Cell-Surface Receptors Transactivation Mediated by G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) are seven transmembrane-spanning proteins belonging to a large family of cell-surface receptors involved in many intracellular signaling cascades. Despite GPCRs lack intrinsic tyrosine kinase activity, tyrosine phosphorylation of a tyrosine kinase receptor (RTK) occurs in response to binding of specific agonists of several such receptors, triggering intracellular mitogenic cascades. This suggests that the notion that GPCRs are associated with the regulation of post-mitotic cell functions is no longer believable. Crosstalk between GPCR and RTK may occur by different molecular mechanism such as the activation of metalloproteases, which can induce the metalloprotease-dependent release of RTK ligands, or in a ligand-independent manner involving membrane associated non-receptor tyrosine kinases, such as c-Src. Reactive oxygen species (ROS) are also implicated as signaling intermediates in RTKs transactivation. Intracellular concentration of ROS increases transiently in cells stimulated with GPCR agonists and their deliberated and regulated generation is mainly catalyzed by enzymes that belong to nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family. Oxidation and/or reduction of cysteine sulfhydryl groups of phosphatases tightly controls the activity of RTKs and ROS-mediated inhibition of cellular phosphatases results in an equilibrium shift from the non-phosphorylated to the phosphorylated state of RTKs. Many GPCR agonists activate phospholipase C, which catalyze the hydrolysis of phosphatidylinositol 4,5-bis-phosphate to produce inositol 1,4,5-triphosphate and diacylglicerol. The consequent mobilization of Ca²⁺ from endoplasmic reticulum leads to the activation of protein kinase C (PKC) isoforms. PKCα mediates feedback inhibition of RTK transactivation during GPCR stimulation. Recent data have expanded the coverage of transactivation to include Serine/Threonine kinase receptors and Toll-like receptors. Herein, we discuss the main mechanisms of GPCR-mediated cell-surface receptors transactivation and the pathways involved in intracellular responses induced by GPCR agonists. These studies may suggest the design of novel strategies for therapeutic interventions.     

G-Protein Coupled Receptors (GPCRs): Signaling Pathways,Characterization, and Functions in Insect Physiology and Toxicology

G-protein-coupled receptors (GPCRs) are known to play central roles in the physiology of many organisms. Members of this seven α-helical transmembrane protein family transduce the extracellular signals and regulate intracellular second messengers through coupling to heterotrimeric G-proteins, adenylate cyclase, cAMPs, and protein kinases. As a result of the critical function of GPCRs in cell physiology and biochemistry, they not only play important roles in cell biology and the medicines used to treat a wide range of human diseases but also in insects’ physiological functions. Recent studies have revealed the expression and function of GPCRs in insecticide resistance, improving our understanding of the molecular complexes governing the development of insecticide resistance. This article focuses on the review of G-protein coupled receptor (GPCR) signaling pathways in insect physiology, including insects’ reproduction, growth and development, stress responses, feeding, behaviors, and other physiological processes. Hormones and polypeptides that are involved in insect GPCR regulatory pathways are reviewed. The review also gives a brief introduction of GPCR pathways in organisms in general. At the end of the review, it provides the recent studies on the function of GPCRs in the development of insecticide resistance, focusing in particular on our current knowledge of the expression and function of GPCRs and their downstream regulation pathways and their roles in insecticide resistance and the regulation of resistance P450 gene expression. The latest insights into the exciting technological advances and new techniques for gene expression and functional characterization of the GPCRs in insects are provided.     

G Protein-Coupled Receptors in Cancer

Despite the fact that G protein-coupled receptors (GPCRs) are the largest signal-conveying receptor family and mediate many physiological processes, their role in tumor biology is underappreciated. Numerous lines of evidence now associate GPCRs and their downstream signaling targets in cancer growth and development. Indeed, GPCRs control many features of tumorigenesis, including immune cell-mediated functions, proliferation, invasion and survival at the secondary site. Technological advances have further substantiated GPCR modifications in human tumors. Among these are point mutations, gene overexpression, GPCR silencing by promoter methylation and the number of gene copies. At this point, it is imperative to elucidate specific signaling pathways of “cancer driver” GPCRs. Emerging data on GPCR biology point to functional selectivity and “biased agonism”; hence, there is a diminishing enthusiasm for the concept of “one drug per GPCR target” and increasing interest in the identification of several drug options. Therefore, determining the appropriate context-dependent conformation of a functional GPCR as well as the contribution of GPCR alterations to cancer development remain significant challenges for the discovery of dominant cancer genes and the development of targeted therapeutics.     

Gαq Is the Specific Mediator of PAR-1 Transactivation of Kinase Receptors in Vascular Smooth Muscle Cells

Aims: G protein-coupled receptor (GPCR) transactivation of kinase receptors greatly expands the actions attributable to GPCRs. Thrombin, via its cognate GPCR, protease-activated receptor (PAR)-1, transactivates tyrosine and serine/threonine kinase receptors, specifically the epidermal growth factor receptor and transforming growth factor-β receptor, respectively. PAR-1 transactivation-dependent signalling leads to the modification of lipid-binding proteoglycans involved in the retention of lipids and the development of atherosclerosis. The mechanisms of GPCR transactivation of kinase receptors are distinct. We aimed to investigate the role of proximal G proteins in transactivation-dependent signalling. Main Methods: Using pharmacological and molecular approaches, we studied the role of the G⍺ subunits, G⍺q and G⍺11, in the context of PAR-1 transactivation-dependent signalling leading to proteoglycan modifications. Key Findings: Pan G⍺q subunit inhibitor UBO-QIC/{"type":"entrez-nucleotide","attrs":{"text":"FR900359","term_id":"525221046"}}FR900359 inhibited PAR-1 transactivation of kinase receptors and proteoglycans modification. The G⍺q/11 inhibitor YM254890 did not affect PAR-1 transactivation pathways. Molecular approaches revealed that of the two highly homogenous G⍺q members, G⍺q and G⍺11, only the G⍺q was involved in regulating PAR-1 mediated proteoglycan modification. Although G⍺q and G⍺11 share approximately 90% homology at the protein level, we show that the two isoforms exhibit different functional roles. Significance: Our findings may be extrapolated to other GPCRs involved in vascular pathology and highlight the need for novel pharmacological tools to assess the role of G proteins in GPCR signalling to expand the preeminent position of GPCRs in human therapeutics.     

A Rapid and Efficient Immunoenzymatic Assay to Detect Receptor Protein Interactions: G Protein-Coupled Receptors

G protein-coupled receptors (GPCRs) represent one of the largest families of cell surface receptors, and are the target of at least one-third of the current therapeutic drugs on the market. Along their life cycle, GPCRs are accompanied by a range of specialized GPCR-interacting proteins (GIPs), which take part in receptor proper folding, targeting to the appropriate subcellular compartments and in receptor signaling tasks, and also in receptor regulation processes, such as desensitization and internalization. The direction of protein-protein interactions and multi-protein complexes formation is crucial in understanding protein function and their implication in pathological events. Although several methods have been already developed to assay protein complexes, some of them are quite laborious, expensive, and, more important, they do not generate fully quantitative results. Herein, we show a rapid immunoenzymatic assay to quantify GPCR interactionswith its signaling proteins. The recently de-orphanized GPCR, GPR17, was chosen as a GPCR prototype to optimize the assay. In a GPR17 transfected cell line and primary oligodendrocyte precursor cells, GPR17 interaction with proteins involved in the typical GPCR regulation, such as desensitization and internalization machinery, was investigated. The obtained results were validated by co-immunoprecipitation experiments, confirming this new method as a rapid and quantitative assay to study protein-protein interactions.     

Pharmacology of Free Fatty Acid Receptors and Their Allosteric Modulators

The physiological function of free fatty acids (FFAs) has long been regarded as indirect in terms of their activities as educts and products in metabolic pathways. The observation that FFAs can also act as signaling molecules at FFA receptors (FFARs), a family of G protein-coupled receptors (GPCRs), has changed the understanding of the interplay of metabolites and host responses. Free fatty acids of different chain lengths and saturation statuses activate FFARs as endogenous agonists via binding at the orthosteric receptor site. After FFAR deorphanization, researchers from the pharmaceutical industry as well as academia have identified several ligands targeting allosteric sites of FFARs with the aim of developing drugs to treat various diseases such as metabolic, (auto)inflammatory, infectious, endocrinological, cardiovascular, and renal disorders. GPCRs are the largest group of transmembrane proteins and constitute the most successful drug targets in medical history. To leverage the rich biology of this target class, the drug industry seeks alternative approaches to address GPCR signaling. Allosteric GPCR ligands are recognized as attractive modalities because of their auspicious pharmacological profiles compared to orthosteric ligands. While the majority of marketed GPCR drugs interact exclusively with the orthosteric binding site, allosteric mechanisms in GPCR biology stay medically underexploited, with only several allosteric ligands currently approved. This review summarizes the current knowledge on the biology of FFAR1 (GPR40), FFAR2 (GPR43), FFAR3 (GPR41), FFAR4 (GPR120), and GPR84, including structural aspects of FFAR1, and discusses the molecular pharmacology of FFAR allosteric ligands as well as the opportunities and challenges in research from the perspective of drug discovery.     

The GRKs Reactome: Role in Cell Biology and Pathology

G protein-coupled receptor kinases (GRKs) are protein kinases that function in concert with arrestins in the regulation of a diverse class of G protein-coupled receptors (GPCRs) signaling. Although GRKs and arrestins are key participants in the regulation of GPCR cascades, the complex regulatory mechanisms of GRK expression, its alternation, and their function are not thoroughly understood. Several studies together with the work from our lab in recent years have revealed the critical role of these kinases in various physiological and pathophysiological processes, including cardiovascular biology, inflammation and immunity, neurodegeneration, thrombosis, and hemostasis. A comprehensive understanding of the mechanisms underlying functional interactions with multiple receptor proteins and how these interactions take part in the development of various pathobiological processes may give rise to novel diagnostic and therapeutic strategies. In this review, we summarize the current research linking the role of GRKs to various aspects of cell biology, pathology, and therapeutics, with a particular focus on thrombosis and hemostasis.     

Calmodulin Supports TRPA1 Channel Association with Opioid Receptors and Glutamate NMDA Receptors in the Nervous Tissue

Transient receptor potential ankyrin member 1 (TRPA1) belongs to the family of thermo TRP cation channels that detect harmful temperatures, acids and numerous chemical pollutants. TRPA1 is expressed in nervous tissue, where it participates in the genesis of nociceptive signals in response to noxious stimuli and mediates mechanical hyperalgesia and allodynia associated with different neuropathies. The glutamate N-methyl-d-aspartate receptor (NMDAR), which plays a relevant role in allodynia to mechanical stimuli, is connected via histidine triad nucleotide-binding protein 1 (HINT1) and type 1 sigma receptor (σ1R) to mu-opioid receptors (MORs), which mediate the most potent pain relief. Notably, neuropathic pain causes a reduction in MOR antinociceptive efficacy, which can be reversed by blocking spinal NMDARs and TRPA1 channels. Thus, we studied whether TRPA1 channels form complexes with MORs and NMDARs that may be implicated in the aforementioned nociceptive signals. Our data suggest that TRPA1 channels functionally associate with MORs, delta opioid receptors and NMDARs in the dorsal root ganglia, the spinal cord and brain areas. These associations were altered in response to pharmacological interventions and the induction of inflammatory and also neuropathic pain. The MOR-TRPA1 and NMDAR-TRPA1 associations do not require HINT1 or σ1R but appear to be mediated by calcium-activated calmodulin. Thus, TRPA1 channels may associate with NMDARs to promote ascending acute and chronic pain signals and to control MOR antinociception.     

Intersection of the Orphan G Protein-Coupled Receptor,GPR19, with the Aging Process

G protein-coupled receptors (GPCRs) represent one of the most functionally diverse classes of transmembrane proteins. GPCRs and their associated signaling systems have been linked to nearly every physiological process. They also constitute nearly 40% of the current pharmacopeia as direct targets of remedial therapies. Hence, their place as a functional nexus in the interface between physiological and pathophysiological processes suggests that GPCRs may play a central role in the generation of nearly all types of human disease. Perhaps one mechanism through which GPCRs can mediate this pivotal function is through the control of the molecular aging process. It is now appreciated that, indeed, many human disorders/diseases are induced by GPCR signaling processes linked to pathological aging. Here we discuss one such novel member of the GPCR family, GPR19, that may represent an important new target for novel remedial strategies for the aging process. The molecular signaling pathways (metabolic control, circadian rhythm regulation and stress responsiveness) associated with this recently characterized receptor suggest an important role in aging-related disease etiology.     

Monitoring TRPC7 Conformational Changes by BRET Following GPCR Activation

Transient receptor potential canonical (TRPC) channels are membrane proteins involved in regulating Ca²⁺ homeostasis, and whose functions are modulated by G protein-coupled receptors (GPCR). In this study, we developed bioluminescent resonance energy transfer (BRET) biosensors to better study channel conformational changes following receptor activation. For this study, two intramolecular biosensors, GFP10-TRPC7-RLucII and RLucII-TRPC7-GFP10, were constructed and were assessed following the activation of various GPCRs. We first transiently expressed receptors and the biosensors in HEK293 cells, and BRET levels were measured following agonist stimulation of GPCRs. The activation of GPCRs that engage Gα_q led to a Gα_q-dependent BRET response of the functional TRPC7 biosensor. Focusing on the Angiotensin II type-1 receptor (AT₁R), GFP10-TRPC7-RLucII was tested in rat neonatal cardiac fibroblasts, expressing endogenous AT₁R and TRPC7. We detected similar BRET responses in these cells, thus validating the use of the biosensor in physiological conditions. Taken together, our results suggest that activation of Gα_q-coupled receptors induce conformational changes in a novel and functional TRPC7 BRET biosensor.     

G Protein-Coupled Receptors: What a Difference a ‘Partner’ Makes

G protein-coupled receptors (GPCRs) are important cell signaling mediators, involved in essential physiological processes. GPCRs respond to a wide variety of ligands from light to large macromolecules, including hormones and small peptides. Unfortunately, mutations and dysregulation of GPCRs that induce a loss of function or alter expression can lead to disorders that are sometimes lethal. Therefore, the expression, trafficking, signaling and desensitization of GPCRs must be tightly regulated by different cellular systems to prevent disease. Although there is substantial knowledge regarding the mechanisms that regulate the desensitization and down-regulation of GPCRs, less is known about the mechanisms that regulate the trafficking and cell-surface expression of newly synthesized GPCRs. More recently, there is accumulating evidence that suggests certain GPCRs are able to interact with specific proteins that can completely change their fate and function. These interactions add on another level of regulation and flexibility between different tissue/cell-types. Here, we review some of the main interacting proteins of GPCRs. A greater understanding of the mechanisms regulating their interactions may lead to the discovery of new drug targets for therapy.     

Misfolded G Protein-Coupled Receptors and Endocrine Disease. Molecular Mechanisms and Therapeutic Prospects

Misfolding of G protein-coupled receptors (GPCRs) caused by mutations frequently leads to disease due to intracellular trapping of the conformationally abnormal receptor. Several endocrine diseases due to inactivating mutations in GPCRs have been described, including X-linked nephrogenic diabetes insipidus, thyroid disorders, familial hypocalciuric hypercalcemia, obesity, familial glucocorticoid deficiency [melanocortin-2 receptor, MC2R (also known as adrenocorticotropin receptor, ACTHR), and reproductive disorders. In these mutant receptors, misfolding leads to endoplasmic reticulum retention, increased intracellular degradation, and deficient trafficking of the abnormal receptor to the cell surface plasma membrane, causing inability of the receptor to interact with agonists and trigger intracellular signaling. In this review, we discuss the mechanisms whereby mutations in GPCRs involved in endocrine function in humans lead to misfolding, decreased plasma membrane expression of the receptor protein, and loss-of-function diseases, and also describe several experimental approaches employed to rescue trafficking and function of the misfolded receptors. Special attention is given to misfolded GPCRs that regulate reproductive function, given the key role played by these particular membrane receptors in sexual development and fertility, and recent reports on promising therapeutic interventions targeting trafficking of these defective proteins to rescue completely or partially their normal function.     

Structural Characterization of Receptor–Receptor Interactions in the Allosteric Modulation of G Protein-Coupled Receptor (GPCR) Dimers

G protein-coupled receptor (GPCR) oligomerization, while contentious, continues to attract the attention of researchers. Numerous experimental investigations have validated the presence of GPCR dimers, and the relevance of dimerization in the effectuation of physiological functions intensifies the attractiveness of this concept as a potential therapeutic target. GPCRs, as a single entity, have been the main source of scrutiny for drug design objectives for multiple diseases such as cancer, inflammation, cardiac, and respiratory diseases. The existence of dimers broadens the research scope of GPCR functions, revealing new signaling pathways that can be targeted for disease pathogenesis that have not previously been reported when GPCRs were only viewed in their monomeric form. This review will highlight several aspects of GPCR dimerization, which include a summary of the structural elucidation of the allosteric modulation of class C GPCR activation offered through recent solutions to the three-dimensional, full-length structures of metabotropic glutamate receptor and γ-aminobutyric acid B receptor as well as the role of dimerization in the modification of GPCR function and allostery. With the growing influence of computational methods in the study of GPCRs, we will also be reviewing recent computational tools that have been utilized to map protein–protein interactions (PPI).     

Structural Basis of Arrestin Selectivity for Active Phosphorylated G Protein-Coupled Receptors

Arrestins are a small family of proteins that bind G protein-coupled receptors (GPCRs). Arrestin binds to active phosphorylated GPCRs with higher affinity than to all other functional forms of the receptor, including inactive phosphorylated and active unphosphorylated. The selectivity of arrestins suggests that they must have two sensors, which detect receptor-attached phosphates and the active receptor conformation independently. Simultaneous engagement of both sensors enables arrestin transition into a high-affinity receptor-binding state. This transition involves a global conformational rearrangement that brings additional elements of the arrestin molecule, including the middle loop, in contact with a GPCR, thereby stabilizing the complex. Here, we review structural and mutagenesis data that identify these two sensors and additional receptor-binding elements within the arrestin molecule. While most data were obtained with the arrestin-1-rhodopsin pair, the evidence suggests that all arrestins use similar mechanisms to achieve preferential binding to active phosphorylated GPCRs.     

Age-Dependent Contributions of NMDA Receptors and L-Type Calcium Channels to Long-Term Depression in the Piriform Cortex

In the hippocampus, the contributions of N-methyl-D-aspartate receptors (NMDARs) and L-type calcium channels (LTCCs) to neuronal transmission and synaptic plasticity change with aging, underlying calcium dysregulation and cognitive dysfunction. However, the relative contributions of NMDARs and LTCCs in other learning encoding structures during aging are not known. The piriform cortex (PC) plays a significant role in odor associative memories, and like the hippocampus, exhibits forms of long-term synaptic plasticity. Here, we investigated the expression and contribution of NMDARs and LTCCs in long-term depression (LTD) of the PC associational fiber pathway in three cohorts of Sprague Dawley rats: neonatal (1–2 weeks), young adult (2–3 months) and aged (20–25 months). Using a combination of slice electrophysiology, Western blotting, fluorescent immunohistochemistry and confocal imaging, we observed a shift from an NMDAR to LTCC mediation of LTD in aged rats, despite no difference in the amount of LTD expression. These changes in plasticity are related to age-dependent differential receptor expression in the PC. LTCC Cav1.2 expression relative to postsynaptic density protein 95 is increased in the associational pathway of the aged PC layer Ib. Enhanced LTCC contribution in synaptic depression in the PC may contribute to altered olfactory function and learning with aging.     

Structure‐Based and Fragment‐Based GPCR Drug Discovery

G protein‐coupled receptors (GPCRs) are an important family of membrane proteins; historically, drug discovery in this target class has been fruitful, with many of the world’s top‐selling drugs being GPCR modulators. Until recently, the modern techniques of structure‐ and fragment‐based drug discovery had not been fully applied to GPCRs, primarily because of the instability of these proteins when isolated from their cell membrane environments. Recent advances in receptor stabilisation have facilitated major advances in GPCR structural biology over the past six years, with 21 new receptor targets successfully crystallised with one or more ligands. The dramatic increase in GPCR structural information has yielded an increased use of structure‐based methods for hit identification and progression, which are reviewed herein. Additionally, a number of fragment‐based drug discovery techniques have been validated for use with GPCRs in recent years; these approaches and their use in hit identification are reviewed.     

Amyloid Beta-Mediated Changes in Synaptic Function and Spine Number of Neocortical Neurons Depend on NMDA Receptors

Onset and progression of Alzheimer’s disease (AD) pathophysiology differs between brain regions. The neocortex, for example, is a brain region that is affected very early during AD. NMDA receptors (NMDARs) are involved in mediating amyloid beta (Aβ) toxicity. NMDAR expression, on the other hand, can be affected by Aβ. We tested whether the high vulnerability of neocortical neurons for Aβ-toxicity may result from specific NMDAR expression profiles or from a particular regulation of NMDAR expression by Aβ. Electrophysiological analyses suggested that pyramidal cells of 6-months-old wildtype mice express mostly GluN1/GluN2A NMDARs. While synaptic NMDAR-mediated currents are unaltered in 5xFAD mice, extrasynaptic NMDARs seem to contain GluN1/GluN2A and GluN1/GluN2A/GluN2B. We used conditional GluN1 and GluN2B knockout mice to investigate whether NMDARs contribute to Aβ-toxicity. Spine number was decreased in pyramidal cells of 5xFAD mice and increased in neurons with 3-week virus-mediated Aβ-overexpression. NMDARs were required for both Aβ-mediated changes in spine number and functional synapses. Thus, our study gives novel insights into the Aβ-mediated regulation of NMDAR expression and the role of NMDARs in Aβ pathophysiology in the somatosensory cortex.     

Orthogonal Peptide-Templated Labeling Elucidates Lateral ETAR/ETBR Proximity and Reveals Altered Downstream Signaling

Fine-tuning of G protein-coupled receptor (GPCR) signaling is important to maintain cellular homeostasis. Recent studies demonstrated that lateral GPCR interactions in the cell membrane can impact signaling profiles. Here, we report on a one-step labeling method of multiple membrane-embedded GPCRs. Based on short peptide tags, complementary probes transfer the cargo (e. g. a fluorescent dye) by an acyl transfer reaction with high spatial and temporal resolution within 5 min. We applied this approach to four receptors of the cardiovascular system: the endothelin receptor A and B (ET_AR and ET_BR), angiotensin II receptor type 1, and apelin. Wild type-like G protein activation after N-terminal modification was demonstrated for all receptor species. Using FRET-competent dyes, a constitutive proximity between hetero-receptors was limited to ET_AR/ET_BR. Further, we demonstrate, that ET_AR expression regulates the signaling of co-expressed ET_BR. Our orthogonal peptide-templated labeling of different GPCRs provides novel insight into the regulation of GPCR signaling.