G-protein Coupled Membrane Receptor Research Articles

G Protein-Coupled Receptor 37L1 Modulates Epigenetic Changes in Human Renal Proximal Tubule Cells.

Renal luminal sodium transport is essential for physiological blood pressure control, and abnormalities in this process are strongly implicated in the pathogenesis of essential hypertension. Renal G protein-coupled receptors (GPCRs) are critical for the regulation of the reabsorption of essential nutrients, ions, and water from the glomerular filtrate. Recently, we showed that GPCR 37L1 (GPR37L1) is expressed on the apical membrane of renal proximal tubules (RPT) and regulates luminal sodium transport and blood pressure by modulating the function of the sodium proton exchanger 3 (NHE3). However, little is known about GPR37L1 intracellular signaling. Here, we show that GPR37L1 is localized to the nuclear membrane, in addition to the plasma membrane in human RPT cells. Furthermore, GPR37L1 signals via the PI3K/AKT/mTOR pathway to decrease the expression of DNA (cytosine-5)-methyltransferase 1 (DNMT1) and enhance NHE3 transcription. Overall, we demonstrate the direct role of a nuclear membrane GPCR in the regulation of renal sodium through epigenetic gene regulation.

Cholesterol-Dependent Dynamics of the Serotonin1A Receptor Utilizing Single Particle Tracking: Analysis of Diffusion Modes.

G protein-coupled receptors (GPCRs) are signaling hubs in cell membranes that regulate a wide range of physiological processes and are popular drug targets. Serotonin1A receptors are important members of the GPCR family and are implicated in neuropsychiatric disorders. Cholesterol is a key constituent of higher eukaryotic membranes and is believed to contribute to the segregated distribution of membrane constituents into domains. To explore the role of cholesterol in lateral dynamics of GPCRs, we utilized single particle tracking (SPT) to monitor diffusion of serotonin1A receptors under acute and chronic cholesterol-depleted conditions. Our results show that the short-term diffusion coefficient of the receptor decreases upon cholesterol depletion, irrespective of the method of cholesterol depletion. Analysis of SPT trajectories revealed that relative populations of receptors undergoing various modes of diffusion change upon cholesterol depletion. Notably, in cholesterol-depleted cells, we observed an increase in the confined population of the receptor accompanied by a reduction in diffusion coefficient for chronic cholesterol depletion. These results are supported by our recent work and present observations that show polymerization of G-actin in response to chronic cholesterol depletion. Taken together, our results bring out the interdependence of cholesterol and actin cytoskeleton in regulating diffusion of GPCRs in membranes.

Lifetime of muscarinic receptor–G-protein complexes determines coupling efficiency and G-protein subtype selectivity

G-protein-coupled receptors (GPCRs) are essential for the detection of extracellular stimuli by cells and transfer the encoded information via the activation of functionally distinct subsets of heterotrimeric G proteins into intracellular signals. Despite enormous achievements toward understanding GPCR structures, major aspects of the GPCR-G-protein selectivity mechanism remain unresolved. As this can be attributed to the lack of suitable and broadly applicable assays, we set out to develop a quantitative FRET-based assay to study kinetics and affinities of G protein binding to activated GPCRs in membranes of permeabilized cells in the absence of nucleotides. We measured the association and dissociation kinetics of agonist-induced binding of Gi/o, Gq/11, Gs, and G12/13 proteins to muscarinic M1, M2, and M3 receptors in the absence of nucleotides between fluorescently labeled G proteins and receptors expressed in mammalian cells. Our results show a strong quantitative correlation between not the on-rates of G-protein-M3-R interactions but rather the affinities of Gq and Go proteins to M3-Rs, their GPCR-G-protein lifetime and their coupling efficiencies determined in intact cells, suggesting that the G-protein subtype-specific affinity to the activated receptor in the absence of nucleotides is, in fact, a major determinant of the coupling efficiency. Our broadly applicable FRET-based assay represents a fast and reliable method to quantify the intrinsic affinity and relative coupling selectivity of GPCRs toward all G-protein subtypes.

Nuclear Membranes GPCRs: Implication in Cardiovascular Health and Diseases

G-protein coupled receptors (GPCRs) are commonly present at the plasma membrane and their signaling modulates excitation-contraction coupling and excitation-secretion coupling of excitable and non-excitable cells of the cardiovascular system. Their effect on excitation-gene expression coupling was attributed, in part, to the nuclear translocation of their signaling and/or to the entry into the nuclear membrane of the internalized GPCRs. However, the recently established paradigm showed that, in addition to plasma membrane G-proteins, GPCRs exist as native nuclear membranes receptors and they modulate nuclear ionic homeostasis and function. These nuclear membrane GPCRs could function independently of plasma membrane GPCRs. Growing evidence also shows that these nuclear membrane GPCRs contribute to protein synthesis and also undergo changes in pathological conditions. The presence of a GPCR at both the plasma and nuclear membranes and/or only at the nuclear membranes represents a new challenge to better understand their contribution to cell physiology and pathology and, consequently, to the development of new therapeutic drugs targeting this category of receptors.

Intracellular and nuclear bradykinin B2 receptors

Bradykinin is a vasoactive peptide that participates in numerous inflammatory processes, vasodilation, and cell growth/survival; it mainly acts through two receptor subtypes, bradykinin B1 and bradykinin B2 receptors, which are G protein-coupled receptor (GPCR) family members. Details on ubiquitin-dependent degradation via the lysosome and/or proteasome, and the recycling process that directs bradykinin B2 receptor to the cell surface after agonist-induced endocytosis remain unclear; nevertheless, intracellular localization and internalization of GPCRs following stimulation by ligands are well known. Evidence concerning the nuclear localization and functions of GPCRs has been accumulating. The bradykinin B2 receptor has been shown to localize in the nucleus and suggested to function as a transcriptional regulator of specific genes. The transfer of membrane GPCRs (regardless of liganding), including the bradykinin B2 receptor to the nucleus can be attributed to the presence of a peptide sequence referred to as the nuclear localization signal (NLS). More recently, we found that nuclear bradykinin B2 receptors form heterodimers with the nuclear lamina protein, lamin C. The function of heterodimerization of the bradykinin B2 receptor with lamin C is still unclear. However, nuclear proteins lamin A/C are involved in a variety of diseases. Although further studies are required to elucidate the precise functions and mechanisms of intracellular and nuclear bradykinin B2 receptors, here we discuss the role of lamin A/C in laminopathies and examine the clinical significance of the bradykinin B2 receptor heterodimer.

A generalized strategy for immobilizing uniformly oriented membrane proteins at solid interfaces

We have developed a method based on self-assembly of thiols on Au substrates to immobilize membrane proteins at interfaces. Using water soluble nitrilotriacetic acid (NTA)-terminated oligo(ethylene glycol) thiols, a histidine-tagged G protein-coupled membrane receptor (GPCR) was captured in a defined orientation with little nonspecific binding.

Bisphenol A promotes testicular seminoma cell proliferation through GPER/GPR30

Dear Sir, Testicular germ cell tumors (TGCT) are the most frequent cancer affecting young men with an increasing incidence all over the world. Pathogenesis and reasons of this increase remain unknown. Epidemiological, clinical and molecular data have suggested that fetal exposure to environmental endocrine disruptors with estrogenic effects could participate in testicular germ cell carcinogenesis by influencing the fate of germ stem cells that share molecular markers with the malignant germ cells. However, no animal model supports this hypothesis, and even estrogen dependency of TGCT has not yet been fully demonstrated. Using the JKT-1 cell line derived from a human testicular seminoma and seminoma tumors, the most frequent TGCTs, we have previously reported that seminoma cells expressed functional aromatase, which is able to convert testosterone into estradiol as well as estrogen receptor beta (ERb) but not ERa. Estradiol 17b (E2) at physiological concentrations was able to suppress in vitro JKT-1 cell proliferation through ERb. However, when E2 was conjugated to bovine serum albumin (E2-BSA), which does not cross the membrane, the effect observed was completely different. E2BSA was able to stimulate JKT-1 cell proliferation by activating the PKA pathway with a rapid (15 min) phosphorylation of the transcription factor cAMP response element binding protein (CREB), through a membrane G-protein-coupled receptor (GPCR). Similar results were obtained with bisphenol A (BPA) at low (pM to nM) concentrations. BPA, a well-recognized estrogenic endocrine disruptor, is used as the monomer to manufacture polycarbonate plastic, and it is released from resin lining of canned foods or beverages or from dental sealants. It is believed to be involved in developmental, reproductive and malignant diseases by mimicking the natural hormone E2 and interfering with

Nuclear membrane receptors and channels as targets for drug development in cardiovascular diseasesThis article is one of a selection of papers from the NATO Advanced Research Workshop on Translational Knowledge for Heart Health (published in part 1 of a 2-part Special Issue).

The use of confocal microscopy has shown that the nucleus plays an important role in excitation-contraction and excitation-secretion coupling of several excitable and nonexcitable cardiovascular cells. It has shown that the nuclear membranes, like the sarcolemmal membrane, possess ionic transporters as well as G protein-coupled receptors (GPCRs), which play a major role in modulating both cytosolic and nuclear ionic homeostasis and nuclear signalling. During spontaneous contraction of heart cells, the increase in cytosolic Ca2+ was immediately followed by a transient increase in nuclear Ca2+. The nuclear Ca2+ rise during excitation-contraction and excitation-secretion coupling was both dependent and independent of changes in cytosolic Ca2+. Nuclear membrane GPCRs, such as those of angiotensin II, neuropeptide Y, and ET-1, were functional and contributed to modulation of nuclear ionic homeostasis via direct and (or) indirect modulation of nuclear membrane ionic transporters such as channels, pumps, and exchangers. The signalling of nuclear membrane GPCRs may also contribute to modulation of gene expression, which may regulate proliferation and remodelling of cells and, indeed, life and death. Direct or indirect targeting of nuclear membrane ionic transporters and GPCRs may constitute a new target for drug action.

Envolvimento de auto-anticorpos na fisiopatologia da Doença de Chagas

Chagas' disease is a serious health problem in Latin America. Between 25 to 30% of the infected patients develop the chronic form of the disease, with progressive myocardial damage and often, sudden death. Adrenergic or cholinergic antibodies with G-protein coupled membrane receptor activity may be present in the sera of these patients. The present study discusses the etiology and the contribution of antibodies to the physiopathology of Chagas' disease.

Insights from biophysical studies on the role of polyunsaturated fatty acids for function of G-protein coupled membrane receptors

The composition of the lipid matrix is critical for function of membrane proteins. Perhaps one of the best studied examples is the function of the G-protein-coupled membrane receptor (GPCR) rhodopsin which is located in membranes with high content of phospholipids with polyunsaturated docosahexaenoic acid chains (DHA, 22:6n-3). Technological advances enabled a more detailed study of structure and dynamics of DHA chains and their interaction with rhodopsin. It was established that polyunsaturated DHA differs from saturated and monounsaturated hydrocarbon chains by far more rapid structural conversions. Furthermore, DHA chains tend to have higher density near the lipid/water interface while density of saturated chains is higher in the bilayer center. The interface of rhodopsin has a small number of sites for tighter interaction with DHA. Polyunsaturated phosphatidylethanolamines accumulate preferentially near the protein. Surprisingly, the high conformational freedom of most DHA chains is not measurably reduced upon interaction with rhodopsin. While some observations point at an involvement of continuum elastic properties of membranes in modulation of rhodopsin function, there is growing evidence for a role of weakly specific DHA–rhodopsin interactions.

Diverse pathways for nuclear signaling by G protein‐coupled receptors and their ligands

Recent realization that plasma membrane G protein-coupled receptors (GPCRs) may translocate and establish ligand-responsive signaling complexes in other cellular structures has motivated studies of site-specific differences in transductional pathways. GPCRs and their ligands may signal transcription and other nuclear events by two basic mechanisms. The first consists of GPCR-complex activation of messengers that enter the nucleus and there initiate cell-modifying processes without the GPCR leaving the plasma membrane. The second encompasses entry into the nuclear membranes or matrix of either GPCR ligands, which bind to non-GPCR nuclear signaling proteins, proteolytic fragments of GPCRs capable of ligand-independent signaling, or intact GPCRs with transduction-competent factors that directly initiate or regulate transcriptional events. With the second mechanism, often concurrent down-regulation of plasma membrane GPCRs terminates signaling from the cell-surface and moves it into the nuclear domain. Site-dependent differences in signals from the same GPCR provide potentials for unique cellular abnormalities attributable to defective intracellular movement and distribution of a GPCR, site-specific alterations in ligand concentration, and limited intracellular bioavailability of pharmacological agents that can interact specifically with both nuclear and plasma membrane forms of a GPCR.

Functional and Structural Characterization of Rhodopsin Oligomers

A major question in G protein-coupled receptor signaling concerns the quaternary structure required for signal transduction. Do these transmembrane receptors function as monomers, dimers, or larger oligomers? We have investigated the oligomeric state of the model G protein-coupled receptor rhodopsin (Rho), which absorbs light and initiates a phototransduction-signaling cascade that forms the basis of vision. In this study, different forms of Rho were isolated using gel filtration techniques in mild detergents, including n-dodecyl-beta-D-maltoside, n-tetradecyl-beta-D-maltoside, and n-hexadecyl-beta-D-maltoside. The quaternary structure of isolated Rho was determined by transmission electron microscopy, demonstrating that in micelles containing n-dodecyl-beta-D-maltoside, Rho exists as a mixture of monomers and dimers whereas in n-tetradecyl-beta-D-maltoside and n-hexadecyl-beta-D-maltoside Rho forms higher ordered structures. Especially in n-hexadecyl-beta-D-maltoside, most of the particles are present in tightly packed rows of dimers. The oligomerization of Rho seems to be important for interaction with its cognate G protein, transducin. Although the activated Rho (Meta II) monomer or dimers are capable of activating the G protein, transducin, the activation process is much faster when Rho exists as organized dimers. Our studies provide direct comparisons between signaling properties of Meta II in different quaternary complexes.

Towards NMR structural studies on the peripheral cannabinoid receptor CB2 in micellar solution.

Human peripheral-type cannabinoid receptor (CB2) is a G protein-coupled membrane receptor (GPCR) involved in the immune and hormonal response. Because of its importance in signal transduction pathways, this membrane protein has become a target for drug design. Development of novel ligands would benefit greatly from NMR structural studies on ligands in the receptor-bound state and the protein. Functional CB2 was successfully expressed in E.coli as a fusion with maltose-binding protein (MBP), thioredoxin (TrxA) and a (His)10 tag (construct 107). The receptor was purified by Ni-NTA affinity chromatography to about 80% purity as judged by silver staining of SDS-PAGE gels. In order to further purify the protein, several constructs were designed which carry a second tag. The construct 109, which carries a biotin ligase recognition site (BRS) downstream of the CB2 sequence, produced in vivo biotinylated protein. However, introduction of this tag did not significantly increase the purity of the protein after elution from a monomeric avidin column. Insertion of the BRS upstream of the CB2 sequence (construct 112) led to a low level of in vivo biotinylation. This protein was successfully biotinylated in vitro and cleaved with Tev protease. Purification of the protein with a monomeric avidin column resulted in a highly enriched CB2 fraction. The efficiency of Tev protease cleavage was tested on a series of CB2-fusions with one or two cleavage sites. Full cleavage of the fusion at mg-quantities was achieved in micellar solution at 4oC. The cleaved CB2-fusion in bacterial membranes retained functionality as shown by ligand binding. Construct 115, with a second decahistidine tag at the N-terminus of CB2, enabled efficient purification of the cleaved CB2 on a Ni-NTA column.

G-protein-coupled receptors signalling at the cell nucleus: an emerging paradigmThis paper is one of a selection of papers published in this Special Issue, entitled The Nucleus: A Cell Within A Cell.

G-protein-coupled receptors (GPCRs) comprise a wide family of monomeric heptahelical glycoproteins that recognize a broad array of extracellular mediators including cationic amines, lipids, peptides, proteins, and sensory agents. Thus far, much attention has been given towards the comprehension of intracellular signaling mechanisms activated by cell membrane GPCRs, which convert extracellular hormonal stimuli into acute, non-genomic (e.g., hormone secretion, muscle contraction, and cell metabolism) and delayed, genomic biological responses (e.g., cell division, proliferation, and apoptosis). However, with respect to the latter response, there is compelling evidence for a novel intracrine mode of genomic regulation by GPCRs that implies either the endocytosis and nuclear translocation of peripheral-liganded GPCR and (or) the activation of nuclearly located GPCR by endogenously produced, nonsecreted ligands. A noteworthy example of the last scenario is given by heptahelical receptors that are activated by bioactive lipoids (e.g., PGE(2) and PAF), many of which may be formed from bilayer membranes including those of the nucleus. The experimental evidence for the nuclear localization and signalling of GPCRs will be reviewed. We will also discuss possible molecular mechanisms responsible for the atypical compartmentalization of GPCRs at the cell nucleus, along with their role in gene expression.

Mechanisms involved in the control of pheromone production in female moths: recent developments

AbstractSpecies‐specific pheromone blends of nocturnal female moths, derived from fatty acid precursors, are produced and released for mate‐finding, and are initiated by the circadian, trophic hormone, Pheromone Biosynthesis Activating Neuropeptide (PBAN). PBAN, produced in the sub‐oesophageal ganglion, is a 33 amino acid neuropeptide with a minimum active core in its FXPRLamide C‐terminal. PBAN acts directly on pheromone gland cells of mature females by binding to a specific G‐protein‐coupled membrane receptor (GPCR), and thereby initiating a signal transduction cascade involving calcium and cAMP. This discussion will review recent developments concerning the identification of the PBAN GPCR, its regulation by juvenile hormone (JH), and its mode of action at the level of the pheromone biosynthetic pathway. The discussion will also include recent developments concerning events occurring as a result of the transfer of pheromonostatic compounds of male origin after mating.

Abnormality of G-protein-coupled receptor kinases at prodromal and early stages of Alzheimer's disease: an association with early beta-amyloid accumulation.

Overwhelming evidence indicates that the effects of beta-amyloid (Abeta) are dose dependent both in vitro and in vivo, which implies that Abeta is not directly detrimental to brain cells until it reaches a threshold concentration. In an effort to understand early Alzheimer's disease (AD) pathogenesis, this study focused on the effects of subthreshold soluble Abeta and the underlying molecular mechanisms in murine microglial cells and an AD transgenic mouse model. We found that there were two phases of dose-dependent Abeta effects on microglial cells: at the threshold of 5 microm and above, Abeta directly induced tumor necrosis factor-alpha (TNF-alpha) release, and at subthreshold doses, Abeta indirectly potentiated TNF-alpha release induced by certain G-protein-coupled receptor (GPCR) activators. Mechanistic studies revealed that subthreshold Abeta pretreatment in vitro reduced membrane GPCR kinase-2/5 (GRK2/5), which led to retarded GPCR desensitization, prolonged GPCR signaling, and cellular hyperactivity to GPCR agonists. Temporal analysis in an early-onset AD transgenic model, CRND8 mice, revealed that the membrane (functional) GRK2/5 in brain cortices were significantly reduced. More importantly, such a GRK abnormality took place before cognitive decline and changed in a manner corresponding with the mild to moderate soluble Abeta accumulation in these transgenic mice. Together, this study not only discovered a novel link between subthreshold Abeta and GRK dysfunction, it also demonstrated that the GRK abnormality in vivo occurs at prodromal and early stages of AD.

Ca2+ sensing receptor activation by CaCl2 increases [Ca2+]i resulting in enhanced spatial interactions with calbindin-D28k protein

The extracellular calcium ion concentration, [Ca2+]o, sensing receptor CaR is a G-protein coupled membrane receptor and it is involved in regulating cell proliferation, differentiation, secretion, and apoptosis. Calbindin-D28k is a high affinity calcium-binding protein that plays important roles in modulating the intracellular calcium ion concentration, [Ca2+]i, and thus influences signal transduction. The role of CaR in sensing and responding to [Ca2+]o and spatial interactions of CaR with calbindin-D28k in a distal tubule-like renal cell line are described. Fura-2 loaded Madin Darby bovine kidney (MDBK) cells were exposed to increasing concentrations of CaCl2 and the [Ca2+]i was determined by ratio fluorescence microscopy. The step-wise addition of CaCl2 caused continual increase in [Ca2+]i. The thapsigargin induced increase in [Ca2+]i observed in basal medium was eliminated by pretreatment of MDBK cells with 10 mM CaCl2 thereby suggesting the involvement of endoplasmic reticulum Ca2+ stores in increasing the [Ca2+]i. CaR was localized in the plasma membrane by using confocal microscopy. The confocal microscopy data also showed CaR and calbindin-D28k were co-localized when cells were exposed to 40 mM CaCl2. We postulate that sensing and responses to increasing [Ca2+]o that occur through CaR, increase the [Ca2+]i causing the translocation of Ca2+-bound calbindin-D28k towards CaR.

Differential gene expression in tumorigenic and nontumorigenic HeLa x normal human fibroblast hybrid cells.

Fusion of tumorigenic HeLa cells with human skin fibroblasts results in chromosomally stable hybrids that are nontumorigenic and no longer express the HeLa tumor-associated marker intestinal alkaline phosphatase (IAP). Previous studies of spontaneous tumorigenic segregants from the nontumorigenic hybrids implicated the loss of one copy of human fibroblast chromosome 11 in the concomitant reexpression of tumorigenicity. In an attempt to identify genes involved in the control of tumorigenic expression, we performed differential display screening of nontumorigenic hybrid cells and tumorigenic segregants. Subsequent northern blot analyses reproducibly showed 17 differentially expressed genes, eight of which were expressed differentially in the nontumorigenic hybrids and nine of which were expressed differentially in the tumorigenic hybrids. The former were genes for 80K-L protein (a substrate of protein kinase C), AXL/UFO (a receptor tyrosine kinase), insulin-like growth factor binding protein 3, apolipoprotein AI regulatory protein, collagen type I alpha-2 chain, transforming growth factor-beta-induced gene product 3 (BIGH3), pregnancy-specific beta-1-glycoprotein, and fibroblast activation protein alpha. The latter nine genes were genes for serum/glucocorticoid-regulated kinase (SGK; a serine/threonine protein kinase), PTPCAAX1 (a tyrosine phosphatase), CXCR-4 (a G-protein-coupled membrane receptor), L-kynurenine hydrolase, beta-1, 4-galactosyltransferase, keratin 8, keratin 17, and H19 and a novel gene. The differential expression of these genes provided several interesting candidates for regulation of tumorigenic expression, including those involved in signal transduction and the extracellular matrix, cytoskeletal proteins, cell-surface enzyme, and the H19 gene.