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PubMed2001

Regulation of the rhodopsin protein phosphatase, RDGC, through interaction with calmodulin.

Hundreds of G protein-coupled receptors (GPCRs) and at least six GPCR kinases have been identified, but the only GPCR phosphatase that has been definitively demonstrated is the rhodopsin phosphatase encoded by the rdgC locus of Drosophila. Mutations in rdgC result in defects in termination of the light response and cause severe retinal degeneration. In the current work, we demonstrate that RDGC binds to calmodulin, and a mutation in an IQ motif that eliminates the calmodulin/RDGC interaction prevents dephosphorylation of rhodopsin in vivo and disrupts termination of the photoresponse. Our data indicate that RDGC is a novel calmodulin-dependent protein phosphatase and raise the possibility that regulation of other GPCRs through dephosphorylation may be controlled by calmodulin-dependent protein phosphatases related to RDGC.

Amino Acid Sequence↗

PubMed2003

Transmitter modulation of slow, activity-dependent alterations in sodium channel availability endows neurons with a novel form of cellular plasticity.

Voltage-gated Na+ channels are major targets of G protein-coupled receptor (GPCR)-initiated signaling cascades. These cascades act principally through protein kinase-mediated phosphorylation of the channel alpha subunit. Phosphorylation reduces Na+ channel availability in most instances without producing major alterations of fast channel gating. The nature of this change in availability is poorly understood. The results described here show that both GPCR- and protein kinase-dependent reductions in Na+ channel availability are mediated by a slow, voltage-dependent process with striking similarity to slow inactivation, an intrinsic gating mechanism of Na+ channels. This process is strictly associated with neuronal activity and develops over seconds, endowing neurons with a novel form of cellular plasticity shaping synaptic integration, dendritic electrogenesis, and repetitive discharge.

Animals↗

PubMed2004

G protein-coupled receptor kinase function is essential for chemosensation in C. elegans.

G protein-coupled receptors (GPCRs) mediate diverse signaling processes, including olfaction. G protein-coupled receptor kinases (GRKs) are important regulators of G protein signal transduction that specifically phosphorylate activated GPCRs to terminate signaling. Despite previously described roles for GRKs in GPCR signal downregulation, animals lacking C. elegans G protein-coupled receptor kinase-2 (Ce-grk-2) function are not hypersensitive to odorants. Instead, decreased Ce-grk-2 function in adult sensory neurons profoundly disrupts chemosensation, based on both behavioral analysis and Ca(2+) imaging. Although mammalian arrestin proteins cooperate with GRKs in receptor desensitization, loss of C. elegans arrestin-1 (arr-1) does not disrupt chemosensation. Either overexpression of the C. elegans Galpha subunit odr-3 or loss of eat-16, which encodes a regulator of G protein signaling (RGS) protein, restores chemosensation in Ce-grk-2 mutants. These results demonstrate that loss of GRK function can lead to reduced GPCR signal transduction and suggest an important role for RGS proteins in the regulation of chemosensation.

Animals↗

PubMed2001

Beta-Arrestins: new roles in regulating heptahelical receptors' functions.

The last few years have seen a marked expansion in appreciation of the diversity of roles played by the betaArrestins in regulating GPCR functions. Originally discovered as molecules that desensitize such receptors, the roles of betaArrestins have expanded to include acting as signalling adapters or intermediates that recruit other key molecules to the GPCRs in an agonist-regulated fashion. For example, interactions with components of the endocytic machinery, such as clathrin, the adapter protein AP-2 and the N-ethylmaleimide sensitive fusion protein (NSF), demonstrate the ability of betaArrestins to act as adapters to facilitate the clathrin-mediated endocytosis of certain members of the GPCR family. BetaArrestins have also been shown to serve as signalling molecules. The Ras-dependent activation of ERK1/2 may involve the betaArrestin-dependent recruitment of c-Src to the beta2-adrenergic receptor (beta2-AR). More recently, betaArrestins have been shown to act as molecular scaffolds that coordinate the assembly of certain MAP kinase complexes that lead to the stimulation of either ERK1/2 or JNK3. Finally, long-term accumulation of arrestin-rhodopsin complexes, in photoreceptor cells has been shown to trigger apoptosis.

Animals↗

PubMed2004

Dimerization of G-protein-coupled receptors: roles in signal transduction.

Recently, many G-protein-coupled receptors (GPCRs) have been demonstrated to form constitutive dimers consisting of identical or distinct monomeric subunits. The discovery of GPCR dimerization has revealed a new level of molecular cross-talk between signalling molecules and may define a general mechanism that modulates the function of GPCRs under both physiological and pathological conditions. The heterodimerization between distinct GPCRs could be responsible for the generation of pharmacologically defined receptors for which no gene has been identified so far. Elucidating the role of dimerization in the activation processes of GPCRs will lead us to develop novel pharmaceutical agents that allosterically promote activation or inhibition of GPCR signalling.

Animals↗

PubMed2002

Epidermal growth factor receptor (EGFR) transactivation by estrogen via the G-protein-coupled receptor, GPR30: a novel signaling pathway with potential significance for breast cancer.

The biological and biochemical effects of estrogen have been ascribed to its known receptors, which function as ligand-inducible transcription factors. However, estrogen also triggers rapid activation of classical second messengers (cAMP, calcium, and inositol triphosphate) and stimulation of intracellular signaling cascades mitogen-activated protein kinase (MAP K), PI3K and eNOS. These latter events are commonly activated by membrane receptors that either possess intrinsic tyrosine kinase activity or couple to heterotrimeric G-proteins. We have shown that estrogen transactivates the epidermal growth factor receptor (EGFR) to MAP K signaling axis via the G-protein-coupled receptor (GPCR), GPR30, through the release of surface-bound proHB-EGF from estrogen receptor (ER)-negative human breast cancer cells [Molecular Endocrinology 14 (2000) 1649]. This finding is consistent with a growing body of evidence suggesting that transactivation of EGFRs by GPCRs is a recurrent theme in cell signaling. GPCR-mediated transactivation of EGFRs by estrogen provides a previously unappreciated mechanism of cross-talk between estrogen and serum growth factors, and explains prior data reporting the EGF-like effects of estrogen. This novel mechanism by which estrogen activates growth factor-dependent signaling and its implications for breast cancer biology are discussed further in this review.

Breast Neoplasms↗

PubMed2001

Galpha(13) mediates activation of a depolarizing chloride current that accompanies RhoA activation in both neuronal and nonneuronal cells.

Loss of membrane potential (membrane depolarization) is one of the earliest and most striking responses of quiescent cells to stimulation with serum or G protein-coupled receptor (GPCR) agonists such as lysophosphatidic acid and thrombin. Membrane depolarization is due to the activation of a chloride conductance. While this response has received relatively little attention in the past, it is clear that the acute loss of membrane potential may have important physiological consequences. However, the dissection of the underlying G protein pathway and the establishment of cause-effect relationships have remained elusive to date. Here we report that, in neuronal cells, the depolarizing chloride current invariably accompanies GPCR-induced activation of RhoA and subsequent neurite retraction, and neither of these events requires phosphoinositide hydrolysis or Ca2+ mobilization. Through antibody microinjections and a genetic approach, we demonstrate that activation of the chloride conductance is mediated by Galpha(13) in a RhoA-independent manner in both neuronal cells and fibroblasts. We further show that, in neuronal cells, this newly described Galpha(13) pathway may profoundly modulate membrane excitability during RhoA-regulated neurite remodeling.

Action Potentials↗

PubMed1999

Binding of Gbetagamma subunits to cRaf1 downregulates G-protein-coupled receptor signalling.

Receptors of the seven transmembrane domain family are coupled to heterotrimeric G proteins [1]. Binding of ligand to these receptors induces dissociation of the heterotrimeric complex into free GTP-Galpha and Gbetagamma subunits, which then interact with their respective effector molecules to stimulate specific cellular responses. In some cases, these cellular responses involve mitogenic signalling [2]. The mitogen-activated protein (MAP) kinase cascade is initiated by the protein kinase cRaf1 and links growth factor receptor signalling to cell growth and differentiation [3]. The main activator of cRaf1 is the small GTP-binding protein Ras [4], and the binding of cRaf1 to GTP-Ras translocates cRaf1 to the plasma membrane, where it is activated [5]. It has been reported that cRaf1 associates directly with the beta subunit of heterotrimeric G proteins in vitro, and with the betagamma subunit complex in vivo [6], but the role of this association is not yet understood. Here, we show that cRaf1 associates with Gbeta1gamma2, and that this association in mammalian cells is significantly enhanced when active p21(Ras) is present or when cRaf1 is otherwise targeted to the membrane. Association with Gbeta1gamma2 has no effect on the kinase activity of cRaf1, but cRaf1 can affect Gbetagamma-mediated signalling events. Thus, membrane-localised cRaf1 inhibits G-protein-coupled receptor (GPCR)-stimulated activation of phospholipase Cbeta (PLCbeta) by sequestration of Gbetagamma subunits, an effect also observed with endogenous levels of cRaf1. Our data suggest that cRaf1 may be an important regulator of signalling by Gbetagamma, particularly in those GPCR systems that stimulate the MAP kinase cascade through the activation of p21(Ras).

Animals↗

PubMed2000

Activation of MAPK cascades by G-protein-coupled receptors: the case of gonadotropin-releasing hormone receptor.

G-protein-coupled receptors (GPCRs) are a large group of integral membrane receptors that transmit signals from a diverse array of external stimuli, including neurotransmitters, hormones, phospholipids, photons, odorants and taste ligands. In response to ligand binding, the GPCRs initiate diverse downstream signaling pathways through four groups of G proteins and other interacting proteins. Key components in GPCR-induced intracellular signaling are four groups of mitogen-activated protein kinase (MAPK) cascades: extracellular signal-related kinase (ERK), Jun N-terminal kinase (JNK), p38MAPK and big MAPK (BMK). The hallmark of MAPK signaling is the stimulation-dependent nuclear translocation of the involved kinases, which regulate gene expression and the cytoplasmic acute response to mitogenic, stress-related, apoptotic and survival stimuli. A special type of GPCR is the gonadotropin-releasing hormone (GnRH) receptor, which uses primarily the Gq protein for its downstream signaling. GnRH activates all four MAPK cascades by a PKC-dependent mechanism. Common signaling molecules, including the tyrosine kinase c-SRC and the small GTPases CDC42, RAC and RAS, are implicated in various aspects of the GnRH-MAPK pathways. Thus, the activation of MAPK cascades by GnRH opens a new vista in the understanding of the transcriptional regulation of genes encoding gonadotropins. However, additional studies on cell lines and whole animals are required to understand GnRH signaling in the context of other hormones during the reproductive cycle of mouse and human.

Animals↗

PubMed2003

High basal expression and injury-induced down regulation of two regulator of G-protein signaling transcripts, RGS3 and RGS4 in primary sensory neurons.

The regulators of G-protein signaling (RGS) proteins are a family of intracellular modulators of G-protein coupled receptor (GPCR) sensitivity. They act as GTPase accelerating proteins returning the Galpha subunit back to an inactive latent state. We find that RGS3 and RGS4 are constitutively expressed at high levels in C-fiber primary sensory neurons in the adult rat dorsal root ganglion (DRG) and transection of the sciatic nerve results in a substantial down regulation of these transcripts. RGS4 mRNA is expressed only in GDNF-responsive neurons and GDNF supports the expression of this transcript in primary DRG cultures. A PDZ domain containing subtype of RGS3 is the most abundant and regulated form of this protein within the DRG. Decreased levels of RGS3 and RGS4 in injured sensory neurons is likely to result in an increased GPCR sensitivity, and therefore contribute to alterations in cellular function seen after such lesions.

Animals↗

PubMed2001

Akt-mediated phosphorylation of the G protein-coupled receptor EDG-1 is required for endothelial cell chemotaxis.

The role of the protein kinase Akt in cell migration is incompletely understood. Here we show that sphingosine-1-phosphate (S1P)-induced endothelial cell migration requires the Akt-mediated phosphorylation of the G protein-coupled receptor (GPCR) EDG-1. Activated Akt binds to EDG-1 and phosphorylates the third intracellular loop at the T(236) residue. Transactivation of EDG-1 by Akt is not required for G(i)-dependent signaling but is indispensable for Rac activation, cortical actin assembly, and chemotaxis. Indeed, T236AEDG-1 mutant sequestered Akt and acted as a dominant-negative GPCR to inhibit S1P-induced Rac activation, chemotaxis, and angiogenesis. Transactivation of GPCRs by Akt may constitute a specificity switch to integrate rapid G protein-dependent signals into long-term cellular phenomena such as cell migration.

Actins↗

PubMed2002

The Galpha protein Gpa2 controls yeast differentiation by interacting with kelch repeat proteins that mimic Gbeta subunits.

G protein coupled receptors (GPCR) sense diverse ligands and signal via heterotrimeric G proteins. The Saccharomyces cerevisiae GPCR Gpr1 senses glucose and controls filamentous growth via an unusual Galpha protein, Gpa2, which lacks any known Gbetagamma subunits. Our genetic and biochemical studies identify Gpa2 interaction partners (Gpb1/2, Gpg1) and provide evidence that these proteins function as G protein subunit mimics and signaling effectors. Gpb1 and Gpb2 lack the seven WD-40 repeats found in Gbeta subunits and instead contain seven kelch repeats implicated in protein-protein interactions. Gbeta subunits and the kelch repeat protein galactose oxidase fold into strikingly similar seven-bladed beta propellers. Our studies demonstrate that Gpa2 signals in conjunction with Gbeta structural mimics and that homologous G protein subunits or effectors may be conserved in multicellular eukaryotes.

Amino Acid Sequence↗

PubMed2004

Identification of beta-arrestin2 as a G protein-coupled receptor-stimulated regulator of NF-kappaB pathways.

Norepinephrine released by the sympathetic nerve terminals regulates the immune system primarily via its stimulation of beta(2)-adrenergic receptor (beta(2)AR), but the underlying molecular mechanisms remain to be elicited. Beta(2)AR, a well-studied G protein-coupled receptor (GPCR), is functionally regulated by beta-arrestin2, which not only causes receptor desensitization and internalization but also serves as a signaling molecule in GPCR signal transduction. Here we show that beta-arrestin2 directly interacts with IkappaBalpha (inhibitor of NF-kappaB, the key molecule in innate and adaptive immunity) and thus prevents the phosphorylation and degradation of IkappaBalpha. Consequently, beta-arrestin2 effectively modulates activation of NF-kappaB and expression of NF-kappaB target genes. Moreover, stimulation of beta(2)AR significantly enhances beta-arrestin2-IkappaBalpha interaction and greatly promotes beta-arrestin2 stabilization of IkappaBalpha, indicating that beta-arrestin2 mediates a crosstalk between beta(2)AR and NF-kappaB signaling pathways. Taken together, the current study may present a novel mechanism for regulation of the immune system by the sympathetic nervous system.

Arrestins↗

PubMed2001

Endocytosis and downregulation of G protein-coupled receptors.

The number of G protein-coupled receptors (GPCRs) displayed at the cell surface is a critical determinant of physiological responsiveness to native ligands and drugs. Downregulation of the number of adrenergic and dopaminergic receptors present on specific neurons can be induced by receptor agonists or by drugs that increase extracellular concentrations of catecholamines such as dopamine. Thus agonist-induced downregulation of GPCRs is of potentially great importance to the treatment of Parkinson's Disease. However, little is known about biochemical mechanisms that mediate GPCR downregulation. Recent studies of cloned GPCRs have provided exciting insights into specific mechanisms that control endocytosis of receptors from the plasma membrane and regulate proteolytic degradation of receptors. In this review we briefly survey representative studies establishing that multiple mechanisms of GPCR membrane trafficking can function in downregulation function both in neural and non-neural cell types. Then we focus on our present view of mechanisms mediating regulated proteolysis of GPCRs, highlighting recent progress in understanding membrane trafficking of GPCRs from the cell surface to lysosomes. Finally we discuss emerging evidence regarding a specific mechanism that modulates sorting of certain GPCRs between recycling and degradative pathways.

Journal Article↗

PubMed2003

G protein-coupled receptor microarrays for drug discovery.

The dominance of G protein-coupled receptors (GPCRs) as a drug target class, coupled with the increased pace of target identification and expansion of compound libraries, presents a compelling need to develop technologies to screen multiple GPCRs simultaneously. To address this need, GPCR microarrays that require the co-immobilization of lipid molecules and the probe receptors of interest have been fabricated, using conventional robotic printing technologies. Assays to screen compounds for their pharmacological properties (binding affinity, relative potency and selectivity) using GPCR microarrays are discussed.

Drug Delivery Systems↗

PubMed2003

Allosteric modulators of G-protein-coupled receptors.

Allosteric modulators of G-protein-coupled receptors (GPCRs) interact with binding sites on the receptor that are topographically distinct from the orthosteric site recognized by the receptor's endogenous agonist. Allosteric modulators offer several advantages over standard orthosteric drugs, including the potential for greater receptor subtype selectivity. To date, the current paucity of clinically available allosteric drugs reflects the bias of traditional radioligand binding assays towards the detection of orthosteric effects. However, the advent of new cell-based high-throughput functional assays has led to an increased detection of allosteric GPCR ligands. The current challenge for modulator-based GPCR drug discovery is the optimization of both binding and functional assays to better detect and validate allosteric ligands.

Allosteric Regulation↗

PubMed2003

Integration of G-protein coupled receptor signaling pathways for activation of a transcription factor (EGR-3).

We recently reported the use of a gene-trapping approach to isolate cell clones in which a reporter gene had integrated into genes modulated by T-cell activation. We have now tested a panel of clones from that report and identified the one that responds to a variety of G-protein coupled receptors (GPCR). The beta-lactamase tagged EGR-3 Jurkat cell was used to dissect specific GPCR signaling in vivo. Three GPCRs were studied, including the chemokine receptor CXCR4 (Gi-coupled) that was endogenously expressed, the platelet activation factor (PAF) receptor (Gq-coupled), and beta2 adrenergic receptor (Gs-coupled) that was both stably transfected. Agonists for each receptor activated transcription of the beta-lactamase tagged EGR-3 gene. Induction of EGR-3 through CXCR4 was blocked by pertussis toxin and PD58059, a specific inhibitor of MEK (MAPK/ERK kinase). Neither of these inhibitors blocked isoproterenol or PAF-mediated activation of EGR-3. Conversely, beta2- and PAF-mediated EGR-3 activation was blocked by the p38, specific inhibitor SB580. In addition, both beta2- and PAF-mediated EGR-3 activation could be synergistically activated by CXCR4 activation. This combined result indicates that EGR-3 can be activated through distinct signal transduction pathways by different GPCRs and that signals can be integrated and amplified to efficiently tune the level of activation.

Culture Media↗

PubMed1998

Catecholamines stimulate the proliferation and alkaline phosphatase activity of MC3T3-E1 osteoblast-like cells.

A number of factors have been shown to influence osteoblastic proliferation, including fluoride. Recent observations suggest that heterotrimeric G proteins are probably involved in the mitogenic response induced by this agent, further suggesting a role of guanosine 5'-triphosphate (GTP)-binding protein-coupled receptors (GPCR) in the regulation of osteoblastic cell growth. We therefore explored what mitogenic factors known to activate GPCR can influence the replication of mouse osteoblast-like MC3T3-E1 cells. Among several candidates, epinephrine was found to be a potent mitogen for these cells, and its effect on the growth and differentiation of these cells was further investigated. Deoxyribonucleic acid (DNA) synthesis was dose dependently enhanced by this catecholamine in the concentration range of 1 nmol/L-10 micromol/L. Stimulation of DNA synthesis by catecholamines was in the order of epinephrine > norepinephrine >> isoproterenol, indicating that alpha adrenergic receptors mediated this cellular response. Further analysis with specific adrenergic receptor agonists and antagonists suggested that the mitogenic response induced by epinephrine in MC3T3-E1 cells is mediated by alpha1 adrenergic receptors. In addition to its effect on cell replication, epinephrine also enhanced alkaline phosphatase (ALP) activity in these cells but had little effect on collagen synthesis and osteocalcin production. As for the mitogenic response, the change in ALP activity was found to be mediated by alpha1 adrenergic receptors. Both effects of epinephrine on cell replication and ALP activity were markedly reduced by pretreatment of the cells with pertussis toxin (PTX), suggesting a role of Gi proteins. These effects were also completely blocked by pretreatment of the cells with 50 micromol/L genistein, a nonselective inhibitor of tyrosine kinase. In conclusion, the results indicate that epinephrine enhances replication and ALP activity of MC3T3-E1 osteoblast-like cells via alpha1 adrenergic receptors coupled to Gi proteins. The signaling mechanism probably involves a tyrosine phosphorylation mechanism. These observations suggest that PTX-sensitive G proteins are potent mediators of cell proliferation and ALP activity of osteoblast-like cells in response to factors acting through G protein-coupled receptors.

3T3 Cells↗