A G-Protein Subunit Translocation Embedded Network Motif Underlies GPCR Regulation of Calcium Oscillations

G-protein βγ subunits translocate reversibly from the plasma membrane to internal membranes on receptor activation. Translocation rates differ depending on the γ subunit type. There is limited understanding of the role of the differential rates of G_βγ translocation in modulating signaling dynamics in a cell. Bifurcation analysis of the calcium oscillatory network structure predicts that the translocation rate of a signaling protein can regulate the damping of system oscillation. Here, we examined whether the G_βγ translocation rate regulates calcium oscillations induced by G-protein-coupled receptor activation. Oscillations in HeLa cells expressing γ subunit types with different translocation rates were imaged and quantitated. The results show that differential G_βγ translocation rates can underlie the diversity in damping characteristics of calcium oscillations among cells. Mathematical modeling shows that a translocation embedded motif regulates damping of G-protein-mediated calcium oscillations consistent with experimental data. The current study indicates that such a motif may act as a tuning mechanism to design oscillations with varying damping patterns by using intracellular translocation of a signaling component.     

Inositol 1,4,5-Trisphosphate Receptor Subtype-Specific Regulation of Calcium Oscillations

Oscillatory fluctuations in the cytosolic concentration of free calcium ions (Ca²⁺) are considered a ubiquitous mechanism for controlling multiple cellular processes. Inositol 1,4,5-trisphosphate (IP₃) receptors (IP₃R) are intracellular Ca²⁺ release channels that mediate Ca²⁺ release from endoplasmic reticulum (ER) Ca²⁺ stores. The three IP₃R subtypes described so far exhibit differential structural, biophysical, and biochemical properties. Subtype specific regulation of IP₃R by the endogenous modulators IP₃, Ca²⁺, protein kinases and associated proteins have been thoroughly examined. In this article we will review the contribution of each IP₃R subtype in shaping cytosolic Ca²⁺ oscillations.     

G-Protein Regulation of an L-Type Calcium Channel Current in Canine Jejunal Circular Smooth Muscle

Calcium entry into smooth muscle cells is essential to maintain contractility. In canine jejunal circular smooth muscle cells the predominant calcium entry pathway is through L-type calcium channels. The aim of this study was to determine the G-protein regulation of L-type calcium channel current (I _CaL) in isolated canine jejunal circular smooth muscle cells. Barium (80 mm) was used as the charge carrier. GTP-γS and GTP increased maximal inward current from 118.7 ± 12 pA to 227.5 ± 21.5 pA (n= 8) and 174.6 ± 10.1 pA (n= 6) respectively. The increase in inward current was blocked by nifedipine suggesting it was through L-type calcium channels. Pertussis toxin did not alter baseline I _CaL while cholera toxin increased I _CaL from 125 ± 19 pA in controls (n= 6) to 347 ± 30 pA (n= 4). Staurosporine inhibited the increase in current evoked by GTP-γS and calyculin further increased I _CaL over the increase evoked by GTP-γS. The results suggest that cholera toxin sensitive G-proteins activate L-type calcium channels in isolated canine jejunal circular smooth muscle cells through protein phosphorylation. Received: 27 March 1997/Revised: 3 July 1997     

Regulation of GPCR signaling in Hypertension

Hypertension represents a complex, multifactorial disease and contributes to the major causes of morbidity and mortality in industrialized countries: ischemic and hypertensive heart disease, stroke, peripheral atherosclerosis and renal failure. Current pharmacological therapy of essential hypertension focuses on the regulation of vascular resistance by inhibition of hormones such as catecholamines and angiotensin II, blocking them from receptor activation. Interaction of G-protein coupled receptor kinases (GRKs) and regulator of G-protein signaling (RGS) proteins with activated G-protein coupled receptors (GPCRs) effect the phosphorylation state of the receptor leading to desensitization and can profoundly impair signaling. Defects in GPCR regulation via these modulators have severe consequences affecting GPCR-stimulated biological responses in pathological situations such as hypertension, since they fine-tune and balance the major transmitters of vessel constriction versus dilatation, thus representing valuable new targets for anti-hypertensive therapeutic strategies. Elevated levels of GRKs are associated with human hypertensive disease and are relevant modulators of blood pressure in animal models of hypertension. This implies therapeutic perspective in a disease that has a prevalence of 65 million in the United States while being directly correlated with occurrence of major adverse cardiac and vascular events. Therefore, therapeutic approaches using the inhibition of GRKs to regulate GPCRs are intriguing novel targets for treatment of hypertension and heart failure.     

Application of in Utero Electroporation of G-Protein Coupled Receptor (GPCR) Genes,for Subcellular Localization of Hardly Identifiable GPCR in Mouse Cerebral Cortex

Lysophosphatidic acid (LPA) is a lipid growth factor that exerts diverse biological effects through its cognate receptors (LPA₁-LPA₆). LPA₁, which is predominantly expressed in the brain, plays a pivotal role in brain development. However, the role of LPA₁ in neuronal migration has not yet been fully elucidated. Here, we delivered LPA₁ to mouse cerebral cortex using in utero electroporation. We demonstrated that neuronal migration in the cerebral cortex was not affected by the overexpression of LPA₁. Moreover, these results can be applied to the identification of the localization of LPA₁. The subcellular localization of LPA₁ was endogenously present in the perinuclear area, and overexpressed LPA₁ was located in the plasma membrane. Furthermore, LPA₁ in developing mouse cerebral cortex was mainly expressed in the ventricular zone and the cortical plate. In summary, the overexpression of LPA₁ did not affect neuronal migration, and the protein expression of LPA₁ was mainly located in the ventricular zone and cortical plate within the developing mouse cerebral cortex. These studies have provided information on the role of LPA₁ in brain development and on the technical advantages of in utero electroporation.     

植物水通道蛋白的运输和调节

水通道蛋白是细胞间和细胞内水分运输的主要通道,其运输和调控对于植物细胞的水分稳态和胁迫响应具有重要作用。本文综述了水通道蛋白运输的分子机制以及结构修饰、门控、膜转运和异源四聚体等调节机制。     

Phosphorylation-Dependent Regulation of G-Protein Cycle during Nodule Formation in Soybean

Signaling pathways mediated by heterotrimeric G-protein complexes comprising Gα, Gβ, and Gγ subunits and their regulatory RGS (Regulator of G-protein Signaling) protein are conserved in all eukaryotes. We have shown that the specific Gβ and Gγ proteins of a soybean (Glycine max) heterotrimeric G-protein complex are involved in regulation of nodulation. We now demonstrate the role of Nod factor receptor 1 (NFR1)-mediated phosphorylation in regulation of the G-protein cycle during nodulation in soybean. We also show that during nodulation, the G-protein cycle is regulated by the activity of RGS proteins. Lower or higher expression of RGS proteins results in fewer or more nodules, respectively. NFR1 interacts with RGS proteins and phosphorylates them. Analysis of phosphorylated RGS protein identifies specific amino acids that, when phosphorylated, result in significantly higher GTPase accelerating activity. These data point to phosphorylation-based regulation of G-protein signaling during nodule development. We propose that active NFR1 receptors phosphorylate and activate RGS proteins, which help maintain the Gα proteins in their inactive, trimeric conformation, resulting in successful nodule development. Alternatively, RGS proteins might also have a direct role in regulating nodulation because overexpression of their phospho-mimic version leads to partial restoration of nodule formation in nod49 mutants.     

GPCR和CaCC信号传导中的钙信号网络

G蛋白偶联受体(G protein-coupled receptor,GPCR)作为最大的一类人膜蛋白受体家族和最重要的药物靶标而倍受关注,其中钙离子在细胞内信号传导级联放大中起了关键的作用。阐述了GPCR和钙激活的氯离子通道蛋白(calcium-activated chloride channel,CaCC)中的钙信号网络与生理功能以及如何干扰阻断该网络,为药物设计和很多疾病的治疗提供了依据。     

A Translocation Motif in Relaxase TrwC Specifically Affects Recruitment by Its Conjugative Type IV Secretion System

Type IV secretion system (T4SS) substrates are recruited through a translocation signal that is poorly defined for conjugative relaxases. The relaxase TrwC of plasmid R388 is translocated by its cognate conjugative T4SS, and it can also be translocated by the VirB/D4 T4SS of Bartonella henselae, causing DNA transfer to human cells. In this work, we constructed a series of TrwC variants and assayed them for DNA transfer to bacteria and human cells to compare recruitment requirements by both T4SSs. Comparison with other reported relaxase translocation signals allowed us to determine two putative translocation sequence (TS) motifs, TS1 and TS2. Mutations affecting TS1 drastically affected conjugation frequencies, while mutations affecting either motif had only a mild effect on DNA transfer rates through the VirB/D4 T4SS of B. henselae. These results indicate that a single substrate can be recruited by two different T4SSs through different signals. The C terminus affected DNA transfer rates through both T4SSs tested, but no specific sequence requirement was detected. The addition of a Bartonella intracellular delivery (BID) domain, the translocation signal for the Bartonella VirB/D4 T4SS, increased DNA transfer up to 4% of infected human cells, providing an excellent tool for DNA delivery to specific cell types. We show that the R388 coupling protein TrwB is also required for this high-efficiency TrwC-BID translocation. Other elements apart from the coupling protein may also be involved in substrate recognition by T4SSs.     

Spatial Wavelet Analysis of Calcium Oscillations in Developing Neurons

Calcium signals play a major role in the control of all key stages of neuronal development, and in particular in the growth and orientation of neuritic processes. These signals are characterized by high spatial compartmentalization, a property which has a strong relevance in the different roles of specific neuronal regions in information coding. In this context it is therefore important to understand the structural and functional basis of this spatial compartmentalization, and in particular whether the behavior at each compartment is merely a consequence of its specific geometry or the result of the spatial segregation of specific calcium influx/efflux mechanisms. Here we have developed a novel approach to separate geometrical from functional differences, regardless on the assumptions on the actual mechanisms involved in the generation of calcium signals. First, spatial indices are derived with a wavelet-theoretic approach which define a measure of the oscillations of cytosolic calcium concentration in specific regions of interests (ROIs) along a cell, in our case developing chick ciliary ganglion neurons. The resulting spatial profile demonstrates clearly that different ROIs along the neuron are characterized by specific patterns of calcium oscillations. Next we have investigated whether this inhomogeneity is due just to geometrical factors, namely the surface to volume ratio in the different subcompartments (e.g. soma vs. growth cone) or it depends on their specific biophysical properties. To this aim correlation functions are computed between the activity indices and the surface/volume ratio along the cell: the data thus obtained are validated by a statistical analysis on a dataset of different cells. This analysis shows that whereas in the soma calcium dynamics is highly correlated to the surface/volume ratio, correlations drop in the growth cone-neurite region, suggesting that in this latter case the key factor is the expression of specific mechanisms controlling calcium influx/efflux.     

Translocation of Calcium in Relation to Tomato Fruit Growth

Regulation of the uptake and distribution of calcium in thetomato plant was investigated in plants grown in recirculatingnutrient solutions at electrical conductivities of 2,7,12 and17 millisiemens (mS). Despite an increased calcium content inthe nutrient solution at high conductivity (7–17 mS),the accumulation of calcium by fruit was progressively reducedby increasing salinity, particularly in the distal half. Theincidence of blossom-end rot in fruit (BER) also increased withsalinity. The uptake of water and ⁴⁵Ca by plants was substantially reducedin the high salinity treatment (17 mS) and, to a lesser extent,by high relative humidity (90 per cent r.h. at 20 °C). Further,the translocation of ⁴⁵Ca from roots to shoots was reduced byhigh salinity, while the percentage distribution of ⁴⁵Ca tothe apex was reduced by high humidity. Only approx. 2 per centof the ⁴⁵Ca taken up by a plant was imported by the truss. The uptake of ⁴⁵Ca and its distribution among pedicel, calyxand berry by detached fruit in 24 h showed that fruit from highsalinity plants had a reduced uptake and a lower accumulationof ⁴⁵ Ca in the berry than in the calyx. In addition, plants grown at high conductivity had a lower rateof xylem sap exudation from decapitated plants. The fruit ofthese plants had a smaller xylem cross-sectional area in thefruit pedicel and a smaller calyx than those of the low conductivitytreatment. Calcium, translocation, tomato, fruit, blossom-end rot     

Multiple Analyses of G-Protein Coupled Receptor (GPCR) Expression in the Development of Gefitinib-Resistance in Transforming Non-Small-Cell Lung Cancer

There is increasing evidence that functional crosstalk between GPCRs and EGFR contributes to the progression of colon, lung, breast, ovarian, prostate and head and neck tumors. In this study, we performed multiple analyses of GPCR expression in a gefitinib-resistant non-small cell lung cancer (NSCLC) cell line, H1975, which harbors an L858R/T790M mutation. To determine the expression profile of mRNAs encoding 384 GPCRs in normal human lung fibroblast (NHLF) and H1975 cells, a GPCR-specific microarray analysis was performed. A heat-map of the microarray revealed considerable differences in the expression of GPCRs between NHLF and H1975 cells. From the GPCR expression list, we selected some GPCR agonists/antagonist to investigate whether the respective ligands could affect the growth of H1975 cells. Among them, treatment with either a selective antagonist of adenosine A2a receptors, which were highly expressed in H1975 cell and another gefitinib-resistant NSCLC cells, HCC827GR cells or “small interfering RNA” (siRNA) targeting adenosine A2a receptors produced a significant decrease in cell viability of both H1975 and HCC827GR cells. Among up-regulated GPCRs in H1975 cells, Gs-, Gi- and Gq-coupled GPCRs were expressed almost equally. Among down-regulated GPCRs, Gi-coupled GPCRs were dominantly expressed in H1975 cells. The present results suggest that multilayered crosstalk between GPCRs and EGFR may play an important role in orchestrating downstream signaling molecules that are implicated in the development of gefitinib-resistant NSCLC.     

Activation of Endothelial Cell Kinin Receptors Leads to Intracellular Calcium Increases and Filamin Translocation: Regulation by Protein Kinase C

Membrane-associated cytoskeletal proteins provide support for endothelial cell (EC) junctional cell adhesion molecules. Nonmuscle filamin is a dimeric actin cross-linking protein that interacts with F-actin and membrane glycoproteins. Both bradykinin and des-Arg⁹-bradykinin cause filamin redistribution from the plasma membrane to the cytosol of confluent EC. Kinin-induced filamin translocation parallels the dynamics of intracellular Ca²⁺ increases. Pretreatment with kinin receptor antagonists blocks the Ca²⁺ response as well as filamin translocation induced by kinins. Protein kinase C activation prior to kinin stimulation attenuates intracellular Ca²⁺ increases and filamin translocation. BAPTA, a cell-permeable Ca²⁺ chelator, attenuates bradykinin-induced intracellular Ca²⁺ increases and filamin translocation. This study demonstrates that bovine pulmonary artery ECs express both kinin B1 and B2 receptors, and that activation of either receptor leads to intracellular Ca²⁺ increases. This Ca²⁺ signalling, which is downregulated by protein kinase C activation, is essential for kinin-induced filamin translocation.     

Identification of a Novel Protein Interaction Motif in the Regulatory Subunit of Casein Kinase 2

Casein kinase 2 (CK2) regulates multiple cellular processes and can promote oncogenesis. Interactions with the CK2β regulatory subunit of the enzyme target its catalytic subunit (CK2α or CK2α′) to specific substrates; however, little is known about the mechanisms by which these interactions occur. We previously showed that by binding CK2β, the Epstein-Barr virus (EBV) EBNA1 protein recruits CK2 to promyelocytic leukemia (PML) nuclear bodies, where increased CK2-mediated phosphorylation of PML proteins triggers their degradation. Here we have identified a KSSR motif near the dimerization interface of CK2β as forming part of a protein interaction pocket that mediates interaction with EBNA1. We show that the EBNA1-CK2β interaction is primed by phosphorylation of EBNA1 on S393 (within a polyserine region). This phosphoserine is critical for EBNA1-induced PML degradation but does not affect EBNA1 functions in EBV replication or segregation. Using comparative proteomics of wild-type (WT) and KSSR mutant CK2β, we identified an uncharacterized cellular protein, C18orf25/ARKL1, that also binds CK2β through the KSSR motif and show that this involves a polyserine sequence resembling the CK2β binding sequence in EBNA1. Therefore, we have identified a new mechanism of CK2 interaction used by viral and cellular proteins.     

Lipid Raft-Mediated Regulation of G-Protein Coupled Receptor Signaling by Ligands which Influence Receptor Dimerization: A Computational Study

G-protein coupled receptors (GPCRs) are the largest family of cell surface receptors; they activate heterotrimeric G-proteins in response to ligand stimulation. Although many GPCRs have been shown to form homo- and/or heterodimers on the cell membrane, the purpose of this dimerization is not known. Recent research has shown that receptor dimerization may have a role in organization of receptors on the cell surface. In addition, microdomains on the cell membrane termed lipid rafts have been shown to play a role in GPCR localization. Using a combination of stochastic (Monte Carlo) and deterministic modeling, we propose a novel mechanism for lipid raft partitioning of GPCRs based on reversible dimerization of receptors and then demonstrate that such localization can affect GPCR signaling. Modeling results are consistent with a variety of experimental data indicating that lipid rafts have a role in amplification or attenuation of G-protein signaling. Thus our work suggests a new mechanism by which dimerization-inducing or inhibiting characteristics of ligands can influence GPCR signaling by controlling receptor organization on the cell membrane.     

Disruption of Calcium Homeostasis in Cardiomyocytes Underlies Cardiac Structural and Functional Changes in Severe Sepsis

Sepsis, a major cause of morbidity/mortality in intensive care units worldwide, is commonly associated with cardiac dysfunction, which worsens the prognosis dramatically for patients. Although in recent years the concept of septic cardiomyopathy has evolved, the importance of myocardial structural alterations in sepsis has not been fully explored. This study offers novel and mechanistic data to clarify subcellular events that occur in the pathogenesis of septic cardiomyopathy and myocardial dysfunction in severe sepsis. Cultured neonatal mice cardiomyocytes subjected to serum obtained from mice with severe sepsis presented striking increment of [Ca²⁺]_i and calpain-1 levels associated with decreased expression of dystrophin and disruption and derangement of F-actin filaments and cytoplasmic bleb formation. Severe sepsis induced in mice led to an increased expression of calpain-1 in cardiomyocytes. Moreover, decreased myocardial amounts of dystrophin, sarcomeric actin, and myosin heavy chain were observed in septic hearts associated with depressed cardiac contractile dysfunction and a very low survival rate. Actin and myosin from the sarcomere are first disassembled by calpain and then ubiquitinated and degraded by proteasome or sequestered inside specialized vacuoles called autophagosomes, delivered to the lysosome for degradation forming autophagolysosomes. Verapamil and dantrolene prevented the increase of calpain-1 levels and preserved dystrophin, actin, and myosin loss/reduction as well cardiac contractile dysfunction associated with strikingly improved survival rate. These abnormal parameters emerge as therapeutic targets, which modulation may provide beneficial effects on future vascular outcomes and mortality in sepsis. Further studies are needed to shed light on this mechanism, mainly regarding specific calpain inhibitors.