Protein-coupled Signaling Pathways Research Articles

Tuft cells in the intestine, immunity and beyond.

Tuft cells have gained substantial attention over the past 10 years due to numerous reports linking them with type 2 immunity and microorganism-sensing capacity in many mucosal tissues. This heightened interest is fuelled by their unique ability to produce an array of biological effector molecules, including IL-25, allergy-related eicosanoids, and the neurotransmitter acetylcholine, enabling downstream responses in diverse cell types. Operating through G protein-coupled receptor-mediated signalling pathways reminiscent of type II taste cells in oral taste buds, tuft cells emerge as chemosensory sentinels that integrate luminal conditions, eliciting appropriate responses in immune, epithelial and neuronal populations. How tuft cells promote tissue alterations and adaptation to the variety of stimuli at mucosal surfaces has been explored in multiple studies in the past few years. Since the initial recognition of the role of tuft cells, the discovery of diverse tuft cell effector functions and associated feedback loops have also revealed the complexity of tuft cell biology. Although earlier work largely focused on extraintestinal tissues, novel genetic tools and recent mechanistic studies on intestinal tuft cells established fundamental concepts of tuft cell activation and functions. This Review is an overview of intestinal tuft cells, providing insights into their development, signalling and interaction modules in immunity and other states.

Abstract PO3-24-06: Integrative genomic and CRISPR screen analysis identifies synthetic viability interactions in BRCA1/2 cancers

Abstract Background Pathogenic BRCA1 and BRCA2 (BRCA1/2) germline mutations contribute to hereditary breast cancer and are linked with increased risk of other cancers. Paradoxically, bi-allelic inactivation of BRCA1/2 (bBRCA1/2) is embryonically lethal. Although TP53 loss can at least in part mitigated the deleterious impact of Brca1 deficiency in mice, this is insufficient in human cells to compensate for decreased proliferative capacity. What other compensatory mechanisms facilitate oncogenesis remains unclear. Methods We performed an integrative analysis of cancer genomes from bBRCA1/2 tumors and CRISPR/Cas9 screens from cellular models to identify novel gene candidates that facilitate synthetic viability. First, we analyzed cancer genomes from ovarian and breast cancer derived from TCGA and identified genomic loci, which were enriched for recurrent copy number alterations (CNAs) in bBRCA1/2 tumors. We subsequently identified genes from these CNA loci that had corresponding changes in gene expression. Then, we used high-throughput genome-wide CRISPR/Cas9 knockout screens in BRCA1/2−/− cell lines (BRCA1−/- in RPE1 cells, BRCA2−/− in DLD1 cells) to identify which of these genes increased cell proliferation in vitro. Lastly, we experimentally validated these synthetic viable interactions using an orthogonal MCF12a system to evaluate changes in colony formation with a clonogenic cell survival assay. Results In human cancer genomes, we identified that bBRCA1/2 tumors harbored recurrent large-scale genetic deletions significantly more frequent than histologically matched control tumors. We identified 148 cytobands (23 distinct genomic loci) frequently deleted in bBRCA1/2 ovarian cancers (N=92), and 90 cytobands (15 distinct genomic loci) in bBRCA1/2 ER+ breast cancers (N=30). Subsequent computational analysis of gene expression data identified transcriptionally decreased genes in the frequently deleted loci. Cross-referencing with CRISPR/Cas9 screens in BRCA1/2−/− cells, we ultimately identified 277 and 218 genes that were altered in human tumors and putatively enhanced viability in BRCA1/2-null models but not in isogenic wild-type cell lines in BRCA1 and BRCA2 models, respectively. We subsequently selected 17 genes that either increased viability in BRCA1, BRCA2, or both contexts for experimental validation. Of these, about 40% (7 of 17) were experimentally validated (RIC8A, GNA12, ATMIN, IPO7, ATXN2, KDM1A, and NUP98) and had evidence of synthetic viability in this cellular context. Interestingly, RIC8A and GNA12 interact with each other, are both involved in G protein-coupled signaling pathway, and are synthetically viable with both BRCA1−/− and BRCA2−/− cells. In a re-analysis of a recently published genomic dataset on metastatic breast cancer, we found that homologous recombination deficient tumors developed additional loss of function mutations in RIC8A in the metastatic setting. Conclusions This study provides insights into the oncogenesis of BRCA1/2 malignancies and describes a high-throughput framework to identify synthetic viability interactions and causal driver genes affected by large-scale CNAs in human cancers. Citation Format: Yingjie Zhu, Xin Pei, Ardijana Novaj, Jeremy Setton, Fatemeh Derakhshan, Pier Selenica, Niamh McDermott, Sonali Sinha, Atif Khan, Simon Powell, Jorge Reis-Filho, Nadeem Riaz. Integrative genomic and CRISPR screen analysis identifies synthetic viability interactions in BRCA1/2 cancers [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-24-06.

Whole-genome sequencing identified candidate genes associated with high and low litter size in Chuanzhong black goats.

The reproductive performance of goats significantly influences breeding efficiency and economic returns, with litter size serving as a comprehensive indicator. Despite this, research on the genetic control of litter size remains limited. Therefore, we aimed to explore the candidate genes affecting fecundity and compared the whole-genome sequences (WGS) of 15 high-litter (HL) and 15 low-litter (LL) size in Chuanzhong black goats. Then genetic diversity and genomic variation patterns were analyzed by phylogenetic, principal component and population genetic structure analysis, it was found that HL and LL subpopulations diverged. Population evolutionary selection elimination analysis was performed by Fst and θπ resulted in 506 genes were annotated in HL and 528 genes in LL. These genes were mainly related to Hippo signaling pathway, G protein-coupled signaling pathway, G protein-coupled receptor activity, cell surface receptor signaling pathway, gonadal and reproductive structure development. According to the significantly selected genomic regions and important pathways, we found that the g.89172108T > G variant locus in the exon of the AMH gene was significantly associated with litter size (P < 0.05), which could be used as an auxiliary selection gene for the high fertility of Chuanzhong black Goat.

Signalling of Adrenoceptors: Canonical Pathways and New Paradigms.

The concept of G protein-coupled receptors initially arose from studies of the β-adrenoceptor, adenylyl cyclase, and cAMP signalling pathway. Since then both canonical G protein-coupled receptor signalling pathways and emerging paradigms in receptor signalling have been defined by experiments focused on adrenoceptors. Here, we discuss the evidence for G protein coupling specificity of the nine adrenoceptor subtypes. We summarise the ability of each of the adrenoceptors to activate proximal signalling mediators including cAMP, calcium, mitogen-activated protein kinases, and protein kinase C pathways. Finally, we highlight the importance of precise spatial and temporal control of adrenoceptor signalling that is controlled by the localisation of receptors at intracellular membranes and in larger protein complexes.

Selective inhibition of cannabinoid CB1 receptor-evoked signalling by the interacting protein GAP43

Cannabinoids exert pleiotropic effects on the brain by engaging the cannabinoid CB1 receptor (CB1R), a presynaptic metabotropic receptor that regulates key neuronal functions in a highly context-dependent manner. We have previously shown that CB1R interacts with growth-associated protein of 43 kDa (GAP43) and that this interaction inhibits CB1R function on hippocampal excitatory synaptic transmission, thereby impairing the therapeutic effect of cannabinoids on epileptic seizures in vivo. However, the underlying molecular features of this interaction remain unexplored. Here, we conducted mechanistic experiments on HEK293T cells co-expressing CB1R and GAP43 and show that GAP43 modulates CB1R signalling in a strikingly selective manner. Specifically, GAP43 did not affect the archetypical agonist-evoked (i) CB1R/Gi/o protein-coupled signalling pathways, such as cAMP/PKA and ERK, or (ii) CB1R internalization and intracellular trafficking. In contrast, GAP43 blocked an alternative agonist-evoked CB1R-mediated activation of the cytoskeleton-associated ROCK signalling pathway, which relied on the GAP43-mediated impairment of CB1R/Gq/11 protein coupling. GAP43 also abrogated CB1R-mediated ROCK activation in mouse hippocampal neurons, and this process led in turn to a blockade of cannabinoid-evoked neurite collapse. An NMR-based characterization of the CB1R-GAP43 interaction supported that GAP43 binds directly and specifically through multiple amino acid stretches to the C-terminal domain of the receptor. Taken together, our findings unveil a CB1R-Gq/11-ROCK signalling axis that is selectively impaired by GAP43 and may ultimately control neurite outgrowth.

Thallium(I) exposure perturbs the gut microbiota and metabolic profile as well as the regional immune function of C57BL/6J mice.

Intestinal microbes regulate the development of diseases induced by environmental exposure. Thallium (Tl) is a highly toxic heavy metal, and its toxicity is rarely discussed in relation to gut microbes. Herein, we showed that Tl(I) exposure (10ppm for 2weeks) affected the alpha diversity of bacteria in the ileum, colon, and feces, but had little effect on the beta diversity of bacteria through 16S rRNA sequencing. LEfSe analysis revealed that Tl(I) exposure changed the abundance of intestinal microbiota along the digestive tract. Cecum metabolomic detection and analysis showed that Tl(I) exposure altered the abundance and composition of metabolites. In addition, the Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analysis revealed that Tl(I) exposure impaired amino acid, lipid, purine metabolism, and G protein-coupled receptor signalling pathways. A consistency test revealed a strong correlation, and a Pearson's correlation analysis showed an extensive interaction, between microorganisms and metabolites. Analysis of the intestinal immunity revealed that Tl(I) exposure suppressed the immune responses, which also had regional differences. These results identify the perturbation of the intestinal microenvironment by Tl exposure and provide a new explanation for Tl toxicity.

Defining a role of NADPH oxidase in myogenic tone development.

The myogenic response sets the foundation for blood flow control. Recent findings suggest a role for G protein-coupled receptors (GPCR) and signaling pathways tied to the generation of reactive oxygen species (ROS). In this regard, this study ascertained the impact of NADPH oxidase (Nox) on myogenic tone in rat cerebral resistance arteries. The study employed real-time qPCR (RT-qPCR), pressure myography, and immunohistochemistry. Gq blockade abolished myogenic tone in rat cerebral arteries, linking GPCR to mechanosensation. Subsequent work revealed that general (TEMPOL) and mitochondrial specific (MitoTEMPO) ROS scavengers had little impact on myogenic tone, whereas apocynin, a broad spectrum Nox inhibitor, initiated transient dilation. RT-qPCR revealed Nox1 and Nox2 mRNA expression in smooth muscle cells. Pressure myography defined Nox1 rather than Nox2 is facilitating myogenic tone. We rationalized that Nox1-generated ROS was initiating this response by impairing the ability of the CaV 3.2 channel to elicit negative feedback via BKCa . This hypothesis was confirmed in functional experiments. The proximity ligation assay further revealed that Nox1 and CaV 3.2 colocalize within 40nm of one another. Our data highlight that vascular pressurization augments Nox1 activity and ensuing ROS production facilitates myogenic tone by limiting Ca2+ influx via CaV 3.2.

Purification of Endogenous Drosophila Transient Receptor Potential Channels.

Drosophila phototransduction is one of the fastest known G protein-coupled signaling pathways. To ensure the specificity and efficiency of this cascade, the calcium (Ca2+)-permeable cation channel, transient receptor potential (TRP), binds tightly to the scaffold protein, inactivation-no-after-potential D (INAD), and forms a large signaling protein complex with eye-specific protein kinase C (ePKC) and phospholipase Cβ/No receptor potential A (PLCβ/NORPA). However, the biochemical properties of the Drosophila TRP channel remain unclear. Based on the assembling mechanism of INAD protein complex, a modified affinity purification plus competition strategy was developed to purify the endogenous TRP channel. First, the purified histidine (His)-tagged NORPA 863-1095 fragment was bound to Ni-beads and used as bait to pull down the endogenous INAD protein complex from Drosophila head homogenates. Then, excessive purified glutathione S-transferase (GST)-tagged TRP 1261-1275 fragment was added to the Ni-beads to compete with the TRP channel. Finally, the TRP channel in the supernatant was separated from the excessive TRP 1261-1275 peptide by size-exclusion chromatography. This method makes it possible to study the gating mechanism of the Drosophila TRP channel from both biochemical and structural angles. The electrophysiology properties of purified Drosophila TRP channels can also be measured in the future.

Crosstalk between heterotrimeric G protein-coupled signaling pathways and WRKY transcription factors modulating plant responses to suboptimal micronutrient conditions.

Nutrient stresses induce foliar chlorosis and growth defects. Here we propose heterotrimeric G proteins as signaling mediators of various nutrient stresses, through meta-analyses of >20 transcriptomic data sets associated with nutrient stresses or G protein mutations. Systematic comparison of transcriptomic data yielded 104 genes regulated by G protein subunits under common nutrient stresses: 69 genes under Gβ subunit (AGB1) control and 35 genes under Gα subunit (GPA1) control. Quantitative real-time PCR experiments validate that several transcription factors and metal transporters changed in expression level under suboptimal iron, zinc, and/or copper concentrations, while being misregulated in the Arabidopsis Gβ-null (agb1) mutant. The agb1 mutant had altered metal ion profiles and exhibited severe growth arrest under zinc stress, and aberrant root waving under iron and zinc stresses, while the Gα-null mutation attenuated leaf chlorosis under iron deficiency in both Arabidopsis and rice. Our transcriptional network analysis inferred computationally that WRKY-family transcription factors mediate the AGB1-dependent nutrient responses. As corroborating evidence of our inference, ectopic expression of WRKY25 or WRKY33 rescued the zinc stress phenotypes and the expression of zinc transporters in the agb1-2 background. These results, together with Gene Ontology analyses, suggest two contrasting roles for G protein-coupled signaling pathways in micronutrient stress responses: one enhancing general stress tolerance and the other modulating ion homeostasis through WRKY transcriptional regulatory networks. In addition, tolerance to iron stress in the rice Gα mutant provides an inroad to improve nutrient stress tolerance of agricultural crops by manipulating G protein signaling.

Convergence of Wnt, growth factor, and heterotrimeric G protein signals on the guanine nucleotide exchange factor Daple.

Cellular proliferation, differentiation, and morphogenesis are shaped by multiple signaling cascades, and their dysregulation plays an integral role in cancer progression. Three cascades that contribute to oncogenic potential are those mediated by Wnt proteins and the receptor Frizzled (FZD), growth factor receptor tyrosine kinases (RTKs), and heterotrimeric G proteins and associated GPCRs. Daple is a guanine nucleotide exchange factor (GEF) for the G protein Gαi Daple also binds to FZD and the Wnt/FZD mediator Dishevelled (Dvl), and it enhances β-catenin-independent Wnt signaling in response to Wnt5a-FZD7 signaling. We identified Daple as a substrate of multiple RTKs and non-RTKs and, hence, as a point of convergence for the three cascades. We found that phosphorylation near the Dvl-binding motif in Daple by both RTKs and non-RTKs caused Daple/Dvl complex dissociation and augmented the ability of Daple to bind to and activate Gαi, which potentiated β-catenin-independent Wnt signals and stimulated epithelial-mesenchymal transition (EMT) similarly to Wnt5a/FZD7 signaling. Although Daple acts as a tumor suppressor in the healthy colon, the concurrent increased abundance of Daple and epidermal growth factor receptor (EGFR) in colorectal tumors was associated with poor patient prognosis. Thus, the Daple-dependent activation of Gαi and the Daple-dependent enhancement of β-catenin-independent Wnt signals are not only stimulated by Wnt5a/FZD7 to suppress tumorigenesis but also hijacked by growth factor-activated RTKs to enhance tumor progression. These findings identify a cross-talk paradigm among growth factor RTKs, heterotrimeric G proteins, and the Wnt/FZD pathway in cancer.

Profile of King-Wai Yau

Neuroscientist King-Wai Yau of Johns Hopkins University has made fundamental discoveries concerning the mechanisms underlying sensory transduction. His research over the past four decades has focused primarily on vision. “Vision is one of our most precious senses from which come art, science, humanity, beauty, and practically all aspects of life,” says Yau. His findings concerning phototransduction—the process by which light is converted into a neural signal—have led to a sophisticated understanding of many hereditary diseases causing blindness. Additionally, Yau has elucidated the process of olfaction transduction that, like vision, relies on a G protein-coupled cellular signaling pathway. His Inaugural Article, signifying his 2010 election to the National Academy of Sciences, advances understanding of the electrical response of olfactory neurons to odorants. King-Wai Yau. Image courtesy of Johns Hopkins Medicine (Baltimore, MD). Yau was born in China in 1948, the sixth of seven children. His family relocated to Hong Kong within months of his birth. His father died 5 years later. Dedicating himself to scholarship and science, Yau excelled in high school. He entered the University of Hong Kong Faculty of Medicine in 1967. After a year, he set out for the United States, studying at the University of Minnesota before transferring to Princeton University on a full scholarship to study physics and eventually graduating in 1971. “The best decision that I ever made was to leave medical school,” Yau says. “I determined that I did not wish to be a clinician.” Yau then entered Harvard Medical School, where he earned a PhD in neurobiology in 1975. At Harvard, he studied invertebrate neurobiology under John Nicholls in the university’s small but influential neurobiology department. There, he became …

Single cell transcriptome analysis of mouse carotid body glomus cells.

Carotid body (CB) glomus cells mediate acute oxygen sensing and the initiation of the hypoxic ventilatory response, yet the gene expression profile of these cells is not available. We demonstrate that the single cell RNA-Seq method is a powerful tool for identifying highly expressed genes in CB glomus cells. Our single cell RNA-Seq results characterized novel CB glomus cell genes, including members of the G protein-coupled receptor signalling pathway, ion channels and atypical mitochondrial electron transport chain subunits. A heterologous cell-based screening identified acetate (which is known to affect CB glomus cell activity) as an agonist for the most highly abundant G protein-coupled receptor (Olfr78) in CB glomus cells. These data established the first transcriptome profile of CB glomus cells, highlighting genes with potential implications in CB chemosensory function. The carotid body (CB) is a major arterial chemoreceptor containing glomus cells whose activities are regulated by changes in arterial blood content, including oxygen. Despite significant advancements in the characterization of their physiological properties, our understanding of the underlying molecular machinery and signalling pathway in CB glomus cells is still limited. To overcome this, we employed the single cell RNA-Seq method by performing next-generation sequencing on single glomus cell-derived cDNAs to eliminate contamination of genes derived from other cell types present in the CB. Using this method, we identified a set of genes abundantly expressed in glomus cells, which contained novel glomus cell-specific genes. Transcriptome and subsequent in situ hybridization and immunohistochemistry analyses identified abundant G protein-coupled receptor signalling pathway components and various types of ion channels, as well as members of the hypoxia-inducible factors pathway. A short-chain fatty acid olfactory receptor Olfr78, recently implicated in CB function, was the most abundant G protein-coupled receptor. Two atypical mitochondrial electron transport chain subunits (Ndufa4l2 and Cox4i2) were among the most specifically expressed genes in CB glomus cells, highlighting their potential roles in mitochondria-mediated oxygen sensing. The wealth of information provided by the present study offers a valuable foundation for identifying molecules functioning in the CB.

Regulation of Glucokinase by Intracellular Calcium Levels in Pancreatic β Cells

Glucokinase (GCK) controls the rate of glucose metabolism in pancreatic β cells, and its activity is rate-limiting for insulin secretion. Posttranslational GCK activation can be stimulated through either G protein-coupled receptors or receptor tyrosine kinase signaling pathways, suggesting a common mechanism. Here we show that inhibiting Ca(2+) release from the endoplasmic reticulum (ER) decouples GCK activation from receptor stimulation. Furthermore, pharmacological release of ER Ca(2+) stimulates activation of a GCK optical biosensor and potentiates glucose metabolism, implicating rises in cytoplasmic Ca(2+) as a critical regulatory mechanism. To explore the potential for glucose-stimulated GCK activation, the GCK biosensor was optimized using circularly permuted mCerulean3 proteins. This new sensor sensitively reports activation in response to insulin, glucagon-like peptide 1, and agents that raise cAMP levels. Transient, glucose-stimulated GCK activation was observed in βTC3 and MIN6 cells. An ER-localized channelrhodopsin was used to manipulate the cytoplasmic Ca(2+) concentration in cells expressing the optimized FRET-GCK sensor. This permitted quantification of the relationship between cytoplasmic Ca(2+) concentrations and GCK activation. Half-maximal activation of the FRET-GCK sensor was estimated to occur at ∼400 nm Ca(2+). When expressed in islets, fluctuations in GCK activation were observed in response to glucose, and we estimated that posttranslational activation of GCK enhances glucose metabolism by ∼35%. These results suggest a mechanism for integrative control over GCK activation and, therefore, glucose metabolism and insulin secretion through regulation of cytoplasmic Ca(2+) levels.

Deficiency of β-arrestin 2 in mice-induced bone remodeling was associated with down-regulation of GPER1 expression

Objective To determine the role of β-arrestin 2 on bone mineral density and microarchitecture, and its underlying mechanisms. Methods Ex vivo dual-energy-X-ray-absorptiometry(DXA) was used to measure the total body bone mineral density (BMD) and micro-CT scan was performed on mouse femora from female mice of wild type(WT) and β-arrestin 2 knockout(KO) at 16, 26, 30 weeks of age. RT-PCR was applied for the mRNA expression. Results Started from 16 weeks of age, total body BMD and the bone volume/total volume(BV/TV), trabecular number(Tb.N) in the femoral metaphysis of β-arrestin 2 KO female mice decreased dramatically. BMD decreased by 8.10%, 7.53%, 8.56%, BV/TV by 53.3%, 49.4%, 55.7% and Tb.N by 53.2%, 52.1%, 49.3% at 16, 26, 30 weeks of age, respectively, compared with those in WT mice(all P<0.05). In contrast, the trabecular pattern factor(Tb.Pf), trabecular separation(Tb.Sp), and structure model index(SMI) of the mice increased gradually with age, and reached statistically significant at age of 30 weeks(increased by 32.0%, 54.3%, and 23.7%, respectively, all P<0.05). RT-PCR showed that mRNA expression levels of osteoprotegrin, RANKL, and G protein-coupled estrogen receptor 1 in tibia from β-arrestin 2 KO mice were significantly lower than those in WT mice. Conclusion The effects of β-arrestin 2 on bone remodeling might be associated with G protein-coupled estrogen receptor1 signaling pathway. (Chin J Endocrinol Metab, 2015, 31: 897-901) Key words: β-Arrestin 2; Bone mineral density; Microarchitecture; Osteoprotegrin; G protein-coupled estrogen receptor 1

β-Arrestin–biased signaling mediates memory reconsolidation

A long-standing hypothesis posits that a G protein-coupled signaling pathway mediates β-adrenergic nervous system functions, including learning and memory. Here we report that memory retrieval (reactivation) induces the activation of β1-adrenergic β-arrestin signaling in the brain, which stimulates ERK signaling and protein synthesis, leading to postreactivation memory restabilization. β-Arrestin2-deficient mice exhibit impaired memory reconsolidation in object recognition, Morris water maze, and cocaine-conditioned place preference paradigms. Postreactivation blockade of both brain β-adrenergic Gs protein- and β-arrestin-dependent pathways disrupts memory reconsolidation. Unexpectedly, selective blockade of the Gs/cAMP/PKA signaling but not the β-arrestin/ERK signaling by the biased β-adrenergic ligands does not inhibit reconsolidation. Moreover, the expression of β-arrestin2 in the entorhinal cortex of β-arrestin 2-deficient mice rescues β1-adrenergic ERK signaling and reconsolidation in a G protein pathway-independent manner. We demonstrate that β-arrestin-biased signaling regulates memory reconsolidation and reveal the potential for β-arrestin-biased ligands in the treatment of memory-related disorders.

Orphanin FQ-ORL-1 regulation of reproduction and reproductive behavior in the female.

Orphanin FQ (OFQ/N) and its receptor, opioid receptor-like receptor-1 (ORL-1), are expressed throughout steroid-responsive limbic and hypothalamic circuits that regulate female ovarian hormone feedback and reproductive behavior circuits. The arcuate nucleus of the hypothalamus (ARH) is a brain region that expresses OFQ/N and ORL-1 important for both sexual behavior and modulating estradiol feedback loops. Within the ARH, the activation of the OFQ/N-ORL-1 system facilitates sexual receptivity (lordosis) through the inhibition of β-endorphin neuronal activity. Estradiol initially activates ARH β-endorphin neurons to inhibit lordosis. Simultaneously, estradiol upregulates coexpression of OFQ/N and progesterone receptors and ORL-1 in ARH β-endorphin neurons. Ovarian hormones regulate pre- and postsynaptic coupling of ORL-1 to its G protein-coupled signaling pathways. When the steroid-primed rat is nonreceptive, estradiol acts pre- and postsynaptically to decrease the ability of the OFQ/N-ORL-1 system to inhibit ARH β-endorphin neurotransmission. Conversely, when sexually receptive, ORL-1 signaling is restored to inhibit β-endorphin neurotransmission. Although steroid signaling that facilitates lordosis converges to deactivate ARH β-endorphin neurons, estradiol-only facilitation of lordosis requires the activation of ORL-1, but estradiol+progesterone does not, indicating that multiple circuits mediate ovarian hormone signaling to deactivate ARH β-endorphin neurons. Research on the role of OFQ/N-ORL-1 in ovarian hormone feedback loops is just beginning. In the rat, OFQ/N may act to terminate gonadotropin-releasing hormone and luteinizing hormone release under positive and negative feedbacks. In the ewe, it appears to directly inhibit gonadotropin-releasing hormone release to mediate progesterone-negative feedback. As a whole, the localization and actions of OFQ/N-ORL-1 system indicate that it may mediate the actions of estradiol and progesterone to synchronize reproductive behavior and ovarian hormone feedback loops.

Opsin expression in human epidermal skin.

Human skin is constantly exposed to solar light containing visible and ultraviolet radiation (UVR), a powerful skin carcinogen. UVR elicits cellular responses in epidermal cells via several mechanisms: direct absorption of short-wavelength UVR photons by DNA, oxidative damage caused by long-wavelength UVR, and, as we recently demonstrated, via a retinal-dependent G protein-coupled signaling pathway. Because the human epidermis is exposed to a wide range of light wavelengths, we investigated whether opsins, light-activated receptors that mediate photoreception in the eye, are expressed in epidermal skin to potentially serve as photosensors. Here we show that four opsins—OPN1-SW, OPN2, OPN3 and OPN5—are expressed in the two major human epidermal cell types, melanocytes and keratinocytes, and the mRNA expression profile of these opsins does not change in response to physiological UVR doses. We detected two OPN3 splice variants present in similar amounts in both cell types and three OPN5 splice isoforms, two of which encode truncated proteins. Notably, OPN2 and OPN3 mRNA were significantly more abundant than other opsins and encoded full-length proteins. Our results demonstrate that opsins are expressed in epidermal skin cells and suggest that they might initiate light–induced signaling pathways, possibly contributing to UVR phototransduction.

Ligand-selective activation of heterologously-expressed mammalian olfactory receptor

Mammalian olfactory receptors (ORs) appear to have the capacity to couple to multiple G protein-coupled signaling pathways in a ligand-dependent selective manner. To better understand the mechanisms and molecular range of such ligand selectivity, we expressed the mouse eugenol OR (mOR-EG) in HEK293T cells together with Gα15 to monitor activation of the phospholipase-C (PLC) signaling pathway and/or Gαolf to monitor activation of the adenylate cyclase (AC) signaling pathway, resulting in intracellular Ca2+ release and/or Ca2+ influx through a cyclic nucleotide-gated channel, respectively. PLC-dependent responses differed dynamically from AC-dependent responses, allowing them to be distinguished when Gα15 and Gαolf were co-expressed. The dynamic difference in readout was independent of the receptor, the heterologous expression system, and the ligand concentration. Of 17 reported mOR-EG ligands tested, including eugenol, its analogs, and structurally dissimilar compounds (mousse cristal, nootkatone, orivone), some equally activated both signaling pathways, some differentially activated both signaling pathways, and some had no noticeable effect even at 1–5mM. Our findings argue that mOR-EG, when heterologously expressed, can couple to two different signaling pathways in a ligand selective manner. The challenge now is to determine the potential of mOR-EG, and perhaps other ORs, to activate multiple signaling pathways in a ligand selective manner in native ORNs.

Characterizations and functions of regulator of G protein signaling (RGS) in fungi

Proteins that serve as regulator of G protein signaling (RGS) primarily function as GTPase accelerators that promote GTP hydrolysis by the Gα subunits, thereby inactivating the G protein and rapidly switching off G protein-coupled signaling pathways. Since the first RGS protein was identified from the budding yeast Saccharomyces cerevisiae, more than 30 RGS and RGS-like proteins have been characterized from several model fungi, such as Aspergillus nidulans, Beauveria bassiana, Candida albicans, Fusarium verticillioides, Magnaporthe oryzae, and Metarhizium anisopliae. In this review, the partial biochemical properties and functional domains of RGS and RGS-like proteins were predicted and compared, and the roles of RGS and RGS-like proteins in different fungi were summarized. Moreover, the phylogenetic relationship among RGS and RGS-like proteins from various fungi was analyzed and discussed.

Raf kinase inhibitor protein inhibits esophageal cancer cell invasion through downregulation of matrix metalloproteinase expression

Esophageal cancer is the eighth most common malignant tumor in the world and is a common cause of tumor-related death. The development of esophageal cancer is a complex process involving many pathogenetic factors, multiple stages and accumulation of multiple gene mutations and interactions. This study aimed to investigate the effects of Raf kinase inhibitor protein (RKIP) on the proliferation, apoptosis and invasion of TE-1 esophageal cancer cells. Surgical specimens from esophageal cancer patients were classified into esophageal cancer tissues, tumor-adjacent tissues and normal esophageal tissues. The tissues were fixed in 4% paraformaldehyde solution for hematoxylin and eosin and immunohistochemical staining. RKIP expression in esophageal tissues was detected by immunohistochemical staining. The esophageal cancer cell line TE-1 was exposed to four different viruses: RKIP-RNAi-AD, NC-RNAi-GFP-AD, RKIP-AD and GFP-AD. Cell proliferation was detected by MTT assay and cell apoptosis was detected by flow cytometry. Cell invasion was determined by a Transwell coated with Matrigel. RKIP, phospho-RKIP, Raf-1, phospho-Raf-1, ERK1/2, phospho-ERK1/2, GRK-2 and GAPDH expression was assayed by western blotting. LIN28 and MMP-14 mRNA was assayed by qPCR. The results showed that RKIP expression was reduced in esophageal cancer tissues in comparison with expression in normal esophageal epithelium tissues and tumor-adjacent tissues. Reduced RKIP expression was associated with lymph node or distant metastasis in esophageal cancer. RKIP inhibited the invasive and metastatic abilities of esophageal cancer cell line TE-1 by downregulating mRNA expression of LIN28 and MMP-14. RKIP had no effect on the MAPK signaling pathway in the esophageal cancer cell line TE-1, but was involved in the G protein-coupled signaling pathway. Our findings clearly demonstrate that RKIP inhibits esophageal cancer cell invasion by downregulating the expression of GRK-2, LIN28 and MMP-14.