventions in several metabolic disorders due to its substantial efficacy in weight loss achievement. Carbohydrate restriction during KD forces the use of FFA, which are subsequently transformed into KB in hepatocytes to provide energy, leading to a significant increase in ketone levels known as "nutritional ketosis". The recent discovery of KB as ligands of G protein-coupled receptors (GPCR) - cellular transducers implicated in a wide range of body functions - has aroused a great interest in understanding whether some of the clinical effects associated to KD consumption might be mediated by the ketone/GPCR axis. Specifically, anti-inflammatory effects associated to KD regimen are presumably due to GPR109A-mediated inhibition of NLRP3 inflammasome by β-OHB, whilst lipid profile amelioration by KDs could be ascribed to the actions of acetoacetate via GPR43 and of β-OHB via GPR109A on lipolysis. Thus, this review will focus on the effects of KD-induced nutritional ketosis potentially mediated by specific GPCRs in metabolic and endocrinological disorders. To discriminate the effects of ketone bodies per se, independently of weight loss, only studies comparing ketogenic vs isocaloric non-ketogenic diets will be considered as well as short-term tolerability and safety of KDs.

Pharmacological targeting of G protein-coupled receptor heteromers

A main rationale for the role of G protein-coupled receptor (GPCR) heteromers as targets for drug development is the putative ability of selective ligands for specific GPCRs to change their pharmacological properties upon GPCR heteromerization. The present study provides a proof of concept for this rationale by demonstrating that heteromerization of dopamine D1 and D3 receptors (D1R and D3R) influences the pharmacological properties of three structurally similar selective dopamine D3R ligands, the phenylpiperazine derivatives PG01042, PG01037 and VK4–116. By using D1R-D3R heteromer-disrupting peptides, it could be demonstrated that the three D3R ligands display different D1R-D3R heteromer-dependent pharmacological properties: PG01042, acting as G protein-biased agonist, counteracted D1R-mediated signaling in the D1R-D3R heteromer; PG01037, acting as a D3R antagonist cross-antagonized D1R-mediated signaling in the D1R-D3R heteromer; and VK4–116 specifically acted as a ß-arrestin-biased agonist in the D1R-D3R heteromer. Molecular dynamics simulations predicted potential molecular mechanisms mediating these qualitatively different pharmacological properties of the selective D3R ligands that are dependent on D1R-D3R heteromerization. The results of in vitro experiments were paralleled by qualitatively different pharmacological properties of the D3R ligands in vivo. The results supported the involvement of D1R-D3R heteromers in the locomotor activation by D1R agonists in reserpinized mice and L-DOPA-induced dyskinesia in rats, highlighting the D1R-D3R heteromer as a main pharmacological target for L-DOPA-induced dyskinesia in Parkinson’s disease. More generally, the present study implies that when suspecting its pathogenetic role, a GPCR heteromer, and not its individual GPCR units, should be considered as main target for drug development.

Finding the Perfect Fit: Conformational Biosensors to Determine the Efficacy of GPCR Ligands.

G protein-coupled receptors (GPCRs) are highly druggable targets that adopt numerous conformations. A ligand's ability to stabilize specific conformation(s) of its cognate receptor determines its efficacy or ability to produce a biological response. Identifying ligands that produce different receptor conformations and potentially discrete pharmacological effects (e.g., biased agonists, partial agonists, antagonists, allosteric modulators) is a major goal in drug discovery and necessary to develop drugs with better effectiveness and fewer side effects. Fortunately, direct measurements of ligand efficacy, via receptor conformational changes are possible with the recent development of conformational biosensors. In this review, we discuss classical efficacy models, including the two-state model, the ternary-complex model, and multistate models. We describe how nanobody-, transducer-, and receptor-based conformational biosensors detect and/or stabilize specific GPCR conformations to identify ligands with different levels of efficacy. In particular, conformational biosensors provide the potential to identify and/or characterize therapeutically desirable but often difficult to measure conformations of receptors faster and better than current methods. For drug discovery/development, several recent proof-of-principle studies have optimized conformational biosensors for high-throughput screening (HTS) platforms. However, their widespread use is limited by the fact that few sensors are reliably capable of detecting low-frequency conformations and technically demanding assay conditions. Nonetheless, conformational biosensors do help identify desirable ligands such as allosteric modulators, biased ligands, or partial agonists in a single assay, representing a distinct advantage over classical methods.

Molecular characterization and functional analysis of Bxy-octr-1 in Bursaphelenchus xylophilus

Bursaphelenchus xylophilus is an invasive plant-parasitic nematode causing the notorious pine wilt disease (PWD) worldwide, which results in huge economic losses. G protein-coupled receptors (GPCRs) play an essential role in mating and reproduction behavior of animals. As a unique biogenic amine in invertebrates, octopamine (OA) can regulate a variety of physiological and behavioral responses by binding specific GPCRs. These specific GPCRs are also called octopamine receptors (OARs), and octr-1 is one of them. However, Bxy-octr-1 is unknown in B. xylophilus. Therefore, we investigated the expression pattern and biological function of Bxy-octr-1. Bioinformatics analysis indicated that Bxy-octr-1 was evolutionarily conserved. The real-time quantitative PCR data revealed that Bxy-octr-1 expression was required throughout the entire life of B. xylophilus. mRNA in situ hybridization showed that Bxy-octr-1 was mainly located in the cephalopharynx, body wall muscle, intestine, and gonadal organs of B. xylophilus. RNA interference (RNAi) showed that embryo hatching rates and locomotion speeds were both dramatically decreased. Obvious abnormal phenotypes were observed in the second-stage of juveniles after RNAi treated. Furthermore, its ontogenesis was stunting. Lack of Bxy-octr-1 reduced fecundity of females, of which 31.25% of them could not successfully ovulate. In addition, the error positioning ratio of the nematode was significantly increased. Our study suggests that Bxy-octr-1 is indispensable for locomotion, early ontogenesis and mating behavior in B. xylophilus.

Identification of Dysregulated Expression of G Protein Coupled Receptors in Endocrine Tumors by Bioinformatics Analysis: Potential Drug Targets?

Background: Many studies link G protein-coupled receptors (GPCRs) to cancer. Some endocrine tumors are unresponsive to standard treatment and/or require long-term and poorly tolerated treatment. This study explored, by bioinformatics analysis, the tumoral profiling of the GPCR transcriptome to identify potential targets in these tumors aiming at drug repurposing. Methods: We explored the GPCR differentially expressed genes (DEGs) from public datasets (Gene Expression Omnibus (GEO) database and The Cancer Genome Atlas (TCGA)). The GEO datasets were available for two medullary thyroid cancers (MTCs), eighty-seven pheochromocytomas (PHEOs), sixty-one paragangliomas (PGLs), forty-seven pituitary adenomas and one-hundred-fifty adrenocortical cancers (ACCs). The TCGA dataset covered 92 ACCs. We identified GPCRs targeted by approved drugs from pharmacological databases (ChEMBL and DrugBank). Results: The profiling of dysregulated GPCRs was tumor specific. In MTC, we found 14 GPCR DEGs, including an upregulation of the dopamine receptor (DRD2) and adenosine receptor (ADORA2B), which were the target of many drugs. In PGL, seven GPCR genes were downregulated, including vasopressin receptor (AVPR1A) and PTH receptor (PTH1R), which were targeted by approved drugs. In ACC, PTH1R was also downregulated in both the GEO and TCGA datasets and was the target of osteoporosis drugs. Conclusions: We highlight specific GPCR signatures across the major endocrine tumors. These data could help to identify new opportunities for drug repurposing.

Expression and Characterization of Relaxin Family Peptide Receptor 1 Variants.

G-protein coupled receptors (GPCR) transduce extracellular stimuli into the cell interior and are thus centrally involved in almost all physiological-neuronal processes. This essential function and association with many diseases or pathological conditions explain why GPCRs are one of the priority targets in medical and pharmacological research, including structure determination. Despite enormous experimental efforts over the last decade, both the expression and purification of these membrane proteins remain elusive. This is attributable to specificities of each GPCR subtype and the finding of necessary experimental in vitro conditions, such as expression in heterologous cell systems or with accessory proteins. One of these specific GPCRs is the leucine-rich repeat domain (LRRD) containing GPCR 7 (LGR7), also termed relaxin family peptide receptor 1 (RXFP1). This receptor is characterized by a large extracellular region of around 400 amino acids constituted by several domains, a rare feature among rhodopsin-like (class A) GPCRs. In the present study, we describe the expression and purification of RXFP1, including the design of various constructs suitable for functional/biophysical studies and structure determination. Based on available sequence information, homology models, and modern biochemical and genetic tools, several receptor variations with different purification tags and fusion proteins were prepared and expressed in Sf9 cells (small-scale), followed by an analytic fluorescence-detection size-exclusion chromatography (F-SEC) to evaluate the constructs. The most promising candidates were expressed and purified on a large-scale, accompanied by ligand binding studies using surface plasmon resonance spectroscopy (SPR) and by determination of signaling capacities. The results may support extended studies on RXFP1 receptor constructs serving as targets for small molecule ligand screening or structural elucidation by protein X-ray crystallography or cryo-electron microscopy.

Physiological roles of mammalian transmembrane adenylyl cyclase isoforms.

Adenylyl cyclases (ACs) catalyze the conversion of ATP to the ubiquitous second messenger cAMP. Mammals possess nine isoforms of transmembrane ACs, dubbed AC1-9, that serve as major effector enzymes of G protein-coupled receptors (GPCRs). The transmembrane ACs display varying expression patterns across tissues, giving the potential for them to have a wide array of physiological roles. Cells express multiple AC isoforms, implying that ACs have redundant functions. Furthermore, all transmembrane ACs are activated by Gαs, so it was long assumed that all ACs are activated by Gαs-coupled GPCRs. AC isoforms partition to different microdomains of the plasma membrane and form prearranged signaling complexes with specific GPCRs that contribute to cAMP signaling compartments. This compartmentation allows for a diversity of cellular and physiological responses by enabling unique signaling events to be triggered by different pools of cAMP. Isoform-specific pharmacological activators or inhibitors are lacking for most ACs, making knockdown and overexpression the primary tools for examining the physiological roles of a given isoform. Much progress has been made in understanding the physiological effects mediated through individual transmembrane ACs. GPCR-AC-cAMP signaling pathways play significant roles in regulating functions of every cell and tissue, so understanding each AC isoform's role holds potential for uncovering new approaches for treating a vast array of pathophysiological conditions.

Abstract P426: Inactivating Grk5 Impairs Basal Cardiac Function And Survival Via P53 Modulation

Introduction: G protein-coupled receptor (GPCR) kinase 5 (GRK5) is a multifunctional protein and depending on its localization within the cell, it has been shown to elicit either protective or deleterious effects. For instance in the heart, when anchored to the plasma membrane, this kinase can regulate specific GPCRs via canonical phosphorylation that can confer cardioprotection. However, when it accumulates in the nucleus its non-canonical activity can drive pathological hypertrophic gene transcription. Interestingly, the latter effects may not be kinase-dependent. Hypothesis: The role played by GRK5’s catalytic activity in the heart has not been fully elucidated and for that reason we sought to assess the in vivo consequences of inactivating the catalytic site of GRK5 with an initial focus at examining the basal cardiac phenotype and response to stress. Methods: We used CRISPR/Cas9 technology to generate a novel knock-in mouse model, with the ATP binding lysine (K) 215 in the catalytic cleft replaced by arginine (R) (GRK5-K215R) resulting in mice devoid of any GRK5 catalytic activity. We studies baseline cardiac function in these mutant mice compared to wild-type (WT) littermates and then stressed them via transverse aortic constriction (TAC). In vitro, we used H9c2 cardiomyocytes and various GRK5 mutants for mechanistic studies. Results: Compared to age-matched WT littermates, GRK5-K215R mice revealed marked and early (9 weeks) deterioration of cardiac function, with augmented apoptosis and fibrosis basally. Importantly, mutant knock-in mice displayed increased p53 gene expression (both at mRNA and protein levels). Moreover, TAC induced increased dysfunction and fibrosis in GRK5-K215R mice compared to WT. Mechanistically, we transduced H9c2 cells with adenoviruses (Ad), encoding for WT GRK5 (Ad-GRK5) or a mutant GRK5 lacking its nuclear localization signal (Ad-NLS) and when GRK5 was localized only outside the nucleus, there was a significant protection against apoptosis, with reduced p53 protein and mRNA levels. Conversely, when we overexpressed a mutant GRK5 without nuclear export signal (GRK5-ΔNES) to trap GRK5 within the nucleus, we found a significant increase in apoptosis, with high p53 protein expression levels. Conclusions: Inactivating GRK5’s catalytic activity impairs its nuclear regulation of p53. This can result in higher levels of p53 mRNA and protein resulting in higher rates of apoptosis in the heart leading to significant cardiac dysfunction and an intolerance to stress.