Expression Of G Protein-coupled Receptors Research Articles

Using the novel HiBiT tag to label cell surface relaxin receptors for BRET proximity analysis.

Relaxin family peptide 1 (RXFP1) is the receptor for relaxin a peptide hormone with important therapeutic potential. Like many G protein‐coupled receptors (GPCRs), RXFP1 has been reported to form homodimers. Given the complex activation mechanism of RXFP1 by relaxin, we wondered whether homodimerization may be explicitly required for receptor activation, and therefore sought to determine if there is any relaxin‐dependent change in RXFP1 proximity at the cell surface. Bioluminescence resonance energy transfer (BRET) between recombinantly tagged receptors is often used in GPCR proximity studies. RXFP1 targets poorly to the cell surface when overexpressed in cell lines, with the majority of the receptor proteins sequestered within the cell. Thus, any relaxin‐induced changes in RXFP1 proximity at the cell surface may be obscured by BRET signal originating from intracellular compartments. We therefore, utilized the newly developed split luciferase system called HiBiT to specifically label the extracellular terminus of cell surface RXFP1 receptors in combination with mCitrine‐tagged receptors, using the GABAB heterodimer as a positive control. This demonstrated that the BRET signal detected from RXFP1‐RXFP1 proximity at the cell surface does not appear to be due to stable physical interactions. The fact that there is also no relaxin‐mediated change in RXFP1‐RXFP1 proximity at the cell surface further supports these conclusions. This work provides a basis by which cell surface GPCR proximity and expression levels can be specifically studied using a facile and homogeneous labeling technique such as HiBiT.

Gi/o-Protein Coupled Receptors in the Aging Brain.

Cells translate extracellular signals to regulate processes such as differentiation, metabolism and proliferation, via transmembranar receptors. G protein-coupled receptors (GPCRs) belong to the largest family of transmembrane receptors, with over 800 members in the human species. Given the variety of key physiological functions regulated by GPCRs, these are main targets of existing drugs. During normal aging, alterations in the expression and activity of GPCRs have been observed. The central nervous system (CNS) is particularly affected by these alterations, which results in decreased brain functions, impaired neuroregeneration, and increased vulnerability to neuropathologies, such as Alzheimer’s and Parkinson diseases. GPCRs signal via heterotrimeric G proteins, such as Go, the most abundant heterotrimeric G protein in CNS. We here review age-induced effects of GPCR signaling via the Gi/o subfamily at the CNS. During the aging process, a reduction in protein density is observed for almost half of the Gi/o-coupled GPCRs, particularly in age-vulnerable regions such as the frontal cortex, hippocampus, substantia nigra and striatum. Gi/o levels also tend to decrease with aging, particularly in regions such as the frontal cortex. Alterations in the expression and activity of GPCRs and coupled G proteins result from altered proteostasis, peroxidation of membranar lipids and age-associated neuronal degeneration and death, and have impact on aging hallmarks and age-related neuropathologies. Further, due to oligomerization of GPCRs at the membrane and their cooperative signaling, down-regulation of a specific Gi/o-coupled GPCR may affect signaling and drug targeting of other types/subtypes of GPCRs with which it dimerizes. Gi/o-coupled GPCRs receptorsomes are thus the focus of more effective therapeutic drugs aiming to prevent or revert the decline in brain functions and increased risk of neuropathologies at advanced ages.

Neutrophil GPCRs as novel therapeutic targets

Each year in the United States, 2 million people are infected with antibiotic resistant pathogens, such as methicillin‐resistant Staphylococcus aureus (MRSA); 23,000 patients die as a result of such infections. With the growth of antibiotic resistance and the slowing in the development of new antibiotics, the discovery of new, effective approaches to treat infections is an important unmet medical need. G protein‐coupled receptors (GPCRs) in polymorphonuclear neutrophils (PMNs), the first‐line effectors of the innate immune response, are a novel but unexplored potential class of “immune boosting” targets. Using a combination of qPCR based arrays and RNAseq analysis, we defined and quantified GPCR expression in human PMNs and found 174 GPCRs commonly expressed among healthy donors tested. To help determine which GPCRs might be involved in response to pathogens, we assessed the GPCR profile of PMNs incubated with MRSA for 3 h. We found numerous GPCRs whose expression increased at least 2‐fold upon pathogen exposure. Analyses of PMN function (e.g., chemotaxis, extracellular trap production, bacterial killing) using selective agonists/antagonists of several such GPCRs indicated that pharmacological agents can “boost” the function of PMNs, including the killing of bacteria. The results indicate that: 1) Human PMNs express a large profile of GPCRs, expression of a number of which is altered by exposure to pathogenic bacteria. 2) Previously unrecognized PMN‐expressed GPCRs can boost PMN function, including the ability to kill bacteria. These data thus suggest a new paradigm for enhancement of innate immunity: targeting of neutrophil‐expressed GPCRs as a means to treat bacterial infections, including ones caused by antibiotic‐resistant bacteria. Furthermore, such GPCRs may also represent new therapeutic targets for the treatment of inflammatory disorders driven by aberrant neutrophil function.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Phosphoproteomic and Functional Analyses Reveal Sperm-specific Protein Changes Downstream of Kappa Opioid Receptor in Human Spermatozoa

G-protein coupled receptors (GPCRs) belong to the seven transmembrane receptor superfamily that transduce signals via G proteins in response to external stimuli to initiate different intracellular signaling pathways which culminate in specific cellular responses. The expression of diverse GPCRs at the plasma membrane of human spermatozoa suggests their involvement in the regulation of sperm fertility. However, the signaling events downstream of many GPCRs in spermatozoa remain uncharacterized. Here, we selected the kappa-opioid receptor (KOR) as a study model and applied phosphoproteomic approach based on TMT labeling and LC-MS/MS analyses. Quantitative coverage of more than 5000 proteins with over 3500 phosphorylation sites revealed changes in the phosphorylation levels of sperm-specific proteins involved in the regulation of the sperm fertility in response to a specific agonist of KOR, U50488H. Further functional studies indicate that KOR could be involved in the regulation of sperm fertile capacity by modulation of calcium channels. Our findings suggest that human spermatozoa possess unique features in the molecular mechanisms downstream of GPCRs which could be key regulators of sperm fertility and improved knowledge of these specific processes may contribute to the development of useful biochemical tools for diagnosis and treatment of male infertility.

Determining the insulin secretion potential for certain specific G-protein coupled receptors in MIN6 pancreatic beta cells

Background/aimThe polypeptide hormone insulin is essential for the maintenance of whole-body fuel homeostasis, and defects in insulin secretion and/or action are associated with the development of type 1 and type 2 diabetes. The aim of this study was to assess the role of some G-protein coupled receptors (GPCRs), GPR54, GPR56, and GPR75, and cannabinoid receptors CB1R and CB2R, in the regulation of pancreatic β-cell function.Materials and methods Insulin secretion from mouse insulinoma β-cell line (MIN6) monolayers was assessed via insulin radioimmunoassay (RIA). Reverse transcription-polymerase chain reaction (RT-PCR) was used to assess the expression of some specific GPCRs and the other receptors by MIN6 pancreatic β-cells. Results The agonists were not found to be toxic for the MIN6 pancreatic β-cells within the range of the doses used in this study, whereas insulin secretion altered depending on the ligands and receptors. In addition, arachidonyl-2’-chloroethylamide (ACEA), carbachol, chemokine (C-C motif) ligand-5 (CCL5), and exendin as well as phorbol myristate acetate (PMA) ligands showed significant increases in the insulin secretion of MIN6 pancreatic β-cells.Conclusion Understanding the normal β-cell function and identifying the defects in β-cell function that lead to the development of diabetes will generate new therapeutic targets.

G protein-coupled receptor trafficking and signaling: new insights into the enteric nervous system.

G protein-coupled receptors (GPCRs) are essential for the neurogenic control of gastrointestinal (GI) function and are important and emerging therapeutic targets in the gut. Detailed knowledge of both the distribution and functional expression of GPCRs in the enteric nervous system (ENS) is critical toward advancing our understanding of how these receptors contribute to GI function during physiological and pathophysiological states. Equally important, but less well defined, is the complex relationship between receptor expression, ligand binding, signaling, and trafficking within enteric neurons. Neuronal GPCRs are internalized following exposure to agonists and under pathological conditions, such as intestinal inflammation. However, the relationship between the intracellular distribution of GPCRs and their signaling outputs in this setting remains a "black box". This review will briefly summarize current knowledge of agonist-evoked GPCR trafficking and location-specific signaling in the ENS and identifies key areas where future research could be focused. Greater understanding of the cellular and molecular mechanisms involved in regulating GPCR signaling in the ENS will provide new insights into GI function and may open novel avenues for therapeutic targeting of GPCRs for the treatment of digestive disorders.

Emerging structural biology of lipid G protein-coupled receptors.

The first crystal structure of a G protein-coupled receptor (GPCR) was that of the bovine rhodopsin, solved in 2000, and is a light receptor within retina rode cells that enables vision by transducing a conformational signal from the light-induced isomerization of retinal covalently bound to the receptor. More than 7 years after this initial discovery and following more than 20 years of technological developments in GPCR expression, stabilization, and crystallography, the high-resolution structure of the adrenaline binding β2 -adrenergic receptor, a ligand diffusible receptor, was discovered. Since then, high-resolution structures of more than 53 unique GPCRs have been determined leading to a significant improvement in our understanding of the basic mechanisms of ligand-binding and ligand-mediated receptor activation that revolutionized the field of structural molecular pharmacology of GPCRs. Recently, several structures of eight unique lipid-binding receptors, one of the most difficult GPCR families to study, have been reported. This review presents the outstanding structural and pharmacological features that have emerged from these new lipid receptor structures. The impact of these findings goes beyond mechanistic insights, providing evidence of the fundamental role of GPCRs in the physiological integration of the lipid signaling system, and highlighting the importance of sustained research into the structural biology of GPCRs for the development of new therapeutics targeting lipid receptors.

Loss of balance in normal GPCR-mediated cell trafficking.

G protein-coupled receptors (GPCRs) form a most diverse family of integral membrane proteins that mediate homeostatic and pathological processes, most notably by orchestrating cell distribution throughout the body, their infiltration, and time of presence in inflamed tissues. Here we discuss loss-of-orientation-effects in GPCR-mediated cell trafficking and migration and their impact on the phenotype of autoimmune diseases. In this context, we provide a systemic and integrative view of the contribution of abnormal GPCR expression as well as the levels of natural ligands and functional autoantibodies to the phenotype of autoimmune diseases. Currently, several studies propose that functional autoantibodies (including those targeting GPCRs) constitute an exclusively pathogenic or pathognomonic phenomenon. Here we reinforce the need of revising this point of view, and suggest that functional autoantibodies primary play a role in normal human physiology, while dysregulation of their functions causes autoimmune disease. Because patients with autoimmune diseases still suffer from severe morbidity and mortality rates, we consider expanding our knowledge on (patho)physiological roles of GPCR as a prerequisite for the development of novel specific therapeutic modalities.

Measuring surface expression and endocytosis of GPCRs using whole-cell ELISA.

G protein-coupled receptors (GPCRs) constitute a large class of cell surface receptors that recognize a wide array of ligands and mediate a diverse spectrum of signaling pathways. Measuring their surface expression in cellular context is a critical aspect of studying their signaling pathways and cellular outcomes. Upon addition of agonist, GPCRs typically undergo internalization and traffic from the plasma membrane to endosomal compartments. Although radioligand binding has been the primary assay to measure GPCR surface expression and internalization, whole-cell ELISA has now emerged as a powerful alternative approach. Here, we present a step-by-step whole-cell ELISA protocol for measuring relative surface expression and agonist-induced internalization of GPCRs containing engineered N-terminal epitope tag and recombinantly expressed in heterologous cells.

CARMA3 Is a Critical Mediator of G Protein-Coupled Receptor and Receptor Tyrosine Kinase-Driven Solid Tumor Pathogenesis.

The CARMA–Bcl10–MALT1 (CBM) signalosome is an intracellular protein complex composed of a CARMA scaffolding protein, the Bcl10 linker protein, and the MALT1 protease. This complex was first recognized because the genes encoding its components are targeted by mutation and chromosomal translocation in lymphoid malignancy. We now know that the CBM signalosome plays a critical role in normal lymphocyte function by mediating antigen receptor-dependent activation of the pro-inflammatory, pro-survival NF-κB transcription factor, and that deregulation of this signaling complex promotes B-cell lymphomagenesis. More recently, we and others have demonstrated that a CBM signalosome also operates in cells outside of the immune system, including in several solid tumors. While CARMA1 (also referred to as CARD11) is expressed primarily within lymphoid tissues, the related scaffolding protein, CARMA3 (CARD10), is more widely expressed and participates in a CARMA3-containing CBM complex in a variety of cell types. The CARMA3-containing CBM complex operates downstream of specific G protein-coupled receptors (GPCRs) and/or growth factor receptor tyrosine kinases (RTKs). Since inappropriate expression and activation of GPCRs and/or RTKs underlies the pathogenesis of several solid tumors, there is now great interest in elucidating the contribution of CARMA3-mediated cellular signaling in these malignancies. Here, we summarize the key discoveries leading to our current understanding of the role of CARMA3 in solid tumor biology and highlight the current gaps in our knowledge.

LncGPR107 drives the self-renewal of liver tumor initiating cells and liver tumorigenesis through GPR107-dependent manner

BackgroundWith self-renewal and differentiation properties, liver tumor initiating cells (TICs) are the reasons for tumor initiation, metastasis and drug resistance. G protein coupled receptors (GPCR) are critical modulators in many physiological and pathological processes. While, their roles in liver TICs are unknown.MethodsAn unbiased screening was performed using online-available data dataset. Liver TICs were sorted by FACS with surface marker CD133, or enriched by oncosphere formation. TIC self-renewal was examined by oncosphere formation and tumor initiation assay. Loss of function and gain of function assays were performed to examine the role of lncRNA. RNA pulldown, RNA immunoprecipitation, ChIP, western blot and double FISH were used explore the molecular mechanism of lncRNA.ResultsWe performed an unbiased screening for GPCR expression in liver cancers, and found GPR107 was the most highly expressed GPCR in liver cancer and liver TICs. GPR107 was essential for the self-renewal of liver TICs. The expression of GPR107 was regulated by a long noncoding RNA lncGPR107. LncGPR107 was also highly expressed in liver cancers and liver TICs. LncGPR107 drove the self-renewal of liver TICs through GPR107. Moreover, lncGPR107 recruited SRCAP complex to GPR107 promoter to drive its transcriptional activation. LncGPR107 depletion inhibited the binding of SRCAP complex and GPR107 promoter and subsequent GPR107 expression. Moreover, LncGPR107-SRCAP-GPR107 can be targeted for liver TIC elimination.ConclusionGPR107 was the most highly expressed GPCR in liver cancer and liver TICs. LncGPR107 participated in the transcriptional regulation of GPR107 in cis, through recruiting SRCAP remodeling complex to GPR107 promoter. This work revealed the important role of GPCR signaling in liver TIC self-renewal and added a new layer for liver TIC and GPCR regulation.

GPCRomics: GPCR Expression in Cancer Cells and Tumors Identifies New, Potential Biomarkers and Therapeutic Targets.

G protein-coupled receptors (GPCRs), the largest family of targets for approved drugs, are rarely targeted for cancer treatment, except for certain endocrine and hormone-responsive tumors. Limited knowledge regarding GPCR expression in cancer cells likely has contributed to this lack of use of GPCR-targeted drugs as cancer therapeutics. We thus undertook GPCRomic studies to define the expression of endoGPCRs (which respond to endogenous molecules such as hormones, neurotransmitters and metabolites) in multiple types of cancer cells. Using TaqMan qPCR arrays to quantify the mRNA expression of ∼340 such GPCRs, we found that human chronic lymphocytic leukemia (CLL) cells/stromal cells associated with CLL, breast cancer cell lines, colon cancer cell lines, pancreatic ductal adenocarcinoma (PDAC) cells, cancer associated fibroblasts (CAFs), and PDAC tumors express 50 to >100 GPCRs, including many orphan GPCRs (which lack known physiologic agonists). Limited prior data exist regarding the expression or function of most of the highly expressed GPCRs in these cancer cells and tumors. Independent results from public cancer gene expression databases confirm the expression of such GPCRs. We propose that highly expressed GPCRs in cancer cells (for example, GPRC5A in PDAC and colon cancer cells and GPR68 in PDAC CAFs) may contribute to the malignant phenotype, serve as biomarkers and/or may be novel therapeutic targets for the treatment of cancer.

G Protein‐Coupled Receptors in Human Lung Fibroblasts: Novel Therapeutic Approaches for Idiopathic Pulmonary Fibrosis?

Idiopathic Pulmonary Fibrosis (IPF) is a progressive, often fatal interstitial lung disease and thus in need of new, effective therapies. A feature of IPF is fibroblast‐to‐myofibroblast transition and resultant increased deposition of extracellular matrix (ECM) proteins, which can lead to loss of functional lung tissue. Previous studies have shown that increases in cyclic AMP (cAMP) can decrease the number and function of myofibroblasts. Cellular synthesis of cAMP is mediated by adenylyl cyclase, the activity of which is regulated by G‐protein signaling and G‐protein coupled receptors (GPCRs). We hypothesized that alteration in GPCR expression in lung fibroblasts from IPF patients may identify new therapeutic targets for lung fibrosis. Using RNA‐seq, we have identified and quantified GPCRs of lung fibroblasts isolated from six IPF patients and six controls grown in low passage primary cultures. RNA‐seq analysis revealed the expression of ~120 GPCRs (of ~380 endoGPCRs [regulated by endogenous ligands]). On average ~20 highly expressed GPCR were detected in all samples, with small differences in biological replicates; these may be “housekeeping” GPCRs. We also identified a group of GPCRs with highly variable expression and found that lung fibroblasts from IPF patients selectively expressed a subset of GPCRs. To assess changes in GPCR expression during fibroblast‐to‐myofibroblast transition, we treated fibroblasts with TGFβ1 (10 ng/ml, 48 hrs or 20 ng/ml, 16 hrs). As expected, TGFβ1 treatment increased the expression of pro‐fibrotic markers (e.g., collagens Iα1, Iα2, III; α‐smooth muscle actin; plasminogen activator inhibitor‐1). In addition, TGFβ1 altered (increased or decreased) the expression of numerous GPCRs. TGFβ1 treatment of control fibroblasts did not fully mimic the GPCR expression changes of fibroblasts from IPF patients. We conclude that GPCR expression (GPCRomic) analysis identifies GPCRs that may contribute to the fibrotic state in the lung and thus, may be novel therapeutic targets for lung fibrosis.Support or Funding InformationSupported by NIH T32 HL 098062‐04, Bristol Myers Squibb and CIHR 201403MOP‐RS‐323925.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Expression of G protein‐coupled receptors in pancreatic cancer cells

Pancreatic ductal adenocarcinoma (PDAC) is one of the most lethal forms of cancer and has an overall 5‐year survival rate ~7%. PDAC has poor response to chemotherapy and radiotherapy; thus new treatments for PDAC are an important, unmet medical need. G protein‐coupled receptor (GPCR) regulate many biological functions, including in cancer cells, but GPCR expression and the role of GPCRs in PDAC cells is unclear. We generated two primary PDAC cell lines (79E and 34E) and an immortalized normal pancreatic ductal epithelial cell line (HPDE6). Hypothesizing that GPCRs may regulate PDAC cells, we used Taqman GPCR array to assess GPCR expression in four PDAC cell lines, 79E, 34E, Miapaca, and AsPC. We identified 68 commonly expressed GPCRs in all four PDAC cell lines, among which 22 GPCRs were significantly up‐regulated (at least 2‐fold) in PDAC cells compared to normal control cell HPDE6; 13 GPCRs were significantly down‐regulated and 3 GPCRs were uniquely expressed in PDACs. We further tested the 13 most up‐regulated GPCRs using real time qPCR and confirmed that LPHN1, CHRM4, FZD4, HRH1 and CD97 have much higher expression in PDACs compared to HPED6, suggesting that these GPCRs might contribute to the malignant phenotype and perhaps could be novel treatment targets for pancreatic cancer, in particular for PDAC.Support or Funding InformationThis work is supported by NIH/NCI Research Grants R21 CA189477, R01 CA155620, and Padres Pedal the Cause PTC2017.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

GPCRs profiling and identification of GPR110 as a potential new target in HER2+ breast cancer.

G protein-coupled receptors (GPCRs) represent the largest family of druggable targets in human genome. Although several GPCRs can cross-talk with the human epidermal growth factor receptors (HERs), the expression and function of most GPCRs remain unknown in HER2+ breast cancer (BC). In this study, we aimed to evaluate gene expression of GPCRs in tumorigenic or anti-HER2 drug-resistant cells and to understand the potential role of candidate GPCRs in HER2+ BC. Gene expression of 352 GPCRs was profiled in Aldeflur+ tumorigenic versus Aldeflur- population and anti-HER2 therapy-resistant derivatives versus parental cells of HER2+ BT474 cells. The GPCR candidates were confirmed in 7 additional HER2+ BC cell line models and publicly available patient dataset. Anchorage-dependent and anchorage-independent cell growth, mammosphere formation, and migration/invasion were evaluated upon GPR110 knockdown by siRNA in BT474 and SKBR3 parental and lapatinib+ trastuzumab-resistant (LTR) cells. Adhesion and class A GPCRs were overexpressed in Aldeflur+ and anti-HER2 therapy-resistant population of BT474 cells, respectively. GPR110 was the only GPCR overexpressed in Aldeflur+ and anti-HER2 therapy-resistant population in BT474, SKBR3, HCC1569, MDA-MB-361, AU565, and/or HCC202 cells and in HER2+ BC subtype in patient tumors. Using BT474 and SKBR3 parental and LTR cells, we found that GPR110 knockdown significantly reduced anchorage-dependent/independent cell growth as well as migration/invasion of parental and LTR cells and mammosphere formation in LTR derivatives and not in parental cells. Our data suggest a potential role of GPR110 in tumorigenicity and in tumor cell dissemination in HER2+ BC.

Selective Photoaffinity Probe That Enables Assessment of Cannabinoid CB2 Receptor Expression and Ligand Engagement in Human Cells.

Chemical tools and methods that report on G protein-coupled receptor (GPCR) expression levels and receptor occupancy by small molecules are highly desirable. We report the development of LEI121 as a photoreactive probe to study the type 2 cannabinoid receptor (CB2R), a promising GPCR to treat tissue injury and inflammatory diseases. LEI121 is the first CB2R-selective bifunctional probe that covalently captures CB2R upon photoactivation. An incorporated alkyne serves as ligation handle for the introduction of reporter groups. LEI121 enables target engagement studies and visualization of endogenously expressed CB2R in HL-60 as well as primary human immune cells using flow cytometry. Our findings show that strategically functionalized probes allow monitoring of endogenous GPCR expression and engagement in human cells using tandem photoclick chemistry and hold promise as biomarkers in translational drug discovery.

Deregulation of Frizzled Receptors in Hepatocellular Carcinoma.

G protein-coupled receptors (GPCRs) have a substantial role in tumorigenesis and are described as a “cancer driver”. Aberrant expression or activation of GPCRs leads to the deregulation of downstream signaling pathways, thereby promoting cancer progression. In hepatocellular carcinoma (HCC), the Wnt signaling pathway is frequently activated and it is associated with an aggressive HCC phenotype. Frizzled (FZD) receptors, a family member of GPCRs, are known to mediate Wnt signaling. Accumulating findings have revealed the deregulation of FZD receptors in HCC and their functional roles have been implicated in HCC progression. Given the important role of FZD receptors in HCC, we summarize here the expression pattern of FZD receptors in HCC and their corresponding functional roles during HCC progression. We also further review and highlight the potential targeting of FZD receptors as an alternative therapeutic strategy in HCC.

An atlas of Caenorhabditis elegans chemoreceptor expression.

One goal of modern day neuroscience is the establishment of molecular maps that assign unique features to individual neuron types. Such maps provide important starting points for neuron classification, for functional analysis, and for developmental studies aimed at defining the molecular mechanisms of neuron identity acquisition and neuron identity diversification. In this resource paper, we describe a nervous system-wide map of the potential expression sites of 244 members of the largest gene family in the C. elegans genome, rhodopsin-like (class A) G-protein-coupled receptor (GPCR) chemoreceptors, using classic gfp reporter gene technology. We cover representatives of all sequence families of chemoreceptor GPCRs, some of which were previously entirely uncharacterized. Most reporters are expressed in a very restricted number of cells, often just in single cells. We assign GPCR reporter expression to all but two of the 37 sensory neuron classes of the sex-shared, core nervous system. Some sensory neurons express a very small number of receptors, while others, particularly nociceptive neurons, coexpress several dozen GPCR reporter genes. GPCR reporters are also expressed in a wide range of inter- and motorneurons, as well as non-neuronal cells, suggesting that GPCRs may constitute receptors not just for environmental signals, but also for internal cues. We observe only one notable, frequent association of coexpression patterns, namely in one nociceptive amphid (ASH) and two nociceptive phasmid sensory neurons (PHA, PHB). We identified GPCRs with sexually dimorphic expression and several GPCR reporters that are expressed in a left/right asymmetric manner. We identified a substantial degree of GPCR expression plasticity; particularly in the context of the environmentally-induced dauer diapause stage when one third of all tested GPCRs alter the cellular specificity of their expression within and outside the nervous system. Intriguingly, in a number of cases, the dauer-specific alterations of GPCR reporter expression in specific neuron classes are maintained during postdauer life and in some case new patterns are induced post-dauer, demonstrating that GPCR gene expression may serve as traits of life history. Taken together, our resource provides an entry point for functional studies and also offers a host of molecular markers for studying molecular patterning and plasticity of the nervous system.

The Placental Secretome: Identifying Potential Cross-Talk Between Placenta and Islet β-Cells

Background/Aims: Insulin-secreting islet β-cells adapt to the insulin resistance associated with pregnancy by increasing functional β-cell mass, but the placental signals involved in this process are not well defined. In the current study, we analysed expression of G-protein coupled receptor (GPCR) mRNAs in mouse islets and islet GPCR ligand mRNAs in placenta during pregnancy to generate an atlas of potential interactions between the placenta and β-cells to inform future functional studies of islet adaptive responses to pregnancy. Methods: Quantative RT-PCR arrays were used to measure mRNA expression levels of: (i) 342 GPCRs in islets from non-pregnant mice, and in islets isolated from mice on gestational days 12 and 18; (ii) 126 islet GPCR ligands in mouse placenta at gestational days 12 and 18. Results: At gestational day 12, a time of rapid expansion of the β-cell mass, 189 islet GPCR mRNAs were quantifiable, while 79 of the 126 known islet GPCR ligand mRNAs were detectable in placental extracts. Approximately half of the quantifiable placental GPCR ligand genes were of unknown function in β-cells. The expression of some islet GPCR and placental ligand mRNAs varied during pregnancy, with altered expression of both GPCR and ligand mRNAs by gestational day 18. Conclusion: The current study has revealed numerous potential routes for interaction between the placenta and islets, and offers an atlas to inform further functional studies of their roles in adaptive responses to pregnancy, and in the regulation of the β-cell mass.

Rac1-stimulated macropinocytosis enhances Gβγ activation of PI3Kβ.

Phosphoinositide 3-kinases (PI 3-kinases) are regulated by a diverse range of upstream activators, including receptor tyrosine kinases (RTKs), G-protein-coupled receptors (GPCRs), and small GTPases from the Ras, Rho and Rab families. For the Class IA PI 3-kinase PI3Kβ, two mechanisms for GPCR-mediated regulation have been described: direct binding of Gβγ subunits to the C2-helical domain linker of p110β, and Dock180/Elmo1-mediated activation of Rac1, which binds to the Ras-Binding Domain of p110β. We now show that the integration of these dual pathways is unexpectedly complex. In breast cancer cells, expression of constitutively activated Rac1 (CA-Rac1) along with either GPCR stimulation or expression of Gβγ led to an additive PI3Kβ-dependent activation of Akt. Whereas CA-Rac1-mediated activation of Akt was blocked in cells expressing a mutated PI3Kβ that cannot bind Gβγ, Gβγ and GPCR-mediated activation of Akt was preserved when Rac1 binding to PI3Kβ was blocked. Surprisingly, PI3Kβ-dependent CA-Rac1 signaling to Akt was still seen in cells expressing a mutant p110β that cannot bind Rac1. Instead of directly binding to PI3Kβ, CA-Rac1 acts by enhancing Gβγ coupling to PI3Kβ, as CA-Rac1-mediated Akt activation was blocked by inhibitors of Gβγ. Cells expressing CA-Rac1 exhibited a robust induction of macropinocytosis, and inhibitors of macropinocytosis blocked the activation of Akt by CA-Rac1 or lysophosphatidic acid. Our data suggest that Rac1 can potentiate the activation of PI3Kβ by GPCRs through an indirect mechanism, by driving the formation of macropinosomes that serve as signaling platforms for Gβγ coupling to PI3Kβ.