Blocking receptors on the inside: pepducin-based intervention of PAR signaling and thrombosis

Transmembrane signaling through G-protein coupled receptors (GPCRs) controls a remarkably diverse array of cellular processes including metabolism, growth, motility, adhesion, neuronal signaling, and blood coagulation. The large number of GPCRs and their important roles in normal physiology and in disease have made them the target for more than 50% of prescribed drugs. GPCR agonists and antagonists invariably act on the extracellular surface of the receptors, whereas the intracellular surface has not yet been exploited for development of new therapeutic agents. Here, we demonstrate the utility of novel cell-penetrating peptides, termed pepducins, that act as intracellular inhibitors and/or agonists of signal transference from receptor to G protein. The pepducins require the presence of their cognate receptor for activity and are highly selective for receptor type. Mutational analysis of both intact receptor and pepducins demonstrates that the cell-penetrating agonists do not activate G proteins by the same mechanism as the intact receptor i3 loop, but instead require the C-tail of the receptor. Attachment of a palmitate lipid to shorter i3 loop peptides derived from protease-activated receptors PAR1 and PAR4 created potent inhibitors of thrombin-mediated aggregation of human platelets. Infusion of the anti-PAR4 pepducin into mice extended bleeding time and protected against systemic platelet activation, consistent with the phenotype of a mouse with genetic deficiency of PAR4. These data show that pepducins may be used to ascertain the physiological roles of GPCRs and rapidly determine the potential therapeutic value of blockade of a particular signaling pathway.     

Signaling by sensory receptors

Sensory systems detect small molecules, mechanical perturbations, or radiation via the activation of receptor proteins and downstream signaling cascades in specialized sensory cells. In vertebrates, the two principal categories of sensory receptors are ion channels, which mediate mechanosensation, thermosensation, and acid and salt taste; and G-protein-coupled receptors (GPCRs), which mediate vision, olfaction, and sweet, bitter, and umami tastes. GPCR-based signaling in rods and cones illustrates the fundamental principles of rapid activation and inactivation, signal amplification, and gain control. Channel-based sensory systems illustrate the integration of diverse modulatory signals at the receptor, as seen in the thermosensory/pain system, and the rapid response kinetics that are possible with direct mechanical gating of a channel. Comparisons of sensory receptor gene sequences reveal numerous examples in which gene duplication and sequence divergence have created novel sensory specificities. This is the evolutionary basis for the observed diversity in temperature- and ligand-dependent gating among thermosensory channels, spectral tuning among visual pigments, and odorant binding among olfactory receptors. The coding of complex external stimuli by a limited number of sensory receptor types has led to the evolution of modality-specific and species-specific patterns of retention or loss of sensory information, a filtering operation that selectively emphasizes features in the stimulus that enhance survival in a particular ecological niche. The many specialized anatomic structures, such as the eye and ear, that house primary sensory neurons further enhance the detection of relevant stimuli.     

Amphioxus (Branchiostoma floridae) has orthologs of vertebrate odorant receptors

Background  

A common feature of chemosensory systems is the involvement of G protein-coupled receptors (GPCRs) in the detection of environmental stimuli. Several lineages of GPCRs are involved in vertebrate olfaction, including trace amine-associated receptors, type 1 and 2 vomeronasal receptors and odorant receptors (ORs). Gene duplication and gene loss in different vertebrate lineages have lead to an enormous amount of variation in OR gene repertoire among species; some fish have fewer than 100 OR genes, while some mammals possess more than 1000. Fascinating features of the vertebrate olfactory system include allelic exclusion, where each olfactory neuron expresses only a single OR gene, and axonal guidance where neurons expressing the same receptor project axons to common glomerulae. By identifying homologous ORs in vertebrate and in non-vertebrate chordates, we hope to expose ancestral features of the chordate olfactory system that will help us to better understand the evolution of the receptors themselves and of the cellular components of the olfactory system.     

A novel surface acoustic wave-based biosensor for highly sensitive functional assays of olfactory receptors

Olfactory receptors, which are responsible for sensing odor molecules, form the largest G protein-coupled receptor (GPCR) family in mammalian animals. These proteins play an important role in the detection of chemical signals and signal transduction to the brain. Currently, only a limited number of olfactory receptors have been characterized, which is mainly due to the lack of sensitive and efficient tools for performing functional assays of these receptors. This paper describes a novel surface acoustic wave (SAW)-based biosensor for highly sensitive functional assays of olfactory receptors. An olfactory receptor of Caenorhabditis elegans, ODR-10, was expressed on the plasma membrane of human breast cancer MCF-7 cells, which was used as a model system for this study. For specific odorant response assays, the membrane fraction of MCF-7 cells containing ODR-10 was extracted and integrated with our SAW sensors. The response of ODR-10 to various odorants was monitored by recording the resonance frequency shifts of SAWs applied to the sensor. Our results show that heterologously expressed ODR-10 receptors can specifically respond to diacetyl, its natural ligand. Dose-dependent responses were obtained by performing measurements using various concentrations of diacetyl. The sensitivity of this biosensor is 2 kHz/ng and can detect concentrations as low as 10⁻¹⁰ mM, which is 10× lower than what has previously been reported. This biosensor can be used to characterize odorant response profiles of olfactory receptors and provide information rich data for functional assays of olfactory receptors. In addition to providing a greater understanding of the biological mechanisms of GPCRs, such data holds great potential in many other fields such as food industry, biomedicine, and environmental protection.     

An overview of the diverse roles of G-protein coupled receptors (GPCRs) in the pathophysiology of various human diseases

G-protein coupled receptors (GPCRs) modulate diverse cellular responses to the majority of neurotransmitters and hormones within the human body. They exhibit much structural and functional diversity, and are responsive to a plethora of endogenous (biogenic amines, cations, lipids, peptides, and glycoproteins) and exogenous (therapeutic drugs, photons, tastants, and odorants) ligands and stimuli. Due to the key roles of GPCRs in tissue/cell physiology and homeostasis, signaling pathways associated with GPCRs are implicated in the pathophysiology of various diseases, ranging from metabolic, immunological, and neurodegenerative disorders, to cancer and infectious diseases. Approximately 40% of clinically approved drugs mediate their effects by modulating GPCR signaling pathways, which makes them attractive targets for drug screening and discovery. The pace of discovery of new GPCR-based drugs has recently accelerated due to rapid advancements in high-resolution structure determination, high-throughput screening technology and in silico computational modeling of GPCR binding interaction with potential drug molecules. This review aims to provide an overview of the diverse roles of GPCRs in the pathophysiology of various diseases that are the major focus of biopharmaceutical research as potential drug targets.     

Opsins and clusters of sensory G-protein-coupled receptors in the sea urchin genome

Rhodopsin-type G-protein-coupled receptors (GPCRs) contribute the majority of sensory receptors in vertebrates. With 979 members, they form the largest GPCR family in the sequenced sea urchin genome, constituting more than 3% of all predicted genes. The sea urchin genome encodes at least six Opsin proteins. Of these, one rhabdomeric, one ciliary and two G(o)-type Opsins can be assigned to ancient bilaterian Opsin subfamilies. Moreover, we identified four greatly expanded subfamilies of rhodopsin-type GPCRs that we call sea urchin specific rapidly expanded lineages of GPCRs (surreal-GPCRs). Our analysis of two of these groups revealed genomic clustering and single-exon gene structures similar to the most expanded group of vertebrate rhodopsin-type GPCRs, the olfactory receptors. We hypothesize that these genes arose by rapid duplication in the echinoid lineage and act as chemosensory receptors of the animal. In support of this, group B surreal-GPCRs are most prominently expressed in distinct classes of pedicellariae and tube feet of the adult sea urchin, structures that have previously been shown to react to chemical stimuli and to harbor sensory neurons in echinoderms. Notably, these structures also express different opsins, indicating that sea urchins possess an intricate molecular set-up to sense their environment.     

GPCRs: Emerging anti-cancer drug targets

G protein-coupled receptors (GPCRs) constitute the largest and most diverse protein family in the human genome with over 800 members identified to date. They play critical roles in numerous cellular and physiological processes, including cell proliferation, differentiation, neurotransmission, development and apoptosis. Consequently, aberrant receptor activity has been demonstrated in numerous disorders/diseases, and as a result GPCRs have become the most successful drug target class in pharmaceuticals treating a wide variety of indications such as pain, inflammation, neurobiological and metabolic disorders. Many independent studies have also demonstrated a key role for GPCRs in tumourigenesis, establishing their involvement in cancer initiation, progression, and metastasis. Given the growing appreciation of the role(s) that GPCRs play in cancer pathogenesis, it is surprising to note that very few GPCRs have been effectively exploited in pursuit of anti-cancer therapies. The present review provides a broad overview of the roles that various GPCRs play in cancer growth and development, highlighting the potential of pharmacologically modulating these receptors for the development of novel anti-cancer therapeutics.     

RTP family members induce functional expression of mammalian odorant receptors

Transport of G protein-coupled receptors (GPCRs) to the cell surface membrane is critical in order for the receptors to recognize their ligands. However, mammalian GPCR odorant receptors (ORs), when heterologously expressed in cells, are poorly expressed on the cell surface. Here we show that the transmembrane proteins RTP1 and RTP2 promote functional cell surface expression of ORs expressed in HEK293T cells. Genes encoding these proteins are expressed specifically in olfactory neurons. These proteins are associated with OR proteins and enhance the OR responses to odorants. Similar although weaker effects were seen with a third protein, REEP1. These findings suggest that RTP1 and RTP2 in particular play significant roles in the translocation of ORs to the plasma membrane as well as in the functioning of ORs. We have used this approach to identify active odorant ligands for ORs, providing a platform for screening the chemical selectivity of the large OR family.     

昆虫非典型嗅觉受体Orco的功能和分子结构研究进展

嗅觉受体是参与昆虫嗅觉识别过程的一类重要蛋白。在昆虫的众多嗅觉受体中, 有一类受体明显不同于其他受体, 被称为Orco。该受体基因在不同昆虫种间高度保守, 且表达广泛。Orco在昆虫嗅觉识别过程中发挥关键作用。采用基因突变或RNAi等技术使Orco基因沉默后, 昆虫会出现严重的嗅觉缺陷, 但Orco本身不与气味配体结合, 它与传统嗅觉受体形成复合体Or-Orco, 促进传统嗅觉受体在神经元树突膜上的定位并维持其稳定性, 提高传统嗅觉受体对气味反应的效率。昆虫嗅觉受体的结构与脊椎动物的G蛋白偶联受体相似, 均有7个跨膜区, 但二者的膜拓扑结构相反, 昆虫嗅觉受体的N末端位于细胞质膜内, C末端在细胞质膜外, Orco与传统嗅觉受体通过保守的C末端区域相互作用形成一种新型的配体门控离子通道--Or-Orco复合体。阐明Orco在昆虫嗅觉识别中的功能机制, 可为开创基于昆虫嗅觉行为干扰的新的害虫防治措施提供基础。     

G protein-coupled receptors in energy homeostasis

G-protein coupled receptors (GPCRs) compromise the largest membrane protein superfamily which play vital roles in physiological and pathophysiological processes including energy homeostasis. Moreover, they also represent the up-to-date most successful drug target. The gut hormone GPCRs, such as glucagon receptor and GLP-1 receptor, have been intensively studied for their roles in metabolism and respective drugs have developed for the treatment of metabolic diseases such as type 2 diabetes (T2D). Along with the advances of biomedical research, more GPCRs have been found to play important roles in the regulation of energy homeostasis from nutrient sensing, appetite control to glucose and fatty acid metabolism with various mechanisms. The investigation of their biological functions will not only improve our understanding of how our body keeps the balance of energy intake and expenditure, but also highlight the possible drug targets for the treatment of metabolic diseases. The present review summarizes GPCRs involved in the energy control with special emphasis on their pathophysiological roles in metabolic diseases and hopefully triggers more intensive and systematic investigations in the field so that a comprehensive network control of energy homeostasis will be revealed, and better drugs will be developed in the foreseeable future.     

The cell biology of smell

The olfactory system detects and discriminates myriad chemical structures across a wide range of concentrations. To meet this task, the system utilizes a large family of G protein-coupled receptors-the odorant receptors-which are the chemical sensors underlying the perception of smell. Interestingly, the odorant receptors are also involved in a number of developmental decisions, including the regulation of their own expression and the patterning of the olfactory sensory neurons' synaptic connections in the brain. This review will focus on the diverse roles of the odorant receptor in the function and development of the olfactory system.     

Characteristic amino acid combinations in olfactory G protein-coupled receptors

The human olfactory subgenome has recently been fully characterized with over 1000 genes. Although as many as two thirds of them are expected to be pseudogenes, it still leaves us with about half of all human G protein-coupled receptors being olfactory. It is therefore of great interest to characterize olfactory receptors with high precision. Usually it is done through sequence motifs that are not fully conserved, making an exact characterization difficult. In this paper, we propose a rule-based characterization of olfactory receptors derived from a multiple sequence alignment of human GPCRs. We show that just seven alignment sites are sufficient to characterize 99% of human olfactory GPCRs with one feature, a tyrosine at site 7.41, being of particular importance. We also show dependencies between sites near the extracellular and intracellular region of a membrane-embedded receptor, indicating that olfactory receptors are characterized by a combination of important residues in these two areas, whereas nonolfactory receptors tend to have residues of lower importance at the same sites.     

Functional evolution of the trace amine associated receptors in mammals and the loss of TAAR1 in dogs

Background  

The trace amine associated receptor family is a diverse array of GPCRs that arose before the first vertebrates walked on land. Trace amine associated receptor 1 (TAAR1) is a wide spectrum aminergic receptor that acts as a modulator in brain monoaminergic systems. Other trace amine associated receptors appear to relate to environmental perception and show a birth-and-death pattern in mammals similar to olfactory receptors.     

G protein-mediated stretch reception

Mechanosensation and -transduction are important for physiological processes like the senses of touch, hearing, and balance. The mechanisms underlying the translation of mechanical stimuli into biochemical information by activating various signaling pathways play a fundamental role in physiology and pathophysiology but are only poorly understood. Recently, G protein-coupled receptors (GPCRs), which are essential for the conversion of light, olfactory and gustatory stimuli, as well as of primary messengers like hormones and neurotransmitters into cellular signals and which play distinct roles in inflammation, cell growth, and differentiation, have emerged as potential mechanosensors. The first candidate for a mechanosensitive GPCR was the angiotensin-II type-1 (AT(1)) receptor. Agonist-independent mechanical receptor activation of AT(1) receptors induces an active receptor conformation that appears to differ from agonist-induced receptor conformations and entails the activation of G proteins. Mechanically induced AT(1) receptor activation plays an important role for myogenic vasoconstriction and for the initiation of cardiac hypertrophy. A growing body of evidence suggests that other GPCRs are involved in mechanosensation as well. These findings highlight physiologically relevant, ligand-independent functions of GPCRs and add yet another facet to the polymodal activation spectrum of this ubiquitous protein family.     

Employing Rhodobacter sphaeroides to functionally express and purify human G protein-coupled receptors

G protein-coupled receptors (GPCRs) represent the largest class of cell surface receptors and play crucial roles in many cellular and physiological processes. Functional production of recombinant GPCRs is one of the main bottlenecks to obtaining structural information. Here, we report the use of a novel bacterial expression system based on the photosynthetic bacterium Rhodobacter sphaeroides for the production of human recombinant GPCRs. The advantage of employing R. sphaeroides as a host lies in the fact that it provides much more membrane surface per cell compared to other typical expression hosts. The system was tailored to overexpress recombinant receptors under the control of the moderately strong and highly regulated superoperonic photosynthetic promoter pufQ. We tested this system for the expression of some class A GPCRs, namely, the human adenosine A2a receptor (A2aR), the human angiotensin AT1a receptor (AT1aR) and the human bradykinin B2 receptor (B2R). Several different constructs were examined and functional production of the recombinant receptors was achieved. The best-expressed receptor, AT1aR, was solubilized and affinity-purified. To the best of our knowledge, this is the first report of successful use of a bacterial host--R. sphaeroides--to produce functional recombinant GPCRs under the control of a photosynthetic gene promoter.     

Ubiquitously expressed GPCR membrane-trafficking orthologs

Olfactory receptors are a diverse set of G-protein-coupled receptors (GPCRs) that localize to cellular plasma membranes in the olfactory epithelium. Associated trafficking proteins often assist in targeting these GPCRs to the membrane, facilitating function. One such trafficking protein has been isolated as a mutant defective for both odorant response and proper receptor localization in Caenorhabditis elegans. This gene (ODR-4) allows for functional expression of olfactory receptors in heterologous cells that are otherwise incapable of targeting. We have isolated a full-length human cDNA that is homologous to the C. elegans gene at the protein level across nearly the entire gene by using a novel RecA-based gene enrichment procedure. This sequence is homologous to a family of orthologs that share predicted structural features, indicating a conserved function. The gene was expressed in 41 of 44 human, mouse, and rat tissues, suggesting an important role in trafficking olfactory and other GPCRs.     

Cloning of a novel neuronally expressed orphan G-protein-coupled receptor which is up-regulated by erythropoietin, interacts with microtubule-associated protein 1b and colocalizes with the 5-hydroxytryptamine 2a receptor

G-protein-coupled receptors (GPCRs) are the largest group of cell surface molecules involved in signal transduction and are receptors for a wide variety of stimuli ranging from light, calcium and odourants to biogenic amines and peptides. It is assumed that systematic genomic data-mining has identified the overwhelming majority of all remaining GPCRs in the genome. Here we report the cloning of a novel orphan GPCR which was identified in a search for erythropoietin-induced genes in the brain as a strongly up-regulated gene. This unknown gene coded for a protein which had a seven-transmembrane topology and key features typical of GPCRs of the A family but a low overall identity to all known GPCRs. The protein, coded ee3, has an unusually high evolutionary conservation and is expressed in neurons in diverse areas of the CNS with relation to integrative functions or motor tasks. A yeast two-hybrid screen for interacting proteins revealed binding to the microtubule-associated protein (MAP) 1b. Coupling to MAP1a has been described for another cognate GPCR, the 5-hydroxytryptamine (5HT) 2a receptor. Surprisingly, we found complete colocalization of ee3 and the 5HT2a receptor. The interaction with MAP1b proved to be critical for the stability or folding of ee3 as in mice lacking MAP1b the ee3 protein was undetectable by immunohistochemistry, although messenger RNA levels remained unchanged. We propose that ee3 is a highly interesting new orphan GPCR with potential connections to erythropoietin and 5HT2a receptor signalling.     

Phylogenetic analysis of 277 human G-protein-coupled receptors as a tool for the prediction of orphan receptor ligands

Background

G-protein-coupled receptors (GPCRs) are the largest and most diverse family of transmembrane receptors. They respond to a wide range of stimuli, including small peptides, lipid analogs, amino-acid derivatives, and sensory stimuli such as light, taste and odor, and transmit signals to the interior of the cell through interaction with heterotrimeric G proteins. A large number of putative GPCRs have no identified natural ligand. We hypothesized that a more complete knowledge of the phylogenetic relationship of these orphan receptors to receptors with known ligands could facilitate ligand identification, as related receptors often have ligands with similar structural features.

Results

A database search excluding olfactory and gustatory receptors was used to compile a list of accession numbers and synonyms of 81 orphan and 196 human GPCRs with known ligands. Of these, 241 sequences belonging to the rhodopsin receptor-like family A were aligned and a tentative phylogenetic tree constructed by neighbor joining. This tree and local alignment tools were used to define 19 subgroups of family A small enough for more accurate maximum-likelihood analyses. The secretin receptor-like family B and metabotropic glutamate receptor-like family C were directly subjected to these methods.

Conclusions

Our trees show the overall relationship of 277 GPCRs with emphasis on orphan receptors. Support values are given for each branch. This approach may prove valuable for identification of the natural ligands of orphan receptors as their relation to receptors with known ligands becomes more evident.     

Homo- and hetero-oligomerization of G protein-coupled receptors

G protein-coupled receptors (GPCRs) form homo-oligomeric and hetero-oligomeric complexes. This understanding has prompted a re-evaluation of many aspects of GPCR biology, however the concept of receptor complexes has not been fully integrated into the current thinking about GPCR structure and function. Nevertheless, receptor oligomerization is a pivotal aspect of the structure and function of GPCRs that has been shown to have implications for receptor trafficking, signaling, and pharmacology and more intricate models for understanding the physiological roles of these receptors are emerging. Here, we summarize some of the advances made in understanding the structural basis and the functional roles of homo- and hetero- oligomerization in this important group of receptors. Although this discussion focuses primarily on the dopamine receptors, particularly the D2 dopamine receptor, and the opioid and serotonin receptors, we discuss the principles governing the oligomerization of all rhodopsin-like GPCRs and potentially of the entire superfamily of these receptors.