ABSTRACT
G proteins play a central role in signal transduction and pharmacology. Signaling is initiated by cell-surface receptors, which promote guanosine triphosphate (GTP) binding and dissociation of Gα from the Gßγ subunits. Structural studies have revealed the molecular basis of subunit association with receptors, RGS proteins, and downstream effectors. In contrast, the mechanism of subunit dissociation is poorly understood. We use cell signaling assays, molecular dynamics (MD) simulations, and biochemistry and structural analyses to identify a conserved network of amino acids that dictates subunit release. In the presence of the terminal phosphate of GTP, a glycine forms a polar network with an arginine and glutamate, putting torsional strain on the subunit binding interface. This "G-R-E motif" secures GTP and, through an allosteric link, discharges the Gßγ dimer. Replacement of network residues prevents subunit dissociation regardless of agonist or GTP binding. These findings reveal the molecular basis of the final committed step of G protein activation.
Subject(s)
Guanosine Triphosphate , Heterotrimeric GTP-Binding Proteins , Molecular Dynamics Simulation , Saccharomyces cerevisiae Proteins , Saccharomyces cerevisiae/enzymology , Allosteric Regulation , Amino Acid Motifs , Guanosine Triphosphate/chemistry , Guanosine Triphosphate/metabolism , HEK293 Cells , Heterotrimeric GTP-Binding Proteins/chemistry , Heterotrimeric GTP-Binding Proteins/metabolism , Humans , Saccharomyces cerevisiae Proteins/chemistry , Saccharomyces cerevisiae Proteins/metabolismABSTRACT
T-helper (Th) cell differentiation drives specialized gene programs that dictate effector T cell function at sites of infection. Here, we have shown Th cell differentiation also imposes discrete motility gene programs that shape Th1 and Th2 cell navigation of the inflamed dermis. Th1 cells scanned a smaller tissue area in a G protein-coupled receptor (GPCR) and chemokine-dependent fashion, while Th2 cells scanned a larger tissue area independent of GPCR signals. Differential chemokine reliance for interstitial migration was linked to STAT6 transcription-factor-dependent programming of integrin αVß3 expression: Th2 cell differentiation led to high αVß3 expression relative to Th1 cells. Th1 and Th2 cell modes of motility could be switched simply by manipulating the amount of αVß3 on the cell surface. Deviating motility modes from those established during differentiation impaired effector function. Thus, programmed expression of αVß3 tunes effector T cell reliance on environmental cues for optimal exploration of inflamed tissues.
Subject(s)
Inflammation/immunology , Th1 Cells/immunology , Th2 Cells/immunology , Adoptive Transfer , Animals , Cell Differentiation , Cell Movement , Cells, Cultured , Cellular Reprogramming Techniques , Chemokines/metabolism , Humans , Integrin alphaVbeta3/metabolism , Lymphocyte Activation , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , STAT6 Transcription Factor/metabolismABSTRACT
Phospholipase Cε (PLCε) is a multifunctional enzyme implicated in cardiovascular, pancreatic, and inflammatory functions. Here we show that conditional deletion of PLCε in mouse cardiac myocytes protects from stress-induced pathological hypertrophy. PLCε small interfering RNA (siRNA) in ventricular myocytes decreases endothelin-1 (ET-1)-dependent elevation of nuclear calcium and activation of nuclear protein kinase D (PKD). PLCε scaffolded to muscle-specific A kinase-anchoring protein (mAKAP), along with PKCε and PKD, localizes these components at or near the nuclear envelope, and this complex is required for nuclear PKD activation. Phosphatidylinositol 4-phosphate (PI4P) is identified as a perinuclear substrate in the Golgi apparatus for mAKAP-scaffolded PLCε. We conclude that perinuclear PLCε, scaffolded to mAKAP in cardiac myocytes, responds to hypertrophic stimuli to generate diacylglycerol (DAG) from PI4P in the Golgi apparatus, in close proximity to the nuclear envelope, to regulate activation of nuclear PKD and hypertrophic signaling pathways.
Subject(s)
Cardiomegaly/metabolism , Cardiomegaly/pathology , Phosphatidylinositol Phosphates/metabolism , Phosphoinositide Phospholipase C/metabolism , Animals , Aorta/pathology , GTP-Binding Protein alpha Subunits, Gq-G11/metabolism , Golgi Apparatus/metabolism , Heart , Heart Ventricles/cytology , Male , Mice , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Nuclear Envelope/metabolism , Phosphoinositide Phospholipase C/genetics , Rats , Signal TransductionABSTRACT
BACKGROUND: Chronically elevated neurohumoral drive, and particularly elevated adrenergic tone leading to ß-adrenergic receptor (ß-AR) overstimulation in cardiac myocytes, is a key mechanism involved in the progression of heart failure. ß1-AR (ß1-adrenergic receptor) and ß2-ARs (ß2-adrenergic receptor) are the 2 major subtypes of ß-ARs present in the human heart; however, they elicit different or even opposite effects on cardiac function and hypertrophy. For example, chronic activation of ß1-ARs drives detrimental cardiac remodeling while ß2-AR signaling is protective. The underlying molecular mechanisms for cardiac protection through ß2-ARs remain unclear. METHODS: ß2-AR signaling mechanisms were studied in isolated neonatal rat ventricular myocytes and adult mouse ventricular myocytes using live cell imaging and Western blotting methods. Isolated myocytes and mice were used to examine the roles of ß2-AR signaling mechanisms in the regulation of cardiac hypertrophy. RESULTS: Here, we show that ß2-AR activation protects against hypertrophy through inhibition of phospholipaseCε signaling at the Golgi apparatus. The mechanism for ß2-AR-mediated phospholipase C inhibition requires internalization of ß2-AR, activation of Gi and Gßγ subunit signaling at endosome and ERK (extracellular regulated kinase) activation. This pathway inhibits both angiotensin II and Golgi-ß1-AR-mediated stimulation of phosphoinositide hydrolysis at the Golgi apparatus ultimately resulting in decreased PKD (protein kinase D) and histone deacetylase 5 phosphorylation and protection against cardiac hypertrophy. CONCLUSIONS: This reveals a mechanism for ß2-AR antagonism of the phospholipase Cε pathway that may contribute to the known protective effects of ß2-AR signaling on the development of heart failure.
Subject(s)
Myocytes, Cardiac , Receptors, Adrenergic, beta-2 , Signal Transduction , Animals , Male , Mice , Rats , Animals, Newborn , Cardiomegaly/metabolism , Cardiomegaly/pathology , Cells, Cultured , Endocytosis , Golgi Apparatus/metabolism , Mice, Inbred C57BL , Mice, Knockout , Myocytes, Cardiac/metabolism , Phosphoinositide Phospholipase C/metabolism , Protein Kinase C/metabolism , Rats, Sprague-Dawley , Receptors, Adrenergic, beta-2/metabolismABSTRACT
Highly homologous members of the Gαi family, Gαi1-3, have distinct tissue distributions and physiological functions, yet their biochemical and functional properties are very similar. We recently identified PDZ-RhoGEF (PRG) as a novel Gαi1 effector that is poorly activated by Gαi2. In a proteomic proximity labeling screen we observed a strong preference for Gαi1 relative to Gαi2 with respect to engagement of a broad range of potential targets. We investigated the mechanistic basis for this selectivity using PRG as a representative target. Substitution of either the helical domain (HD) from Gαi1 into Gαi2 or substitution of a single amino acid, A230 in Gαi2 with the corresponding D in Gαi1, largely rescues PRG activation and interactions with other potential Gαi targets. Molecular dynamics simulations combined with Bayesian network models revealed that in the GTP bound state, separation at the HD-Ras-like domain (RLD) interface is more pronounced in Gαi2 than Gαi1. Mutation of A230 to D in Gαi2 stabilizes HD-RLD interactions via ionic interactions with R145 in the HD which in turn modify the conformation of Switch III. These data support a model where D229 in Gαi1 interacts with R144 and stabilizes a network of interactions between HD and RLD to promote protein target recognition. The corresponding A230 in Gαi2 is unable to stabilize this network leading to an overall lower efficacy with respect to target interactions. This study reveals distinct mechanistic properties that could underly differential biological and physiological consequences of activation of Gαi1 or Gαi2 by G protein-coupled receptors.
Subject(s)
GTP-Binding Protein alpha Subunits, Gi-Go , Signal Transduction , Humans , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/chemistry , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , Molecular Dynamics Simulation , GTP-Binding Protein alpha Subunit, Gi2/metabolism , GTP-Binding Protein alpha Subunit, Gi2/genetics , GTP-Binding Protein alpha Subunit, Gi2/chemistry , HEK293 Cells , Protein Domains , Protein Stability , Protein BindingABSTRACT
Opioid analgesics are widely used as a treatment option for pain management and relief. However, the misuse of opioid analgesics has contributed to the current opioid epidemic in the United States. Prescribed opioids such as morphine, codeine, oxycodone, and fentanyl are mu-opioid receptor (MOR) agonists primarily used in the clinic to treat pain or during medical procedures, but development of tolerance limits their utility for treatment of chronic pain. Here we explored the effects of biasing Gßγ signaling on tolerance development after chronic morphine treatment in vivo. We hypothesized that biasing Gßγ signaling with gallein could prevent activation of regulatory signaling pathways that result in tolerance to antinociceptive effects of MOR agonists. Gallein has been shown to bind to Gßγ and inhibit interactions of Gßγ with phospholipase-Cß3 (PLCß3) or G-protein-coupled receptor kinase 2 (GRK2) but not G-protein inwardly rectifying potassium (GIRK) channels. In mice, morphine-induced antinociception was evaluated in the 55°C warm water tail withdrawal assay. We used two paradigms for gallein treatment: administration during and after three times-daily morphine administration. Our results show that gallein cotreatment during repeated administration of morphine decreased opioid tolerance development and that gallein treatment in an opioid-tolerant state enhanced the potency of morphine. Mechanistically, our data suggest that PLCß3 is necessary for potentiating effects of gallein in an opioid-tolerant state but not in preventing the development of tolerance. These studies demonstrate that small molecules that target Gßγ signaling could reduce the need for large doses of opioid analgesics to treat pain by producing an opioid-sparing effect. SIGNIFICANCE STATEMENT: Biasing Gßγ signaling prevents tolerance to repeated morphine administration in vivo and potentiates the antinociceptive effects of morphine in an opioid-tolerant state. Mechanistically, phospholipase-Cß is necessary for potentiating effects of gallein in an opioid-tolerant state but not in preventing the development of tolerance. This study identifies a novel treatment strategy to decrease the development of tolerance to the analgesic effects of mu-opioid receptor agonists, which are necessary to improve pain treatment and decrease the incidence of opioid use disorder.
Subject(s)
Analgesics, Opioid , Drug Tolerance , GTP-Binding Protein beta Subunits , GTP-Binding Protein gamma Subunits , Mice, Inbred C57BL , Morphine , Nociception , Signal Transduction , Animals , Morphine/pharmacology , Drug Tolerance/physiology , Signal Transduction/drug effects , Mice , GTP-Binding Protein beta Subunits/metabolism , Male , Analgesics, Opioid/pharmacology , GTP-Binding Protein gamma Subunits/metabolism , Nociception/drug effects , Receptors, Opioid, mu/metabolism , Receptors, Opioid, mu/agonists , Phospholipase C beta/metabolism , XanthenesABSTRACT
Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) infects up to a quarter of the world's population. Although immune responses can control Mtb infection, 5%-10% of infected individuals can progress to active TB disease (progressors). A myriad of host factors regulate disease progression in TB and a better understanding of immune correlates of protection and disease is pivotal for the development of new therapeutics. Comparison of human whole blood transcriptomic metadata with that of macaque TB progressors and Mtb-infected diversity outbred mice (DO) led to the identification of differentially regulated gene (DEG) signatures, associated with TB progression or control. The current study assessed the function of Phospholipase C epsilon (PLCÆ1), the top downregulated gene across species in TB progressors, using a gene-specific knockout mouse model of Mtb infection and in vitro Mtb-infected bone marrow-derived macrophages. PLCÆ1 gene expression was downregulated in TB progressors across species. PLCε1 deficiency in the mouse model resulted in increased susceptibility to Mtb infection, coincident accumulation of lung myeloid cells, and reduced ability to mount antibacterial responses. However, PLCε1 was not required for the activation and accumulation of T cells in mice. Our results suggest an important early role for PLCÆ1 in shaping innate immune response to TB and may represent a putative target for host-directed therapy.
Subject(s)
Mycobacterium tuberculosis , Phosphoinositide Phospholipase C , Tuberculosis , Humans , Mice , Animals , Macrophage Activation , Immunity, InnateABSTRACT
Extra-large stimulatory Gα (XLαs) is a large variant of G protein αs subunit (Gαs) that uses an alternative promoter and thus differs from Gαs at the first exon. XLαs activation by G protein-coupled receptors mediates cAMP generation, similarly to Gαs; however, Gαs and XLαs have been shown to have distinct cellular and physiological functions. For example, previous work suggests that XLαs can stimulate inositol phosphate production in renal proximal tubules and thereby regulate serum phosphate levels. In this study, we show that XLαs directly and specifically stimulates a specific isoform of phospholipase Cß (PLCß), PLCß4, both in transfected cells and with purified protein components. We demonstrate that neither the ability of XLαs to activate cAMP generation nor the canonical G protein switch II regions are required for PLCß stimulation. Furthermore, this activation is nucleotide independent but is inhibited by Gßγ, suggesting a mechanism of activation that relies on Gßγ subunit dissociation. Surprisingly, our results indicate that enhanced membrane targeting of XLαs relative to Gαs confers the ability to activate PLCß4. We also show that PLCß4 is required for isoproterenol-induced inositol phosphate accumulation in osteocyte-like Ocy454 cells. Taken together, we demonstrate a novel mechanism for activation of phosphoinositide turnover downstream of Gs-coupled receptors that may have a critical role in endocrine physiology.
Subject(s)
GTP-Binding Protein alpha Subunits, Gs , Inositol Phosphates , Phospholipase C beta , Cell Membrane/metabolism , GTP-Binding Protein alpha Subunits, Gs/metabolism , Inositol Phosphates/metabolism , Isoenzymes/metabolism , Isoproterenol/pharmacology , Phospholipase C beta/metabolismABSTRACT
Pain management is an important problem worldwide. The current frontline approach for pain management is the use of opioid analgesics. The primary analgesic target of opioids is the µ-opioid receptor (MOR). Deletion of phospholipase Cß3 (PLCß3) or selective inhibition of Gßγ regulation of PLCß3 enhances the potency of the antinociceptive effects of morphine suggesting a novel strategy for achieving opioid-sparing effects. Here we investigated a potential mechanism for regulation of PLC signaling downstream of MOR in human embryonic kidney 293 cells and found that MOR alone could not stimulate PLC but rather required a coincident signal from a Gq-coupled receptor. Knockout of PLCß3 or pharmacological inhibition of its upstream regulators, Gßγ or Gq, ex vivo in periaqueductal gray slices increased the potency of the selective MOR agonist [D-Ala2, N-Me-Phe4, Gly5-ol]-enkephalin acetate salt in inhibiting presynaptic GABA release. Finally, inhibition of Gq- G protein-coupled receptor coupling in mice enhanced the antinociceptive effects of morphine. These data support a model where Gq and Gßγ-dependent signaling cooperatively regulate PLC activation to decrease MOR-dependent antinociceptive potency. Ultimately, this could lead to identification of new non-MOR targets that would allow for lower-dose utilization of opioid analgesics. SIGNIFICANCE STATEMENT: Previous work demonstrated that deletion of phospholipase Cß3 (PLCß3) in mice potentiates µ-opioid receptor (MOR)-dependent antinociception. How PLCß3 is regulated downstream of MOR had not been clearly defined. We show that PLC-dependent diacylglycerol generation is cooperatively regulated by MOR-Gßγ and Gq-coupled receptor signaling through PLCß3 and that blockade of either Gq-signaling or Gßγ signaling enhances the potency of opioids in ex vivo brain slices and in vivo. These results reveal potential novel strategies for improving opioid analgesic potency and safety.
Subject(s)
Analgesics, Opioid , Receptors, Opioid, mu , Animals , Mice , Humans , Analgesics, Opioid/pharmacology , Phospholipase C beta , Mice, Knockout , Receptors, Opioid, mu/physiology , Morphine/pharmacology , Analgesics , Enkephalin, Ala(2)-MePhe(4)-Gly(5)-/pharmacologyABSTRACT
Phospholipase Cε (PLCε) is a phospholipase C isoform with a wide range of physiological functions. It has been implicated in aortic valve disorders, but its role in frequently associated aortic disease remains unclear. To determine the role of PLCε in thoracic aortic aneurysm and dissection (TAAD) we used PLCε-deficient mice, which develop aortic valve insufficiency and exhibit aortic dilation of the ascending thoracic aorta and arch without histopathological evidence of injury. Fourteen days of infusion of Plce1+/+ and Plce1-/- mice with angiotensin II (ANG II), which induces aortic dilation and dissection, led to sudden death secondary to ascending aortic dissection in 43% of Plce1-/- versus 5% of Plce1+/+ mice (P < 0.05). Medial degeneration and TAAD were detected in 80% of Plce1-/- compared with 10% of Plce1+/+ mice (P < 0.05) after 4 days of ANG II. Treatment with ANG II markedly increased PLCε expression within the ascending aortic adventitia. Total RNA sequencing demonstrated marked upregulation of inflammatory and fibrotic pathways mediated by interleukin-1ß, interleukin-6, and tumor necrosis factor-α. In silico analysis of whole exome sequences of 258 patients with type A dissection identified 5 patients with nonsynonymous PLCE1 variants. Our data suggest that PLCε deficiency plays a role in the development of TAAD and aortic insufficiency.NEW & NOTEWORTHY We describe a novel phenotype by which PLCε deficiency predisposes to aortic valve insufficiency and ascending aortic aneurysm, dissection, and sudden death in the setting of ANG II-mediated hypertension. We demonstrate PLCE1 variants in patients with type A aortic dissection and aortic insufficiency, suggesting that PLCE1 may also play a role in human aortic disease. This finding is of very high significance because it has not been previously demonstrated that PLCε directly mediates aortic dissection.
Subject(s)
Aneurysm, Ascending Aorta , Aortic Aneurysm, Thoracic , Aortic Aneurysm , Aortic Dissection , Aortic Valve Insufficiency , Hypertension , Humans , Mice , Animals , Aortic Valve Insufficiency/genetics , Mice, Inbred C57BL , Aortic Aneurysm/genetics , Aortic Aneurysm/pathology , Aortic Dissection/genetics , Angiotensin II , Death, Sudden , Aortic Aneurysm, Thoracic/genetics , Aortic Aneurysm, Thoracic/metabolismABSTRACT
ABSTRACT: Adrenergic receptors are critical regulators of cardiac function with profound effects on cardiac output during sympathetic stimulation. Chronic stimulation of the adrenergic system of the heart under conditions of cardiac stress leads to cardiac dysfunction, hypertrophy, and ultimately failure. Emerging data have revealed that G protein-coupled receptors in intracellular compartments are functionally active and regulate distinct cellular processes from those at the cell surface. ß2 adrenergic receptors internalize onto endosomes in various cell types where they have recently been shown to continue to stimulate cAMP production to selectively regulate gene expression. Other studies have identified ß1 adrenergic receptors at the nuclear envelope and the Golgi apparatus. Here, we discuss data on signaling by ß1 and ß2 adrenergic receptors in the heart and the possible influence of their subcellular locations on their divergent physiological functions in cardiac myocytes and in cardiac pathology. Understanding the relative roles of these receptors at these locations could have a significant impact on pharmacological targeting of these receptors for the treatment of heart failure and cardiac diseases.
Subject(s)
Heart Failure , Receptors, Adrenergic, beta , Heart Failure/drug therapy , Heart Failure/metabolism , Humans , Myocytes, Cardiac , Receptors, Adrenergic, beta/metabolism , Receptors, Adrenergic, beta-1/genetics , Receptors, Adrenergic, beta-1/metabolism , Receptors, Adrenergic, beta-2/metabolism , Signal TransductionABSTRACT
Phosphatidylinositol (3,4,5) trisphosphate (PIP3)-dependent Rac exchanger 1 (P-Rex1) is a Rho guanine-nucleotide exchange factor that was originally discovered in neutrophils and is regulated by G protein ßγ subunits and the lipid PIP3 in response to chemoattractants. P-Rex1 has also become increasingly recognized for its role in promoting metastasis of breast cancer, prostate cancer, and melanoma. Recent structural, biochemical, and biologic work has shown that binding of PIP3 to the pleckstrin homology (PH) domain of P-Rex1 is required for its activation in cells. Here, differential scanning fluorimetry was used in a medium-throughput screen to identify six small molecules that interact with the P-Rex1 PH domain and block binding of and activation by PIP3 Three of these compounds inhibit N-formylmethionyl-leucyl-phenylalanine induced spreading of human neutrophils as well as activation of the GTPase Rac2, both of which are downstream effects of P-Rex1 activity. Furthermore, one of these compounds reduces neutrophil velocity and inhibits neutrophil recruitment in response to inflammation in a zebrafish model. These results suggest that the PH domain of P-Rex1 is a tractable drug target and that these compounds might be useful for inhibiting P-Rex1 in other experimental contexts. SIGNIFICANCE STATEMENT: A set of small molecules identified in a thermal shift screen directed against the phosphatidylinositol (3,4,5) trisphosphate-dependent Rac exchanger 1 (P-Rex1) pleckstrin homology domain has effects consistent with P-Rex1 inhibition in neutrophils.
Subject(s)
Drug Discovery/methods , Guanine Nucleotide Exchange Factors/antagonists & inhibitors , Guanine Nucleotide Exchange Factors/metabolism , Neutrophils/metabolism , Phosphatidylinositol Phosphates/metabolism , Animals , Binding Sites/drug effects , Binding Sites/physiology , Cells, Cultured , Crystallography, X-Ray/methods , Dose-Response Relationship, Drug , Drug Delivery Systems/methods , Guanine Nucleotide Exchange Factors/chemistry , Humans , Neutrophils/drug effects , Phosphatidylinositol Phosphates/chemistry , Protein Structure, Secondary , Protein Structure, Tertiary , ZebrafishABSTRACT
Loss-of-function mutations in phospholipase C-ε1 (PLCE1) have been detected in patients with nephrotic syndrome, but other family members with the same mutation were asymptomatic, suggesting additional stressor are required to cause the full phenotype. Consistent with these observations, we determined that global Plce1-deficient mice have histologically normal glomeruli and no albuminuria at baseline. Angiotensin II (ANG II) is known to induce glomerular damage in genetically susceptible individuals. Therefore, we tested whether ANG II enhances glomerular damage in Plce1-deficient mice. ANG II increased blood pressure equally in Plce1-deficient and wild-type littermates. Additionally, it led to 20-fold increased albuminuria and significantly more sclerotic glomeruli in Plce1-deficient mice compared with wild-type littermates. Furthermore, Plce1-deficient mice demonstrated diffuse mesangial expansion, podocyte loss, and focal podocyte foot process effacement. To determine whether these effects are mediated by hypertension and hyperfiltration, rather than directly through ANG II, we raised blood pressure to a similar level using DOCA + salt + uninephrectomy and norepinephrine. This caused a fivefold increase in albuminuria in Plce1-deficient mice and a significant increase in the number of sclerotic glomeruli. Consistent with previous findings in mice, we detected strong PLCE1 transcript expression in podocytes using single cell sequencing of human kidney tissue. In hemagglutinin-tagged Plce1 transgenic mice, Plce1 was detected in podocytes and also in glomerular arterioles using immunohistochemistry. Our data demonstrate that Plce1 deficiency in mice predisposes to glomerular damage secondary to hypertensive insults.
Subject(s)
Blood Pressure , Glomerulonephritis/enzymology , Hypertension/enzymology , Kidney Glomerulus/enzymology , Phosphoinositide Phospholipase C/deficiency , Albuminuria/enzymology , Albuminuria/genetics , Albuminuria/physiopathology , Animals , Desoxycorticosterone Acetate , Disease Models, Animal , Female , Glomerulonephritis/genetics , Glomerulonephritis/pathology , Glomerulonephritis/physiopathology , Hypertension/genetics , Hypertension/physiopathology , Kidney Glomerulus/pathology , Kidney Glomerulus/physiopathology , Male , Mice, Inbred C57BL , Mice, Knockout , Nephrectomy , Phosphoinositide Phospholipase C/genetics , Sodium Chloride, DietaryABSTRACT
G-protein ßγ subunits are key participants in G-protein signaling. These subunits facilitate interactions between receptors and G proteins that are critical for the G protein activation cycle at the plasma membrane. In addition, they play roles in directly transducing signals to an ever expanding range of downstream targets, including integral membrane and cytosolic proteins. Emerging data indicate that Gßγ may play additional roles at intracellular compartments including endosomes, the Golgi apparatus, and the nucleus. Here, we discuss the molecular and structural basis for their ability to coordinate this wide range of cellular activities.
Subject(s)
GTP-Binding Protein beta Subunits/metabolism , GTP-Binding Protein gamma Subunits/metabolism , Receptors, G-Protein-Coupled/metabolism , Signal Transduction/physiology , Animals , Cell Membrane/metabolism , Cell Membrane/physiology , Cell Nucleus/metabolism , Cell Nucleus/physiology , Endosomes/metabolism , Endosomes/physiology , Golgi Apparatus/metabolism , Golgi Apparatus/physiology , HumansABSTRACT
Regulation of integrins is critical for lymphocyte adhesion to endothelium and migration throughout the body. Inside-out signaling to integrins is mediated by the small GTPase Ras-proximate-1 (Rap1). Using an RNA-mediated interference screen, we identified phospholipase Cε 1 (PLCε1) as a crucial regulator of stromal cell-derived factor 1 alpha (SDF-1α)-induced Rap1 activation. We have shown that SDF-1α-induced activation of Rap1 is transient in comparison with the sustained level following cross-linking of the antigen receptor. We identified that PLCε1 was necessary for SDF-1α-induced adhesion using shear stress, cell morphology alterations, and crawling on intercellular adhesion molecule 1 (ICAM-1)-expressing cells. Structure-function experiments to separate the dual-enzymatic function of PLCε1 uncover necessary contributions of the CDC25, Pleckstrin homology, and Ras-associating domains, but not phospholipase activity, to this pathway. In the mouse model of delayed type hypersensitivity, we have shown an essential role for PLCε1 in T-cell migration to inflamed skin, but not for cytokine secretion and proliferation in regional lymph nodes. Our results reveal a signaling pathway where SDF-1α induces T-cell adhesion through activation of PLCε1, suggesting that PLCε1 is a specific potential target in treating conditions involving migration of T cells to inflamed organs.
Subject(s)
Chemokine CXCL12/genetics , Inflammation/genetics , Phosphoinositide Phospholipase C/genetics , Telomere-Binding Proteins/genetics , Animals , Cell Adhesion/genetics , Cell Adhesion/immunology , Cell Movement/genetics , Cell Movement/immunology , Chemokine CXCL12/immunology , Humans , Inflammation/immunology , Inflammation/pathology , Intercellular Adhesion Molecule-1/immunology , Lymphocytes/immunology , Lymphocytes/pathology , Mice , Phosphoinositide Phospholipase C/immunology , Receptors, Antigen/genetics , Receptors, Antigen/immunology , Shelterin Complex , Signal Transduction , T-Lymphocytes/immunology , T-Lymphocytes/pathology , Telomere-Binding Proteins/immunology , ras-GRF1/immunologyABSTRACT
Our recent work uncovered novel roles for activated Gαi signaling in the regulation of neutrophil polarity and adhesion. GαiGTP-mediated enhancement of neutrophil polarization was dependent on inhibition of cAMP/PKA signaling, whereas reversal of Gßγ-stimulated adhesion was cAMP/PKA independent. To uncover the mechanism for Gαi regulation of adhesion, we analyzed the effects of constitutively active Gαi1(Q204L) expression on adhesion driven by constitutively active Rap1a(G12V) or its downstream effector Radil in neutrophil-like HL-60 cells, or in HT-1080 fibrosarcoma cells. In HT-1080 cells, Rap1a(G12V) or Radil cause an increase in cell spreading and adhesion to fibronectin, which are both reversed by Gαi1(Q204L) but not WT Gαi1 In contrast, Gαi1(Q204L) did not reverse Rap1-GTP-interacting adaptor molecule (RIAM)-dependent increases in cell adhesion. This indicates that adhesion regulation by Gαi-GTP occurs downstream of Rap1a and Radil, but is upstream of components such as integrins and talin that are regulated by both Radil and RIAM. HL-60 neutrophil-like cells expressing Rap1a(G12V) or Radil have an elongated phenotype because of enhanced uropod adhesion as they attempt to migrate on fibronectin. This elongated phenotype driven by Rap1a(G12V) or Radil is reversed by Gαi1(Q204L), but not by WT Gαi1 expression, suggesting that Gαi-GTP also regulates adhesion in immune cells at the level of, or downstream of, Radil. These data identify a novel role of Gαi-GTP in regulation of cell adhesion and migration. Cell migration involves cycles of adhesion and de-adhesion, and we propose that the dynamic spatiotemporal balance between Gßγ-promoted adhesion and Gαi-GTP reversal of adhesion is important for this process.
Subject(s)
Carrier Proteins/metabolism , Cell Movement , GTP-Binding Protein alpha Subunits, Gi-Go/metabolism , Mutation, Missense , rap1 GTP-Binding Proteins/metabolism , Amino Acid Substitution , Carrier Proteins/genetics , Cell Adhesion , Fibronectins/metabolism , GTP-Binding Protein alpha Subunits, Gi-Go/genetics , GTP-Binding Protein beta Subunits/genetics , GTP-Binding Protein beta Subunits/metabolism , GTP-Binding Protein gamma Subunits/genetics , GTP-Binding Protein gamma Subunits/metabolism , HL-60 Cells , Humans , rap1 GTP-Binding Proteins/geneticsABSTRACT
Phospholipase C (PLC) enzymes hydrolyze membrane phosphatidylinositol 4,5 bisphosphate (PIP2) and regulate Ca2+ and protein kinase signaling in virtually all mammalian cell types. Chronic activation of the PLCϵ isoform downstream of G protein-coupled receptors (GPCRs) contributes to the development of cardiac hypertrophy. We have previously shown that PLCϵ-catalyzed hydrolysis of Golgi-associated phosphatidylinositol 4-phosphate (PI4P) in cardiac myocytes depends on G protein ßγ subunits released upon stimulation with endothelin-1. PLCϵ binds and is directly activated by Ras family small GTPases, but whether they directly interact with Gßγ has not been demonstrated. To identify PLCϵ domains that interact with Gßγ, here we designed various single substitutions and truncations of WT PLCϵ and tested them for activation by Gßγ in transfected COS-7 cells. Deletion of only a single domain in PLCϵ was not sufficient to completely block its activation by Gßγ, but blocked activation by Ras. Simultaneous deletion of the C-terminal RA2 domain and the N-terminal CDC25 and cysteine-rich domains completely abrogated PLCϵ activation by Gßγ, but activation by the GTPase Rho was retained. In vitro reconstitution experiments further revealed that purified Gßγ directly interacts with a purified fragment of PLCϵ (PLCϵ-PH-RA2) and increases PIP2 hydrolysis. Deletion of the RA2 domain decreased Gßγ binding and eliminated Gßγ stimulation of PIP2 hydrolysis. These results provide first evidence that Gßγ directly interacts with PLCϵ and yield insights into the mechanism by which ßγ subunits activate PLCϵ.
Subject(s)
GTP-Binding Protein beta Subunits/metabolism , GTP-Binding Protein gamma Subunits/metabolism , Golgi Apparatus/enzymology , Myocytes, Cardiac/enzymology , Phosphoinositide Phospholipase C/metabolism , cdc25 Phosphatases/metabolism , Animals , COS Cells , Chlorocebus aethiops , Endothelin-1/genetics , Endothelin-1/metabolism , GTP-Binding Protein beta Subunits/genetics , GTP-Binding Protein gamma Subunits/genetics , Golgi Apparatus/genetics , Myocytes, Cardiac/cytology , Phosphatidylinositol Phosphates/genetics , Phosphatidylinositol Phosphates/metabolism , Phosphoinositide Phospholipase C/genetics , Protein Domains , Rats , cdc25 Phosphatases/genetics , rho GTP-Binding Proteins/genetics , rho GTP-Binding Proteins/metabolismABSTRACT
Exchange protein directly activated by cAMP (Epac) is a direct effector for the ubiquitous second messenger cAMP. Epac activates the phospholipase Cε (PLCε) pathway. PLCß has been linked to the synthesis of the endocannabinoid 2-arachidonoylglycerol (2-AG). Here, we report that Epac facilitates endocannabinoid-mediated retrograde synaptic depression through activation of PLCε. Intracellular loading of a selective Epac agonist 8-CPT-2Me-cAMP into ventral tegmental area (VTA) dopamine neurons enabled previously ineffective stimuli to induce depolarization-induced suppression of inhibition (DSI) and long-term depression of IPSCs (I-LTD) in the VTA. DSI and I-LTD are mediated by 2-AG since they were blocked by a diacylglycerol lipase inhibitor. The effects of 8-CPT-2Me-cAMP on DSI and I-LTD were absent in Epac2 and PLCε knock-out mice, but remained intact in Epac1 knock-out mice. These results identify a novel mechanism for on-demand synthesis of retrograde signaling 2-AG by the Epac2-PLCε pathway. We investigated the functional significance of Epac2-PLCε-2-AG signaling in regulating inhibitory synaptic plasticity in VTA dopamine neurons induced by in vivo cocaine exposure. We showed that cocaine place conditioning led to a decrease in the frequency and amplitude of spontaneous IPSCs and an increase in action potential firing in wild-type mice, but not in Epac2 or PLCε knock-out mice. Together, these results indicate that the Epac2-PLCε-2-AG signaling cascade contributes to cocaine-induced disinhibition of VTA dopamine neurons.SIGNIFICANCE STATEMENT 2-arachidonoylglycerol (2-AG) is an endogenous cannabinoid that depresses synaptic transmission through stimulation of CB1 receptors. Among the six isoforms of phospholipase C (PLC; PLCß, PLCγ, PLCδ, PLCε, PLCζ, PLCη), only PLCß has been linked to 2-AG synthesis. Here we demonstrate that 8-CPT-2Me-cAMP, a selective agonist of the cAMP sensor protein Epac, enhances 2-AG-mediated synaptic depression in ventral tegmental area (VTA) dopamine neurons via activation of PLCε. These results identify a novel mechanism for 2-AG synthesis via activation of the Epac-PLCε pathway. Furthermore, we show that cocaine-induced conditioned place preference and disinhibition of VTA dopamine neurons were impaired in mice lacking Epac or PLCε. Thus, the Epac-PLCε signaling pathway contributes to cocaine-induced disinhibition of VTA dopamine neurons and formation of drug-associated memories.
Subject(s)
Cocaine/administration & dosage , Dopaminergic Neurons/physiology , Endocannabinoids/metabolism , Neural Inhibition/physiology , Phosphoinositide Phospholipase C/metabolism , Ventral Tegmental Area/physiology , Animals , Dopaminergic Neurons/drug effects , Female , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Neural Inhibition/drug effects , Signal Transduction/drug effects , Signal Transduction/physiology , Ventral Tegmental Area/cytology , Ventral Tegmental Area/drug effectsABSTRACT
In cardiac myocytes activation of an exchange factor activated by cAMP (Epac) leads to activation of phospholipase Cε (PLCε)-dependent hydrolysis of phosphatidylinositol 4-phosphate (PI4P) in the Golgi apparatus a process critical for development of cardiac hypertrophy. Here we show that ß-adrenergic receptor (ßAR) stimulation does not stimulate this pathway in the presence of the broad spectrum phosphodiesterase (PDE) inhibitor IBMX, but selective PDE3 inhibition revealed ßAR-dependent PI4P depletion. On the other hand, selective inhibition of PDE2 or PDE9A blocked endothelin-1 (ET-1) and cAMP-dependent PI4P hydrolysis by PLCε. Direct activation of protein kinase A (PKA), protein kinase G (PKG), or the atrial natriuretic factor (ANF) receptor abolished PI4P hydrolysis in response to multiple upstream stimuli. These results reveal distinct pools of cyclic nucleotides that either inhibit PLCε at the Golgi through PKA/PKG, or activate PLCε at the Golgi through Epac. These data together reveal a new mechanism by which ANF and selective PDE inhibitors can protect against cardiac hypertrophy.
Subject(s)
Cardiomegaly/genetics , Cell Compartmentation/genetics , Golgi Apparatus/genetics , Nucleotides/genetics , Phosphoinositide Phospholipase C/genetics , 1-Methyl-3-isobutylxanthine/administration & dosage , A Kinase Anchor Proteins/genetics , A Kinase Anchor Proteins/metabolism , Animals , Atrial Natriuretic Factor/genetics , Cardiomegaly/metabolism , Cardiomegaly/pathology , Cardiomyopathy, Hypertrophic/genetics , Cardiomyopathy, Hypertrophic/pathology , Cyclic AMP/metabolism , Cyclic AMP-Dependent Protein Kinases/genetics , Golgi Apparatus/drug effects , Humans , Myocytes, Cardiac/metabolism , Myocytes, Cardiac/pathology , Nucleotides/metabolism , Phosphatidylinositol Phosphates/metabolism , Phosphoinositide Phospholipase C/metabolism , Phosphoric Diester Hydrolases/genetics , Rats , Rats, Sprague-Dawley , Receptors, Adrenergic, beta/genetics , Signal Transduction/geneticsABSTRACT
Adhesion G protein-coupled receptors (aGPCRs) have emerged as potential therapeutic targets in multiple cancers and in neurologic diseases. However, there are few modulatory compounds that act on these receptors. The majority of aGPCRs are orphans and a general activation mechanism has only recently been defined: aGPCRs are activated by a tethered agonist. aGPCRs constitutively cleave themselves during biosynthesis to generated two-part receptors comprising an extracellular domain (ECD) and a 7-transmembrane spanning domain (7TM). ECD dissociation reveals the tethered agonist initiating G protein signaling. Synthetic peptides that mimic the tethered agonist region can activate aGPCRs. We hypothesized that small molecules could act in the same way as peptide agonists. High throughput screening of the 2000-compound Spectrum Collection library using the serum response element luciferase gene reporter assay revealed two related classes of small molecules that could activate the aGPCR GPR56/ADGRG1. The most potent compound identified was 3-α-acetoxydihydrodeoxygedunin, or 3-α-DOG. 3-α-DOG activated engineered, low-activity GPR56 7TM in independent biochemical and cell-based assays with an EC50 of â¼5 µM. The compound also activated a subset of aGPCRs but not two class A GPCRs tested. The mode of 3-α-DOG-mediated receptor activation is that of partial agonist. 3-α-DOG activated GPR56 less efficaciously than peptide agonist and could antagonize both the peptide agonist and the endogenous tethered agonist, which are pharmacological hallmarks of partial agonists. Taken together, we have uncovered a novel group of aGPCR partial agonists that will serve as invaluable resources for understanding this unique class receptors.