Cyclic strain induces reorganization of integrin α5β1 and α2β1 in human umbilical vein endothelial cells

Cyclic strain has been shown to modulate endothelial cell (EC) morphology, proliferation, and function. We have recently reported that the focal adhesion proteins focal adhesion kinase (pp125^FAK) and paxillin, are tyrosine phosphorylated in EC exposed to strain and these events regulate the morphological change and migration induced by cyclic strain. Integrins are also localized on focal adhesion sites and have been reported to induce tyrosine phosphorylation of pp125^FAK under a variety of stimuli. To study the involvement of different integrins in signaling induced by cyclic strain, we first observed the redistribution of α and β integrins in EC subjected to 4 h cyclic strain. Human umbilical vein endothelial cells (HUVEC) seeded on either fibronectin or collagen surfaces were subjected to 10% average strain at a frequency 60 cycles/min. Confocal microscopy revealed that β₁ integrin reorganized in a linear pattern parallel with the long axis of the elongated cells creating a fusion of focal adhesion plaques in EC plated on either fibronectin (a ligand for α₅β₁) or collagen (a ligand for α₂β₁) coated plates after 4 h exposure to cyclic strain. β₃ integrin, which is a vitronectin receptor, did not redistribute in EC exposed to cyclic strain. Cyclic strain also led to a reorganization of α₅ and α₂ integrins in a linear pattern in HUVEC seeded on fibronectin or collagen, respectively. The expression of integrins α₅, α₂, and β₁ did not change even after 24 h exposure to strain when assessed by immunoprecipitation of these integrins. Cyclic strain-induced tyrosine phosphorylation of pp125^FAK occurred concomitant with the reorganization of β₁ integrin. We concluded that α₅β₁ and α₂β₁ integrins play an important role in transducing mechanical stimuli into intracellular signals. J. Cell. Biochem. 64:505–513. © 1997 Wiley-Liss, Inc.     

The role of PLDα1 in providing specificity to signal‐response coupling by heterotrimeric G‐protein components in Arabidopsis

Heterotrimeric G‐proteins comprised of Gα, Gβ and Gγ subunits are important signal transducers in all eukaryotes. In plants, G‐proteins affect multiple biotic and abiotic stress responses, as well as many developmental processes, even though their repertoire is significantly limited compared with that in metazoan systems. One canonical and three extra‐large Gα, 1 Gβ and 3 Gγ proteins represent the heterotrimeric G‐protein complex in Arabidopsis, and a single regulatory protein, RGS1, is one of the few known biochemical regulators of this signaling complex. This quantitative disparity between the number of signaling components and the range of processes they influence is rather intriguing. We now present evidence that the phospholipase Dα1 protein is a key component and modulator of the G‐protein complex in affecting a subset of signaling pathways. We also show that the same G‐protein subunits and their modulators exhibit distinct physiological and genetic interactions depending on specific signaling and developmental pathways. Such developmental plasticity and interaction specificity likely compensates for the lack of multiplicity of individual subunits, and helps to fine tune the plants' responses to constantly changing environments.     

Sphingosylphosphorylcholine-induced contraction of feline ileal smooth muscle cells is mediated by Gαi3 protein and MAPK

We studied the mechanism of sphingosylphosphorylcholine (SPC)-induced contraction in feline ileal smooth muscle cells. Western blotting revealed that G protein subtypes of Gα_i1, Gα_i3 and Gα_o existed in feline ileum. Gα_i3 antibody penetration into permeabilized cells decreased SPC-induced contraction. In addition, incubation of [³⁵S]guanosine 5′-O-(3-thiotriphosphate) ([³⁵S]GTPγS) with membrane fraction increased its binding to Gα_i3 subtype after SPC treatment, suggesting that the signalling pathways invoked by SPC were mediated by Gα_i3 protein. MAPK kinase (MEK) inhibitor PD98059 blocked the contraction significantly, but p38 mitogen-activated protein kinase (MAPK) inhibitor SB202190 did not. Chelerythrine and neomycin also inhibited the contraction. However, cotreatment of PD98059 and chelerythrine showed no significant difference. Phosphorylation of p44/42 MAPK was increased by SPC treatment, which was reversed by pretreatment of inhibitors of signalling molecules that decreased SPC-induced contraction previously. The same result was obtained in the assay of MAPK activity.     

The transforming activity of activated Gα12

Heterotrimeric GTP-binding proteins transduce receptor-mediated extracellular signals to their effectors. Several activated mutations of Gsα and Giα have been shown to be associated with endocrine tumors. In this report, we have evaluated the transforming activity of an putative activated form of Gα12 subunit. We found that transient expression in NIH3T3 cells of the Gα12 mutant with substitution of glutamine-229 for leucine could lead to focus formation and that stably transfected NIH3T3 cells could form colonies in soft agar and tumors in nude mice.     

Illuminating the role of the Gα heterotrimeric G protein subunit,RGA1, in regulating photoprotection and photoavoidance in rice

We studied physiological mechanisms of photoavoidance and photoprotection of a dwarf rice mutant with erect leaves, d1, in which the RGA1 gene, which encodes the Gα subunit of the heterotrimeric G protein, is non‐functional. Leaves of d1 exhibit lower leaf temperature and higher photochemical reflectance index relative to wild type (WT), indicative of increased photoavoidance and more efficient light harvesting. RNA sequencing analysis of flag leaves revealed that messenger RNA levels of genes encoding heat shock proteins, enzymes associated with chlorophyll breakdown, and ROS scavengers were down‐regulated in d1. By contrast, genes encoding proteins associated with light harvesting, Photosystem II, cyclic electron transport, Photosystem I, and chlorophyll biosynthesis were up‐regulated in d1. Consistent with these observations, when WT and d1 plants were experimentally subjected to the same light intensity, d1 plants exhibited a greater capacity to dissipate excess irradiance (increased nonphotochemical quenching) relative to WT. The increased capacity in d1 for both photoavoidance and photoprotection reduced sustained photoinhibitory damage, as revealed by a higher F_v/F_m. We therefore propose RGA1 as a regulator of photoavoidance and photoprotection mechanisms in rice and highlight the prospect of exploiting modulation of heterotrimeric G protein signalling to increase these characteristics and improve the yield of cereals in the event of abiotic stress.     

机械拉伸下人肺上皮细胞增殖及整联蛋白再分布

应用体外周期性拉伸装置研究机械拉伸对人肺上皮细胞A5 4 9增殖及其膜表面受体———整联蛋白α5、β1再分布的调控作用。结果表明 :在应变为 15 % ,频率为 2 0次 /min、4 0次 /min的拉伸刺激下 ,4 8h后 ,应用流式细胞技术检测细胞的增殖活性指数明显降低 ,A5 4 9细胞的DNA合成受到显著抑制。在 4 0次 /min的拉伸频率下 ,整联蛋白α5、β1的分布发生重组并向基底层转移 ,形成局部粘附连接。研究表明 :整联蛋白α5、β1可能在肺上皮细胞感应机械应力过程中起了重要的作用。     

Regulator of G protein signaling 2 inhibits Gαq-dependent uveal melanoma cell growth

Activating mutations in Gα_q/11 are a major driver of uveal melanoma (UM), the most common intraocular cancer in adults. While progress has recently been made in targeting Gα_q/11 for UM therapy, the crucial role for these proteins in normal physiology and their high structural similarity with many other important GTPase proteins renders this approach challenging. The aim of the current study was to validate whether a key regulator of Gq signaling, regulator of G protein signaling 2 (RGS2), can inhibit Gα_q-mediated UM cell growth. We used two UM cell lines, 92.1 and Mel-202, which both contain the most common activating mutation Gα_q^Q209L and developed stable cell lines with doxycycline-inducible RGS2 protein expression. Using cell viability assays, we showed that RGS2 could inhibit cell growth in both of these UM cell lines. We also found that this effect was independent of the canonical GTPase-activating protein activity of RGS2 but was dependent on the association between RGS2 and Gα_q. Furthermore, RGS2 induction resulted in only partial reduction in cell growth as compared to siRNA-mediated Gαq knockdown, perhaps because RGS2 was only able to reduce mitogen-activated protein kinase signaling downstream of phospholipase Cβ, while leaving activation of the Hippo signaling mediators yes-associated protein 1/TAZ, the other major pathway downstream of Gα_q, unaffected. Taken together, our data indicate that RGS2 can inhibit UM cancer cell growth by associating with Gα_q^Q209L as a partial effector antagonist.     

Effects of Mutations in the Cytoplasmic Domain of Integrin β1 to Talin Binding and Cell Spreading

Integrins are transmembrane proteins linking the extracellular matrix or certain cell–cell contacts to the cytoskeleton. To study integrin–cytoskeleton interactions we wanted to relate talin–integrin interaction to integrin function in cell spreading and formation of focal adhesions. For talin-binding studies we used fusion proteins of glutathione S-transferase and the cytoplasmic domain of integrin β₁ (GST-cytoβ₁) expressed in bacteria. For functional studies chimeric integrins containing the extracellular and transmembrane parts of β₃ linked to the cytoplasmic domain of β₁ were expressed in CHO cells as a dimer with the α_IIb subunit. Point mutations in the amino acid sequence N⁷⁸⁵PIY⁷⁸⁸ of β₁ disrupted both the integrin–talin interaction and the ability of the integrin to mediate cell spreading. COOH-terminal truncation of β₁ at the amino acid position 797 disrupted its ability to mediate cell spreading, whereas the disruption of talin binding required deletion of five more amino acids (truncation at position 792). A synthetic peptide from this region of β₁ (W⁷⁸⁰DTGENPIYKSAV⁷⁹²) bound to purified talin and inhibited talin binding to GST-cytoβ₁. The ability of the mutants to mediate focal adhesion formation or to codistribute to focal adhesions formed by other integrins correlated with their ability to mediate cell spreading. These results confirm the previous finding that a talin-binding site in the integrin β₁ tail resides at or close to the central NPXY motif and suggest that the integrin–talin interaction is necessary but not sufficient for integrin-mediated cell spreading.     

Gαs directly drives PDZ-RhoGEF signaling to Cdc42

Gα proteins promote dynamic adjustments of cell shape directed by actin-cytoskeleton reorganization via their respective RhoGEF effectors. For example, Gα₁₃ binding to the RGS-homology (RH) domains of several RH-RhoGEFs allosterically activates these proteins, causing them to expose their catalytic Dbl-homology (DH)/pleckstrin-homology (PH) regions, which triggers downstream signals. However, whether additional Gα proteins might directly regulate the RH-RhoGEFs was not known. To explore this question, we first examined the morphological effects of expressing shortened RH-RhoGEF DH/PH constructs of p115RhoGEF/ARHGEF1, PDZ-RhoGEF (PRG)/ARHGEF11, and LARG/ARHGEF12. As expected, the three constructs promoted cell contraction and activated RhoA, known to be downstream of Gα₁₃. Intriguingly, PRG DH/PH also induced filopodia-like cell protrusions and activated Cdc42. This pathway was stimulated by constitutively active Gα_s (Gα_sQ227L), which enabled endogenous PRG to gain affinity for Cdc42. A chemogenetic approach revealed that signaling by G_s-coupled receptors, but not by those coupled to G_i or G_q, enabled PRG to bind Cdc42. This receptor-dependent effect, as well as CREB phosphorylation, was blocked by a construct derived from the PRG:Gα_s-binding region, PRG-linker. Active Gα_s interacted with isolated PRG DH and PH domains and their linker. In addition, this construct interfered with Gα_sQ227L''s ability to guide PRG''s interaction with Cdc42. Endogenous G_s-coupled prostaglandin receptors stimulated PRG binding to membrane fractions and activated signaling to PKA, and this canonical endogenous pathway was attenuated by PRG-linker. Altogether, our results demonstrate that active Gα_s can recognize PRG as a novel effector directing its DH/PH catalytic module to gain affinity for Cdc42.     

Signaling mechanisms and physiological functions of G‐protein Gα13 in blood vessel formation,bone homeostasis,and cancer

Heterotrimeric G‐proteins are cellular signal transducers. They mainly relay signals from G‐protein‐coupled receptors (GPCRs). GPCRs function as guanine nucleotide‐exchange factors to active these G‐proteins. Based on the sequence and functional similarities, these G‐proteins are grouped into four subfamilies: G_s, G_i, G_q, and G_12/13. The G_12/13 subfamily consists of two members: G₁₂ and G₁₃. G_12/13‐mediated signaling pathways play pivotal roles in a variety of physiological processes, while aberrant regulation of this pathway has been identified in various human diseases. Here we summarize the signaling mechanisms and physiological functions of Gα₁₃ in blood vessel formation and bone homeostasis. We further discuss the expanding roles of Gα₁₃ in cancers, serving as oncogenes as well as tumor suppressors.     

The Arabidopsis heterotrimeric G‐protein β subunit,AGB1, is required for guard cell calcium sensing and calcium‐induced calcium release

Cytosolic calcium concentration ([Ca²⁺]_cyt) and heterotrimeric G‐proteins are universal eukaryotic signaling elements. In plant guard cells, extracellular calcium (Ca_o) is as strong a stimulus for stomatal closure as the phytohormone abscisic acid (ABA), but underlying mechanisms remain elusive. Here, we report that the sole Arabidopsis heterotrimeric Gβ subunit, AGB1, is required for four guard cell Ca_o responses: induction of stomatal closure; inhibition of stomatal opening; [Ca²⁺]_cyt oscillation; and inositol 1,4,5‐trisphosphate (InsP3) production. Stomata in wild‐type Arabidopsis (Col) and in mutants of the canonical Gα subunit, GPA1, showed inhibition of stomatal opening and promotion of stomatal closure by Ca_o. By contrast, stomatal movements of agb1 mutants and agb1/gpa1 double‐mutants, as well as those of the agg1agg2 Gγ double‐mutant, were insensitive to Ca_o. These behaviors contrast with ABA‐regulated stomatal movements, which involve GPA1 and AGB1/AGG3 dimers, illustrating differential partitioning of G‐protein subunits among stimuli with similar ultimate impacts, which may facilitate stimulus‐specific encoding. AGB1 knockouts retained reactive oxygen species and NO production, but lost YC3.6‐detected [Ca²⁺]_cyt oscillations in response to Ca_o, initiating only a single [Ca²⁺]_cyt spike. Experimentally imposed [Ca²⁺]_cyt oscillations restored stomatal closure in agb1. Yeast two‐hybrid and bimolecular complementation fluorescence experiments revealed that AGB1 interacts with phospholipase Cs (PLCs), and Ca_o induced InsP3 production in Col but not in agb1. In sum, G‐protein signaling via AGB1/AGG1/AGG2 is essential for Ca_o‐regulation of stomatal apertures, and stomatal movements in response to Ca_o apparently require Ca²⁺‐induced Ca²⁺ release that is likely dependent on Gβγ interaction with PLCs leading to InsP3 production.     

Receptor‐Like Kinase Phosphorylation of Arabidopsis Heterotrimeric G‐Protein Gα ‐Subunit AtGPA1

As molecular on–off switches, heterotrimeric G protein complexes, comprised of a Gα subunit and an obligate Gβγ dimer, transmit extracellular signals received by G protein–coupled receptors (GPCRs) to cytoplasmic targets that respond to biotic and abiotic stimuli. Signal transduction is modulated by phosphorylation of GPCRs and G protein complexes. In Arabidopsis thaliana, the Gα subunit AtGPA1 is phosphorylated by the receptor‐like kinase (RLK) BRI1‐associated Kinase 1 (BAK1), but the extent that other RLKs phosphorylates AtGPA1 is unknown. Twenty‐two trans‐phosphorylation sites on AtGPA1 are mapped by 12 RLKs hypothesized to act in the Arabidopsis G protein signaling pathway. Cis‐phosphorylation sites are also identified on these RLKs, some newly shown to be dual specific kinases. Multiple sites are present in the core AtGPA1 functional units, including pSer52 and/or pThr53 of the conserved P‐loop that directly binds nucleotide/phosphate, pThr164, and pSer175 from αE helix in the intramolecular domain interface for nucleotide exchange and GTP hydrolysis, and pThr193 and/or pThr194 in Switch I (SwI) that coordinates nucleotide exchange and protein partner binding. Several AtGPA1 S/T phosphorylation sites are potentially nucleotide‐dependent phosphorylation patterns, such as Ser52/Thr53 in the P‐loop and Thr193 and/or Thr194 in SwI.     

Intact vinculin protein is required for control of cell shape, cell mechanics, and rac-dependent lamellipodia formation.

Studies were carried out using vinculin-deficient F9 embryonic carcinoma (gamma229) cells to analyze the relationship between structure and function within the focal adhesion protein vinculin, in the context of control of cell shape, cell mechanics, and movement. Atomic force microscopy studies revealed that transfection of the head (aa 1-821) or tail (aa 811-1066) domain of vinculin, alone or together, was unable to fully reverse the decrease in cell stiffness, spreading, and lamellipodia formation caused by vinculin deficiency. In contrast, replacement with intact vinculin completely restored normal cell mechanics and spreading regardless of whether its tyrosine phosphorylation site was deleted. Constitutively active rac also only induced extension of lamellipodia when microinjected into cells that expressed intact vinculin protein. These data indicate that vinculin's ability to physically couple integrins to the cytoskeleton, to mechanically stabilize cell shape, and to support rac-dependent lamellipodia formation all appear to depend on its intact three-dimensional structure.     

Tissue inhibitor of metalloproteinase‐2 (TIMP‐2) regulates neuromuscular junction development via a β1 integrin‐mediated mechanism

Extracellular matrix (ECM) molecules play critical roles in muscle function by participating in neuromuscular junction (NMJ) development and the establishment of stable, cytoskeleton‐associated adhesions required for muscle contraction. Matrix metalloproteinases (MMPs) are neutral endopeptidases that degrade all ECM components. While the role of MMPs and their inhibitors, the tissue inhibitor of metalloproteinases (TIMPs), has been investigated in many tissues, little is known about their role in muscle development and mature function. TIMP‐2 ^?/? mice display signs of muscle weakness. Here, we report that TIMP‐2 is expressed at the NMJ and its expression is greater in fast‐twitch (extensor digitorum longus, EDL) than slow‐twitch (soleus) muscle. EDL muscle mass is reduced in TIMP‐2^?/? mice without a concomitant change in fiber diameter or number. The TIMP‐2^?/? phenotype is not likely due to increased ECM proteolysis because net MMP activity is actually reduced in TIMP‐2^?/? muscle. Most strikingly, TIMP‐2 colocalizes with β1 integrin at costameres in the wild‐type EDL and β1 integrin expression is significantly reduced in TIMP‐2^?/? EDL. We propose that reduced β1 integrin in fast‐twitch muscle may be associated with destabilized ECM‐cytoskeletal interactions required for muscle contraction in TIMP‐2^?/? muscle; thus, explaining the muscle weakness. Given that fast‐twitch fibers are lost in muscular dystrophies and age‐related sarcopenia, if TIMP‐2 regulates mechanotransduction in an MMP‐independent manner it opens new potential therapeutic avenues. © 2006 Wiley Periodicals, Inc. J Neurobiol, 2006     

Gαs protein C‐terminal α‐helix at the interface: does the plasma membrane play a critical role in the Gαs protein functionality?

The heterotrimeric guanine nucleotide-binding regulatory proteins (G proteins, Galphabetagamma) mediate the signalling process of a large number of receptors, known as G protein-coupled receptors. The C-terminal domain of the heterotrimeric G protein alpha-subunit plays a key role in the selective activation of G proteins by their cognate receptors. The interaction of this domain can take place at the end of a cascade including several successive conformational modifications. Galpha(s)(350-394) is the 45-mer peptide corresponding to the C-terminal region of the Galpha(s) subunit. In the crystal structure of the Galpha(s) subunit it encompasses the alpha4/beta6 loop, the beta6 beta-sheet segment and the alpha5 helix region. Following a previous study based on the synthesis, biological activity and conformational analysis of shorter peptides belonging to the same Galpha(s) region, Galpha(s)(350-394) was synthesized and investigated. The present study outlines the central role played by the residues involved in the alpha4/beta6 loop and beta6/alpha5 loops in the stabilization of the C-terminal Galpha(s)alpha-helix. H(2)O/(2)H(2)O exchange experiments, and NMR diffusion experiments show interesting evidence concerning the interaction between the SDS micelles and the polypeptide. These data prompt intriguing speculations on the role of the intracellular environment/cellular membrane interface in the stabilization and functionality of the C-terminal Galpha(s) region.     

Modulation of α5β1 and αVβ3 integrins on the cell surface during mitosis

One of the hallmarks of cells undergoing mitotic division is their rounded morphology and reduced adhesion to the substratum. We have studied and compared the attachment of interphase and mitotic cells to substrata coated with fibronectin and vitronectin. We have found that adhesion of mitotic cells, as compared to interphase cells, is significantly reduced to fibronectin, but is higher to vitronectin. These results correlate well with the expression of α5β1 and αVβ3 integrins, the respective receptors for fibronectin and vitronectin, on the cell surface. Mitotic cells show higher levels of αVβ3 and very low levels of α5β1 proteins on the cell surface as compared to interphase cells. This difference in the levels of these integrins also reflects in the total amounts of fibronectin and vitronectin present on the cell surface of these cells. We have further shown, by flow cytometry, that binding of vitronectin, or the synthetic peptide-GRGDSP-, causes an increase in the intracellular levels of Ca²⁻ in mitotic cells, but no change is seen in the interphase cells. Binding of fibronectin to either of these cells fails to elicit any response. One interesting feature of our results is that the levels of total, i.e., cytoplasmic plus membrane bound, α5β1 and αVβ3 integrins of mitotic and interphase cells remain the same, thus implying an alteration in the distribution of integrin chains between the plasma membrane and the cytoplasm during the conversion of interphase cells into the mitotic phase. © 1996 Wiley-Liss, Inc.     

The structure of the integrin αIIbβ3 transmembrane complex explains integrin transmembrane signalling

Heterodimeric integrin adhesion receptors regulate cell migration, survival and differentiation in metazoa by communicating signals bi‐directionally across the plasma membrane. Protein engineering and mutagenesis studies have suggested that the dissociation of a complex formed by the single‐pass transmembrane (TM) segments of the α and β subunits is central to these signalling events. Here, we report the structure of the integrin αIIbβ3 TM complex, structure‐based site‐directed mutagenesis and lipid embedding estimates to reveal the structural event that underlies the transition from associated to dissociated states, that is, TM signalling. The complex is stabilized by glycine‐packing mediated TM helix crossing within the extracellular membrane leaflet, and by unique hydrophobic and electrostatic bridges in the intracellular leaflet that mediate an unusual, asymmetric association of the 24‐ and 29‐residue αIIb and β3 TM helices. The structurally unique, highly conserved integrin αIIbβ3 TM complex rationalizes bi‐directional signalling and represents the first structure of a heterodimeric TM receptor complex.