Orphan Receptor GPR158 Is an Allosteric Modulator of RGS7 Catalytic Activity with an Essential Role in Dictating Its Expression and Localization in the BrainRegulators of G protein signaling control the duration and extent of signaling via G protein-coupled receptor (GPCR) pathways by accelerating the GTP hydrolysis on G protein α subunits thereby promoting termination of GPCR signaling. A member of this family, RGS7, plays a critical role in the nervous system where it regulates multiple neurotransmitter GPCRs that mediate vision, memory, and the action of addictive drugs. Previous studies have established that in vivo RGS7 forms mutually exclusive complexes with the membrane protein RGS7-binding protein or the orphan receptor GPR158.
Protein Kinase A (PKA) Phosphorylation of Shp2 Protein Inhibits Its Phosphatase Activity and Modulates Ligand SpecificityPathological cardiac hypertrophy (an increase in cardiac mass resulting from stress-induced cardiac myocyte growth) is a major factor underlying heart failure. Src homology 2 domain-containing phosphatase (Shp2) is critical for cardiac function because mutations resulting in loss of Shp2 catalytic activity are associated with congenital cardiac defects and hypertrophy. We identified a novel mechanism of Shp2 inhibition that may promote cardiac hypertrophy. We demonstrate that Shp2 is a component of the protein kinase A anchoring protein (AKAP)-Lbc complex.
Self-cleavage of the Pseudomonas aeruginosa Cell-surface Signaling Anti-sigma Factor FoxR Occurs through an N-O Acyl RearrangementThe Fox system of Pseudomonas aeruginosa is a cell-surface signaling (CSS) pathway employed by the bacterium to sense and respond to the presence of the heterologous siderophore ferrioxamine in the environment. This regulatory pathway controls the transcription of the foxA ferrioxamine receptor gene through the extracytoplasmic function sigma factor σFoxI. In the absence of ferrioxamine, the activity of σFoxI is inhibited by the transmembrane anti-sigma factor FoxR. Upon binding of ferrioxamine by the FoxA receptor, FoxR is processed by a complex proteolytic cascade leading to the release and activation of σFoxI.
Distinct Pathways Regulate Syk Protein Activation Downstream of Immune Tyrosine Activation Motif (ITAM) and hemITAM Receptors in PlateletsTyrosine kinase pathways are known to play an important role in the activation of platelets. In particular, the GPVI and CLEC-2 receptors are known to activate Syk upon tyrosine phosphorylation of an immune tyrosine activation motif (ITAM) and hemITAM, respectively. However, unlike GPVI, the CLEC-2 receptor contains only one tyrosine motif in the intracellular domain. The mechanisms by which this receptor activates Syk are not completely understood. In this study, we identified a novel signaling mechanism in CLEC-2-mediated Syk activation.
Opposite Cross-Talk by Oleate and Palmitate on Insulin Signaling in Hepatocytes through Macrophage ActivationChronic low grade inflammation in adipose tissue during obesity is associated with an impairment of the insulin signaling cascade. In this study, we have evaluated the impact of palmitate or oleate overload of macrophage/Kupffer cells in triggering stress-mediated signaling pathways, in lipoapoptosis, and in the cross-talk with insulin signaling in hepatocytes. RAW 264.7 macrophages or Kupffer cells were stimulated with oleate or palmitate, and levels of M1/M2 polarization markers and the lipidomic profile of eicosanoids were analyzed.
Tyrosine Kinase 2-mediated Signal Transduction in T Lymphocytes Is Blocked by Pharmacological Stabilization of Its Pseudokinase DomainBackground: Interleukin-23 mediates pathobiology in many autoimmune disorders.Results: A chemogenomics approach identified small molecule agents that block receptor-mediated activation or tyrosine kinase 2 (Tyk2) and downstream signaling. Compounds stabilize the pseudokinase domain of Tyk2.Conclusion: Small molecule ligands of the Tyk2 pseudokinase domain stabilize an autoinhibitory interaction with the catalytic domain.Significance: This work enables the discovery of selective therapeutics targeting Tyk2-dependent pathways critical in autoimmunity.
Apoptosis Signal-regulating Kinase 1 (ASK1)-p38 Pathway-dependent Cytoplasmic Translocation of the Orphan Nuclear Receptor NR4A2 Is Required for Oxidative Stress-induced NecrosisBackground: The molecular mechanisms of p38 MAPK-mediated necrosis currently have not been well elucidated.Results: During oxidative stress, NR4A2 is phosphorylated and translocated into the cytosol in an ASK1-p38-dependent manner, which ultimately leads to the promotion of necrosis.Conclusion: ASK1-p38 MAPK pathway-dependent phosphorylation and subsequent cytoplasmic translocation of NR4A2 promote oxidative stress-induced necrosis.Significance: We found a novel intracellular signaling pathway that regulates oxidative stress-induced and p38-mediated necrosis.
Group VIA Phospholipase A2 (iPLA2β) Modulates Bcl-x 5′-Splice Site Selection and Suppresses Anti-apoptotic Bcl-x(L) in β-CellsBackground: β-Cell apoptosis, a critical contributor to T1D, involves iPLA2β activation and is suppressed by Bcl-x(L).Results: iPLA2β-derived lipids activate an alternative 5′-splice site, reducing protective Bcl-x(L) protein.Conclusion: Modulation of Bcl-x splicing is another key mechanism by which iPLA2β-derived lipids promote β-cell apoptosis.Significance: Delineation of molecular mechanisms underlying iPLA2β-regulated splicing will elucidate novel strategies to counter β-cell death in T1D.
Bicarbonate Modulates Photoreceptor Guanylate Cyclase (ROS-GC) Catalytic ActivityBackground: ROS-GCs generate cGMP and control phototransduction in rods and cones.Results: Through a unique [Ca2+]i-independent mechanism, bicarbonate stimulates ROS-GC activity to increase circulating current, quicken flash responses, and reduce relative sensitivity.Conclusion: Bicarbonate is a novel modulator of the photoreceptor ROS-GC.Significance: Vision and certain forms of retinal diseases may be affected by the metabolic states of retinal cells.
Heterotrimeric G Proteins Directly Regulate MMP14/Membrane Type-1 Matrix Metalloprotease: A NOVEL MECHANISM FOR GPCR-EGFR TRANSACTIVATIONBackground: Mechanisms underlying GPCR-mediated EGFR transactivation are not fully defined.Results: Heterotrimeric G proteins directly regulate membrane-localized MMP14/MT1-MMP, resulting in HB-EGF release and EGFR transactivation.Conclusion: These results define a previously unrecognized, membrane-delimited mechanism for EGFR transactivation.Significance: This mechanism likely plays a role in settings that involve GPCR-RTK transactivation and may represent new therapeutic opportunities.
Quantitative Analysis of Receptor Tyrosine Kinase-Effector Coupling at Functionally Relevant Stimulus LevelsBackground: The RET receptor activates downstream effectors that mediate function.Results: Large EC50 shifts between receptor and effector dose responses show RET activates ERK and Akt most efficiently at low, functionally relevant ligand concentrations.Conclusion: Experiments at high ligand concentrations can obscure quantitative features of receptor signaling.Significance: Consideration of receptor-effector coupling is important for understanding how stimulus drives response in cell signaling.
Signaling Pathways That Control Rho Kinase Activity Maintain the Embryonic Epicardial Progenitor StateBackground: Epicardial cells are a potential source of progenitor cells for revascularization of the injured heart.Results: Decreased p63RhoGEF and GEF-H1 and increased Epac, p190RhoGAP, and Rnds activities suppress RhoA signaling in epicardial progenitors.Conclusion: The embryonic epicardial progenitor state is maintained by signaling pathways that control RhoA activity.Significance: Manipulation of these signaling molecules might promote cardiac revascularization.
Ubiquitin-associated Domain-containing Ubiquitin Regulatory X (UBX) Protein UBXN1 Is a Negative Regulator of Nuclear Factor κB (NF-κB) SignalingBackground: Excessive NF-κB hyperactivation should be tightly controlled in cells.Results: UBXN1 inhibits TNFα-triggered NF-κB signaling by sequestering cIAPs from being recruited to TNFR1.Conclusion: UBXN1 is a novel negative regulator of TNFα-triggered NF-κB signaling.Significance: Our study identified a novel ubiquitin-linked protein UBXN1 as a negative regulator of NF-κB signaling pathway independent of VCP/p97 and provided important insight into the new regulatory mechanism of the UBX protein family.
Activated α2-Macroglobulin Binding to Human Prostate Cancer Cells Triggers Insulin-like ResponsesBackground: Ligation of cancer cell surface GRP78 by activated α2-macroglobulin (α2M*) promotes proliferation and blocks apoptosis.Results: α2M* treatment of prostate cancer cells enhances the Warburg effect and up-regulates lipid metabolism in an insulin-like manner.Conclusion: α2M* exerts insulin-like effects on prostate cancer cells.Significance: Targeting cell surface GRP78-dependent cancer cell regulation, such as by antibodies, offers a unique potential therapeutic approach. Ligation of cell surface GRP78 by activated α2-macroglobulin (α2M*) promotes cell proliferation and suppresses apoptosis.
A Novel Phosphatidic Acid-Protein-tyrosine Phosphatase D2 Axis Is Essential for ERBB2 Signaling in Mammary Epithelial CellsBackground: The role of protein-tyrosine phosphatases (PTP) in ERBB2 signaling is undefined.Results: Phosphatidic acid (PA) activated PTPD2; inhibition of PA production or PTPD2 expression attenuated ERBB2-mediated morphological changes in mammary epithelial cells.Conclusion: The PLD2-PTPD2 axis is required for ERBB2 signaling.Significance: PA-regulated PTPD2 activity is a novel, positive element of ERBB2 signaling, which may offer a new therapeutic strategy in breast cancer. We used a loss-of-function screen to investigate the role of classical protein-tyrosine phosphatases (PTPs) in three-dimensional mammary epithelial cell morphogenesis and ERBB2 signaling.
ADP Ribosylation Factor 6 (ARF6) Promotes Acrosomal Exocytosis by Modulating Lipid Turnover and Rab3A ActivationBackground: Sperm acrosomal exocytosis requires GTPases, SNAREs, and a complex lipid signaling.Results: Exocytic stimuli promote ARF6 activation, which accomplishes exocytosis by stimulating PLC and Rab3A.Conclusion: ARF6 induces acrosome calcium efflux and assembles the fusion machinery leading to membrane fusion.Significance: This study explores a novel molecular link between ARF6, PLC, and Rab3A and provides insight into the molecular mechanisms of exocytosis and reproduction. Regulated secretion is a central issue for the specific function of many cells; for instance, mammalian sperm acrosomal exocytosis is essential for egg fertilization.
Contributions of Unique Intracellular Domains to Switchlike Biosensing by Toll-like Receptor 4Background: TLR4 is an important receptor, promoting both protective immunity and inflammatory diseases.Results: A revised model of TLR4 signaling is proposed, based upon new observations.Conclusion: TLR4 forms an autoinhibitory complex in the absence of ligand; disrupting this complex induces downstream signaling.Significance: This new mechanism may explain TLR4 responsiveness to diverse ligands and guide the design of novel therapeutics.
Recruitment of β-Catenin to N-Cadherin Is Necessary for Smooth Muscle ContractionBackground: β-Catenin links transmembrane cadherin to actin filaments at cell-cell contacts.Results: Contractile activation increases the association of β-catenin with N-cadherin, which is regulated by actin polymerization.Conclusion: Actin polymerization controls the recruitment of β-catenin to N-cadherin, which is essential for smooth muscle contraction.Significance: The regulated interaction of β-catenin with N-cadherin is a novel mechanism for the control of smooth muscle contraction.
Urotensin-II Receptor Stimulation of Cardiac L-type Ca2+ Channels Requires the βγ Subunits of Gi/o-protein and Phosphatidylinositol 3-Kinase-dependent Protein Kinase C β1 IsoformBackground: Regulation of L-type Ca2+ channels has important roles in determining the electrical properties of cardiomyocytes.Results: U-II potentiates ICa,L via U-IIR that couples to the PI3K-dependent PKCβ1 isoform.Conclusion: U-IIR stimulation of ICa,L contributes to the increase in the amplitude of sarcomere shortening.Significance: Regulation of ICa,L by U-IIR plays important roles in cardiovascular actions including cardiac positive inotropic effects and increasing cardiac output.
Loss of the Polarity Protein PAR3 Activates STAT3 Signaling via an Atypical Protein Kinase C (aPKC)/NF-κB/Interleukin-6 (IL-6) Axis in Mouse Mammary CellsBackground: Loss of PAR3 triggers accelerated growth and invasion of mammary tumors via STAT3 activity.Results: PAR3 silencing induces aPKC activity, which triggers NF-κB-IL-6 signaling.Conclusion: Regulation of aPKC activity is a key tumor suppressor function of PAR3.Significance: This work contributes to our understanding of mechanisms by which polarity proteins restrain tumor progression.
The Matricellular Protein Cyr61 Is a Key Mediator of Platelet-derived Growth Factor-induced Cell MigrationBackground: PDGF is a potent chemoattractant for cells.Results: The PDGF-induced extracellular matrix component Cyr61 bridges the intracellular PDGF-ERK and JNK signaling pathways with integrin/FAK signaling, leading to cell migration.Conclusion: Cyr61 is a key mediator of PDGF-induced cell migration via the Cyr61-integrin-FAK pathway.Significance: Extracellular Cyr61 convergence with growth factor and integrin/FAK signaling is a new cell migration concept.
Huntingtin-associated Protein 1 (HAP1) Is a cGMP-dependent Kinase Anchoring Protein (GKAP) Specific for the cGMP-dependent Protein Kinase Iβ IsoformBackground: Protein kinase compartmentalization through anchoring proteins provides spatiotemporal specificity.Results: Competitive elution combined with cyclic nucleotide affinity enrichment identifies HAP1 as a putative novel PKG anchoring protein (GKAP).Conclusion: Secondary structure predictions, in vitro binding studies, and site-directed mutagenesis define the binding domain and classify HAP1 as a GKAP specifically anchoring PKG Iβ.Significance: The repertoire of PKG anchoring proteins is expanded, enforcing that also PKG signaling is tightly spatiotemporally regulated.
Evaluating the Role of Retinal Membrane Guanylyl Cyclase 1 (RetGC1) Domains in Binding Guanylyl Cyclase-activating Proteins (GCAPs)Background: GCAP1 and GCAP2 regulate cGMP synthesis by RetGC1 in photoreceptors.Results: GCAPs compete for binding to RetGC1 in biochemical assays and in HEK293 cells co-expressing fluorescently labeled GCAPs with different forms of RetGC1.Conclusion: The GCAP1 and GCAP2 binding site(s) overlaps within the kinase homology and/or dimerization domains of RetGC1.Significance: RetGC1 and GCAPs contribute to normal vision and congenital blindness in humans.
Single-cell Analysis of G-protein Signal TransductionThe growing use of fluorescent biosensors to directly probe the spatiotemporal dynamics of biochemical processes in living cells has revolutionized the study of intracellular signaling. In this review, we summarize recent developments in the use of biosensors to illuminate the molecular details of G-protein-coupled receptor (GPCR) signaling pathways, which have long served as the model for our understanding of signal transduction, while also offering our perspectives on the future of this exciting field.
G Protein-coupled Receptor (GPCR) Signaling via Heterotrimeric G Proteins from EndosomesSome G protein-coupled receptors (GPCRs), in addition to activating heterotrimeric G proteins in the plasma membrane, appear to elicit a “second wave” of G protein activation after ligand-induced internalization. We briefly summarize evidence supporting this view and then discuss what is presently known about the functional significance of GPCR-G protein activation in endosomes. Endosomal activation can shape the cellular response temporally by prolonging its overall duration, and may shape the response spatially by moving the location of intracellular second messenger production relative to effectors.