Cytosolic guanine nucledotide binding deficient form of transglutaminase 2 (R580a) potentiates cell death in oxygen glucose deprivation

Transglutaminase 2 (TG2) is a hypoxia-responsive protein that is a calcium-activated transamidating enzyme, a GTPase and a scaffolding/linker protein. Upon activation TG2 undergoes a large conformational change, which likely affects not only its enzymatic activities but its non-catalytic functions as well. The focus of this study was on the role of transamidating activity, conformation and localization of TG2 in ischemic cell death. Cells expressing a GTP binding deficient form of TG2 (TG2-R580A) with high basal transamidation activity and a more extended conformation showed significantly increased cell death in response to oxygen-glucose deprivation; however, targeting TG2-R580A to the nucleus abrogated its detrimental role in oxygen-glucose deprivation. Treatment of cells expressing wild type TG2, TG2-C277S (a transamidating inactive mutant) and TG2-R580A with Cp4d, a reversible TG2 inhibitor, did not affect cell death in response to oxygen-glucose deprivation. These findings indicate that the pro-cell death effects of TG2 are dependent on its localization to the cytosol and independent of its transamidation activity. Further, the conformational state of TG2 is likely an important determinant in cell survival and the prominent function of TG2 in ischemic cell death is as a scaffold to modulate cellular processes.     

Mutation of a critical arginine in the GTP-binding site of transglutaminase 2 disinhibits intracellular cross-linking activity

Transglutaminase type 2 (TG2; also known as G(h)) is a multifunctional protein involved in diverse cellular processes. It has two well characterized enzyme activities: receptor-stimulated signaling that requires GTP binding and calcium-activated transamidation or cross-linking that is inhibited by GTP. In addition to the GDP binding residues identified from the human TG2 crystal structure (Liu, S., Cerione, R. A., and Clardy, J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 2743-2747), we have previously implicated Ser171 in GTP binding, as binding is lost with glutamate substitution (Iismaa, S. E., Wu, M.-J., Nanda, N., Church, W. B., and Graham, R. M. (2000) J. Biol. Chem. 275, 18259-18265). Here, we have shown that alanine substitution of homologous residues in rat TG2 (Phe174 in the core domain or Arg476, Arg478, or Arg579 in barrel 1) does not affect TG activity but reduces or abolishes GTP binding and GTPgammaS inhibition of TG activity in vitro, indicating that these residues are important in GTP binding. Alanine substitution of Ser171 does not impair GTP binding, indicating this residue does not interact directly with GTP. Arg579 is particularly important for GTP binding, as isothermal titration calorimetry demonstrated a 100-fold reduction in GTP binding affinity by the R579A mutant. Unlike wild-type TG2 or its S171E or F174A mutants, which are sensitive to both trypsin and mu-calpain digestion, R579A is inherently more resistant to mu-calpain, but not trypsin, digestion, indicating reduced accessibility and/or flexibility of this mutant in the region of the calpain cleavage site(s). Basal TG activity of intact R579A stable SH-SY5Y neuroblastoma cell transfectants was slightly increased relative to wild-type transfectants and, in contrast to the TG activity of the latter, was further stimulated by muscarinic receptor-activated calcium mobilization. Thus, loss of GTP binding sensitizes TG2 to intracellular calcium concentrations. These findings are consistent with the notion that intracellularly, under physiological conditions, TG2 is maintained largely as a latent enzyme, its calcium-activated cross-linking activity being suppressed allosterically by guanine nucleotide binding.     

Detection of Transglutaminase 2 conformational changes in living cell

Transglutaminase 2 (TG2) is a ubiquitous Ca(2+)-dependent protein cross-linking enzyme that is implicated in a variety of biological disorders. In in vitro experiments when Ca(2+) concentration was increased TG2 changed its conformation and was able to cross-link other proteins via formation of an isopeptide bond. However the mechanisms that regulate TG2 transamidation activity in cells are still unknown. In this study we have developed FRET-based method for monitoring TG2 conformation changes and, probably, cross-linking activity in living cells. Using this approach we have showed that a significant amount of TG2 within the cell is accumulated in perinuclear endosomes and has a cross-linking inactive conformation, while TG2 that is located beneath the cell membrane has a transamidation active conformation. After the induction of apoptosis cytoplasmic TG2 changed its conformation and activates while, TG2 in endosomes retained transamidation inactive conformation even at late stages of apoptosis.     

Fluorescence resonance energy transfer biosensors that detect Ran conformational changes and a Ran x GDP-importin-beta -RanBP1 complex in vitro and in intact cells

The Ran GTPase plays a central role in nucleocytoplasmic transport. Association of Ran x GTP with transport carriers (karyopherins) triggers the loading/unloading of export or import cargo, respectively. The C-terminal tail of Ran x GTP is deployed in an extended conformation when associated with a Ran binding domain or importins. To monitor tail orientation, a Ran-GFP fusion was labeled with the fluorophore Alexa546. Fluorescence resonance energy transfer (FRET) occurs efficiently between the green fluorescent protein (GFP) and Alexa546 for Ran x GDP and Ran x GTP, suggesting that the tail is tethered in both states. However, Ran x GTP complexes with importin-beta, RanBP1, and Crm1 all show reduced FRET consistent with tail extension. Displacement of the C-terminal tail of Ran by karyopherins may be a general mechanism to facilitate RanBP1 binding. A Ran x GDP-RanBP1-importin-beta complex also displayed a low FRET signal. To detect this complex in vivo, a bipartite biosensor consisting of Ran-Alexa546 plus GST-GFP-RanBP1, was co-injected into the cytoplasm of cells. The Ran redistributed predominantly to the nucleus, and RanBP1 remained cytoplasmic. Nonetheless, a robust cytoplasmic FRET signal was detectable, which suggests that a significant fraction of cytoplasmic Ran.GDP may exist in a ternary complex with RanBP1 and importins.     

Tissue Transglutaminase Is an In Situ Substrate of Calpain: Regulation of Activity

Abstract: Tissue transglutaminase (tTG) is a calcium-dependent enzyme that catalyzes the transamidation of specific polypeptide-bound glutamine residues, a reaction that is inhibited by GTP. There is also preliminary evidence that, in situ, calpain and GTP may regulate tTG indirectly by modulating its turnover by the calcium-activated protease calpain. In the present study, the in vitro and in situ proteolysis of tTG by calpain, and modulation of this process by GTP, was examined. tTG is an excellent substrate for calpain and is rapidly degraded. Previously it has been demonstrated that GTP binding protects tTG from degradation by trypsin. In a similar manner, guanosine-5'- O -(3-thiotriphosphate) protects tTG against proteolysis by calpain. Treatment of SH-SY5Y cells with 1 n M maitotoxin, which increases intracellular calcium levels, resulted in a significant increase in in situ TG activity, with only a slight decrease in tTG protein levels. In contrast, when GTP levels were depleted by pretreating the cells with tiazofurin, maitotoxin treatment resulted in an ∼50% decrease in tTG protein levels, and a significant decrease in TG activity, compared with maitotoxin treatment alone. Addition of calpain inhibitors inhibited the degradation of tTG in response to the combined treatment of maitotoxin and tiazofurin and resulted in a significant increase in in situ TG activity. These studies indicate that tTG is an endogenous substrate of calpain and that GTP selectively inhibits the degradation of tTG by calpain.     

Spatial and temporal regulation of focal adhesion kinase activity in living cells

Focal adhesion kinase (FAK) is an essential kinase that regulates developmental processes and functions in the pathology of human disease. An intramolecular autoinhibitory interaction between the FERM and catalytic domains is a major mechanism of regulation. Based upon structural studies, a fluorescence resonance energy transfer (FRET)-based FAK biosensor that discriminates between autoinhibited and active conformations of the kinase was developed. This biosensor was used to probe FAK conformational change in live cells and the mechanism of regulation. The biosensor demonstrates directly that FAK undergoes conformational change in vivo in response to activating stimuli. A conserved FERM domain basic patch is required for this conformational change and for interaction with a novel ligand for FAK, acidic phospholipids. Binding to phosphatidylinositol 4,5-bisphosphate (PIP2)-containing phospholipid vesicles activated and induced conformational change in FAK in vitro, and alteration of PIP2 levels in vivo changed the level of activation of the conformational biosensor. These findings provide direct evidence of conformational regulation of FAK in living cells and novel insight into the mechanism regulating FAK conformation.     

Structural basis for the coordinated regulation of transglutaminase 3 by guanine nucleotides and calcium/magnesium

Transglutaminase 3 (TGase 3) is a member of a family of Ca2+-dependent enzymes that catalyze covalent cross-linking reactions between proteins or peptides. TGase 3 isoform is widely expressed and is important for effective epithelial barrier formation in the assembly of the cell envelope. Among the nine TGase enzyme isoforms known in the human genome, only TGase 2 is known to bind and hydrolyze GTP to GDP; binding GTP inhibits its transamidation activity but allows it to function in signal transduction. Here we present biochemical and crystallographic evidence for the direct binding of GTP/GDP to the active TGase 3 enzyme, and we show that the TGase 3 enzyme undergoes a GTPase cycle. The crystal structures of active TGase 3 with guanosine 5'-O-(thiotriphosphate) (GTPgammaS) and GDP were determined to 2.1 and 1.9 A resolution, respectively. These studies reveal for the first time the reciprocal actions of Ca2+ and GTP with respect to TGase 3 activity. GTPgammaS binding is coordinated with the replacement of a bound Ca2+ with Mg2+ and conformational rearrangements that together close a central channel to the active site. Hydrolysis of GTP to GDP results in two stable conformations, resembling both the GTP state and the non-nucleotide bound state, the latter of which allows substrate access to the active site.     

Protein transamidation by transglutaminase 2 in cells: a disputed Ca2+-dependent action of a multifunctional protein

Transglutaminase 2 (TG2) is the first described cellular member of an enzyme family catalyzing Ca(2+)-dependent transamidation of proteins. During the last two decades its additional enzymatic (GTP binding and hydrolysis, protein disulfide isomerase, protein kinase) and non-enzymatic (multiple interactions in protein scaffolds) activities, which do not require Ca(2+) , have been recognized. It became a prevailing view that TG2 is silent as a transamidase, except in extreme stress conditions, in the intracellular environment characterized by low Ca(2+) and high GTP concentrations. To counter this presumption a critical review of the experimental evidence supporting the role of this enzymatic activity in cellular processes is provided. It includes the structural basis of TG2 regulation through non-canonical Ca(2+) binding sites, mechanisms making it sensitive to low Ca(2+) concentrations, techniques developed for the detection of protein transamidation in cells and examples of basic cellular phenomena as well as pathological conditions influenced by this irreversible post-translational protein modification.     

Intracellular Localization and Conformational State of Transglutaminase 2: Implications for Cell Death

Transglutaminase 2 (TG2) is a multifunctional enzyme that has guanine nucleotide binding and GTP hydrolyzing activity in addition to its transamidating function. Studies show that TG2 is a player in mediating cell death processes. However, there is far from a consensus about the role of this enzyme in cell death processes as it appears to be dependent upon the cell type, stimuli, subcellular localization and conformational state of the enzyme. The purpose of this study was to dissect the role of TG2 in the cell death processes. To this end, we created and characterized 4 distinct point mutants of TG2, each of which differs from the wild type by its conformation or by lacking an important function. We also prepared these mutants as nuclear targeted proteins. By overexpressing mutant or wild type forms of TG2 in HEK 293 cells, we investigated the modulatory role of the protein in the cell death process in response to three stressors: thapsigargin, hyperosmotic stress and oxygen/glucose deprivation (OGD). All of the TG2 constructs, except the R580A mutant (which cannot bind guanine nucleotides and is therefore more prone to exhibit transamidating activity), either did not significantly affect the cell death processes or were protective. However in the case of the R580A mutant, cell death in response to high thapsigargin concentrations, was significantly increased. Intriguingly, nuclear localization of R580A-TG2 was sufficient to counteract the pro-death role of cytoplasmic R580A-TG2. In addition, nuclear localization of TG2 significantly facilitated its protective role against OGD. Our data support the hypothesis that the transamidation activity of TG2, which is mostly quiescent except in extreme stress conditions, is necessary for its pro-death role. In addition, nuclear localization of TG2 generally plays a key role in its protective function against cell death processes, either counteracting the detrimental effect or strengthening the protective role of the protein.     

Regulation of the interaction of inosine monophosphate dehydrogenase with mycophenolic Acid by GTP

Inosine monophosphate dehydrogenase (IMPDH), a rate-limiting enzyme in the de novo synthesis of guanine nucleotides, is a major therapeutic target. A prototypic uncompetitive inhibitor of IMPDH, mycophenolic acid (MPA), is the active form of mycophenolate mofeteil (CellCept), a widely used immunosuppressive drug. We have found that MPA interacts with intracellular IMPDH in vivo to alter its mobility on SDS-polyacrylamide gels. MPA also induces a striking conformational change in IMPDH protein in intact cells, resulting in the formation of annular aggregates of protein with concomitant inhibition of IMPDH activity. These aggregates are not associated with any known intracellular organelles and are reversible by incubating cells with guanosine, which repletes intracellular GTP, or with GTPgammaS. GTP also restores IMPDH activity. Treatment of highly purified IMPDH with MPA also results in the formation of large aggregates of protein, a process that is both prevented and reversed by the addition of GTP. Finally, GTP binds to IMPDH at physiologic concentrations, induces the formation of linear arrays of tetrameric protein, and prevents the aggregation of protein induced by MPA. We conclude that intracellular GTP acts as an antagonist to MPA by directly binding to IMPDH and reversing the conformational changes in the protein.     

satFRET: estimation of Förster resonance energy transfer by acceptor saturation

We demonstrate theoretically and experimentally the quantification of Förster resonance energy transfer (FRET) by direct and systematic saturation of the excited state of acceptor molecules. This version of acceptor depletion methods for FRET estimation, denoted as “satFRET” is reversible and suitable for time-resolved measurements. The technique was investigated theoretically using the steady-state solution of the differential equation system of donor and acceptor molecular states. The influence of acceptor photobleaching during measurement was included in the model. Experimental verification was achieved with the FRET-pair Alexa 546- Alexa 633 loaded on particles in different stoichiometries and measured in a confocal microscope. Estimates of energy transfer efficiency by excited state saturation were compared to those obtained by measurements of sensitised emission and acceptor photobleaching. The results lead to a protocol that allows time-resolved FRET measurements of fixed and living cells on a conventional confocal microscope. This procedure was applied to fixed Chinese hamster ovary cells containing a cyan fluorescent protein and yellow fluorescent protein pair. The time resolution of the technique was demonstrated in a live T cell activation assay comparing the FRET efficiencies measured using a genetically encoded green and red fluorescent protein biosensor for GTP/GDP turnover to those measured by acceptor photobleaching of fixed cells.     

2-Color calcium pump reveals closure of the cytoplasmic headpiece with calcium binding

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) undergoes conformational changes while transporting calcium, but the details of the domain motions are still unclear. The objective of the present study was to measure distances between the cytoplasmic domains of SERCA2a in order to reveal the magnitude and direction of conformational changes. Using fluorescence microscopy of live cells, we measured intramolecular fluorescence resonance energy transfer (FRET) from a donor fluorescent protein fused to the SERCA N-terminus to an acceptor fluorescent protein fused to either the N-, P-, or transmembrane domain. The "2-color" SERCA constructs were catalytically active as indicated by ATPase activity in vitro and Ca uptake in live cells. All constructs exhibited dynamic FRET changes in response to the pump ligands calcium and thapsigargin (Tg). These FRET changes were quantified as an index of SERCA conformational changes. Intramolecular FRET decreased with Tg for the two N-domain fusion sites (at residue 509 or 576), while the P- (residue 661) and TM-domain (C-terminus) fusions showed increased FRET with Tg. The magnitude of the Tg-dependent conformational change was not decreased by coexpression of phospholamban (PLB), nor did PLB slow the kinetics of Tg binding. FRET in ionophore-permeabilized cells was lower in EGTA than in saturating calcium for all constructs, indicating a decrease in domain separation distance with the structural transition from E2 (Ca-free) to E1 (Ca-bound). The data suggest closure of the cytoplasmic headpiece with Ca-binding. The present results provide insight into the structural dynamics of the Ca-ATPase. In addition, the 2-color SERCA constructs developed for this study may be useful for evaluating candidate small molecule regulators of Ca uptake activity.     

Steady-State Acceptor Fluorescence Anisotropy Imaging under Evanescent Excitation for Visualisation of FRET at the Plasma Membrane

We present a novel imaging system combining total internal reflection fluorescence (TIRF) microscopy with measurement of steady-state acceptor fluorescence anisotropy in order to perform live cell Förster Resonance Energy Transfer (FRET) imaging at the plasma membrane. We compare directly the imaging performance of fluorescence anisotropy resolved TIRF with epifluorescence illumination. The use of high numerical aperture objective for TIRF required correction for induced depolarization factors. This arrangement enabled visualisation of conformational changes of a Raichu-Cdc42 FRET biosensor by measurement of intramolecular FRET between eGFP and mRFP1. Higher activity of the probe was found at the cell plasma membrane compared to intracellularly. Imaging fluorescence anisotropy in TIRF allowed clear differentiation of the Raichu-Cdc42 biosensor from negative control mutants. Finally, inhibition of Cdc42 was imaged dynamically in live cells, where we show temporal changes of the activity of the Raichu-Cdc42 biosensor.     

Rho guanine dissociation inhibitors: pivotal molecules in cellular signalling.

The small G proteins of the Ras family act as bimodal relays in the transfer of intracellular signals. This is a dynamic phenomenon involving a cascade of protein-protein interactions modulated by chemical modifications, structural rearrangements and intracellular relocalisations. Most of the small G proteins could be operationally defined as proteins having two conformational states, each of which interacts with different cellular partners. These two states are determined by the nature of the bound nucleotide, GDP or GTP. This capacity to cycle between a GDP-bound conformation and a GTP-bound conformation enables them to filter, to amplify or to temporise the upstream signals that they receive. Thus the control of this cycle is crucial. Membrane anchoring of the proteins in the Ras family is a prerequisite for their activity. Most of the proteins in the Rho/Rac and Rab subfamilies of Ras proteins cycle between cytosol and membranes. Then the control of membrane association/dissociation is an other important regulation level. This review will describe one family of crucial regulators acting on proteins in the Rho/Rac family-the Rho guanine nucleotide dissociation inhibitors, or RhoGDIs. As yet, only three RhoGDIs have been described: RhoGDI-1, RhoGDI-2 (or D4/Ly-GDI) and RhoGDI-3. RhoGDI 1 and 2 are cytosolic and participate in the regulation of both the GDP/GTP cycle and the membrane association/dissociation cycle of Rho/Rac proteins. The non-cytosolic RhoGDI-3 seems to act in a slightly different way.     

Dynamic polymorphism of Ras observed by single molecule FRET is the basis for molecular recognition

Ras regulates signal transduction pathway function by dynamically interacting with various effectors. To understand the basis for Ras function, its conformational dynamics were measured in the absence and presence of effectors using single molecule fluorescence resonance energy transfer (FRET) between probes located on the Switch II region and GTP. The time trajectories of FRET efficiency from GTP-bound Ras showed that this conformation spontaneously varies among multiple states. Among them, a low FRET state was identified as an inactive state. The transition involving the inactive conformational state occurred in the time range of seconds. In contrast, fluctuation occurring most probably between multiple active high FRET conformational states lasted approximately 30 ms but converged to a specific conformational state upon binding to an effector. Thus, Ras conformation spontaneously fluctuates to readily interact with various effectors.     

Phosphorylated Phospholamban Stabilizes a Compact Conformation of?the Cardiac Calcium-ATPase

The sarcoendoplasmic reticulum calcium ATPase (SERCA) plays a key role in cardiac calcium handling and is considered a high-value target for the treatment of heart failure. SERCA undergoes conformational changes as it harnesses the chemical energy of ATP for active transport. X-ray crystallography has provided insight into SERCA structural substates, but it is not known how well these static snapshots describe in vivo conformational dynamics. The goals of this work were to quantify the direction and magnitude of SERCA motions as the pump performs work in live cardiac myocytes, and to identify structural determinants of SERCA regulation by phospholamban. We measured intramolecular fluorescence resonance energy transfer (FRET) between fluorescent proteins fused to SERCA cytoplasmic domains. We detected four discrete structural substates for SERCA expressed in cardiac muscle cells. The relative populations of these discrete states oscillated with electrical pacing. Low FRET states were most populated in low Ca (diastole), and were indicative of an open, disordered structure for SERCA in the E2 (Ca-free) enzymatic substate. High FRET states increased with Ca (systole), suggesting rigidly closed conformations for the E1 (Ca-bound) enzymatic substates. Notably, a special compact E1 state was observed after treatment with β-adrenergic agonist or with coexpression of phosphomimetic mutants of phospholamban. The data suggest that SERCA calcium binding induces the pump to undergo a transition from an open, dynamic conformation to a closed, ordered structure. Phosphorylated phospholamban stabilizes a unique conformation of SERCA that is characterized by a compact architecture.     

External GTP-bound transglutaminase 2 is a molecular switch for chondrocyte hypertrophic differentiation and calcification

Chondrocyte maturation to hypertrophy, associated with up-regulated transglutaminase 2 (TG2) expression, mediates not only physiologic growth plate mineralization but also pathologic matrix calcification and dys-regulated matrix repair in osteoarthritic articular cartilage. TG2-/- mouse chondrocytes demonstrate markedly inhibited progression to hypertrophic differentiation in response to both retinoic acid and the chemokine CXCL1. Here, our objectives were to test if up-regulated TG2 alone is sufficient to promote chondrocyte hypertrophic differentiation and to identify TG2 molecular determinants and potential downstream signals involved. TG2 activities, regulated by nucleotides and calcium, include cross-linking of cartilage matrix proteins, binding of fibronectin, and hydrolysis of GTP and ATP. Following transfection of TG2 site-directed mutants into chondrocytic cells, we observed that wild type TG2, and TG catalytic site and fibronectin-binding mutants promoted type X collagen expression and matrix calcification consistent with chondrocyte hypertrophic differentiation. In contrast, transfected mutants of TG2 GTP binding (K173L) and externalization (Y274A) sites did not stimulate chondrocyte hypertrophy. Recombinant TG2 treatment of bovine cartilage explants demonstrated that extracellular TG2 induced hypertrophy more robustly in the GTP-bound state, confirming an essential role of TG2 GTP binding. Finally, TG2 treatment induced type X collagen in a beta1 integrin-mediated manner, associated with rapid phosphorylation of both Rac1 and p38 kinases that were inhibited by mutation of the TG2 GTP binding site. In conclusion, externalized GTP-bound TG2 serves as a molecular switch for differentiation of chondrocytes to a hypertrophic, calcifying phenotype in a manner that does not require either TG2 transamidation activity or fibronectin binding.     

Identification of a guanosine triphosphate-binding site on guinea pig liver transglutaminase. Role of GTP and calcium ions in modulating activity

Guanosine 5'-triphosphate (GTP) was found to inhibit guinea pig liver transglutaminase activity as measured by [3H]putrescine incorporation into casein. GDP and GTP-gamma-S also inhibited enzyme activity (GTP-gamma-S greater than GTP greater than GDP). Kinetic studies showed that GTP acted as a reversible, noncompetitive inhibitor and that CaCl2 partially reversed GTP inhibition. GTP also inhibited rat liver and adult bovine aortic endothelial cell transglutaminase, but did not inhibit Factor XIIIa activity. Guanosine monophosphate (GMP), cyclic GMP, and polyguanylic acid did not inhibit enzyme activity. Guinea pig liver transglutaminase adsorbed well to GTP-agarose affinity columns, but not to CTP-agarose columns, and the binding was inhibited by the presence of calcium ions. Specific binding of GTP to transglutaminase was demonstrated by photoaffinity labeling with 8-azidoguanosine 5'-[gamma-32P] triphosphate, which was inhibited by the presence of GTP or CaCl2. GTP inhibited trypsin proteolysis of guinea pig liver transglutaminase without affecting the trypsin proteolysis of chromogenic substrates. Proteolytic protection was reversed by the addition of calcium. This study demonstrates that GTP binds to transglutaminase and that both GTP and calcium ions function in concert to regulate transglutaminase structure and function.     

G Proteins in Reverse Mode: RECEPTOR-MEDIATED GTP RELEASE INHIBITS G PROTEIN AND EFFECTOR FUNCTION*

Active G protein-coupled receptors activate heterotrimeric Gαβγ proteins by catalyzing the exchange of GDP by GTP at the Gα subunit. A paradoxical attenuation of G protein-activated inwardly rectifying potassium channels (GIRK) upon stimulation of native cells with high concentrations of agonist is known. However, a deactivation of activated G proteins by active receptors has not been experimentally studied in intact cells. We monitored GIRK currents and G_o protein activation by means of fluorescence resonance energy transfer (FRET) in parallel. The results suggested that GIRK currents were paradoxically attenuated due to an inactivation of G_o proteins by active α_2A-adrenergic receptors. To study the mechanisms, G protein activation and receptor-G protein interactions were analyzed as a function of nucleotide type and nucleotide concentrations by means of FRET, while controlling intracellular nucleotides upon permeabilization of the cell membrane. Results suggested a receptor-catalyzed dissociation of GTP from activated heterotrimeric Gαβγ. Consequently, nucleotide-free G proteins were sequestrated in heterotrimeric conformation at the active receptor, thus attenuating downstream signaling in an agonist-dependent manner.     

Visualization of polarized membrane type 1 matrix metalloproteinase activity in live cells by fluorescence resonance energy transfer imaging

Membrane type 1 matrix metalloproteinase (MT1-MMP) plays a critical role in cancer cell biology by proteolytically remodeling the extracellular matrix. Utilizing fluorescence resonance energy transfer (FRET) imaging, we have developed a novel biosensor, with its sensing element anchoring at the extracellular surface of cell membrane, to visualize MT1-MMP activity dynamically in live cells with subcellular resolution. Epidermal growth factor (EGF) induced significant FRET changes in cancer cells expressing MT1-MMP, but not in MT1-MMP-deficient cells. EGF-induced FRET changes in MT1-MMP-deficient cells could be restored after reconstituting with wild-type MT1-MMP, but not MMP-2, MMP-9, or inactive MT1-MMP mutants. Deletion of the transmembrane domain in the biosensor or treatment with tissue inhibitor of metalloproteinase-2, a cell-impermeable MT1-MMP inhibitor, abolished the EGF-induced FRET response, indicating that MT1-MMP acts at the cell surface to generate FRET changes. In response to EGF, active MT1-MMP was directed to the leading edge of migrating cells along micropatterned fibronectin stripes, in tandem with the local accumulation of the EGF receptor, via a process dependent upon an intact cytoskeletal network. Hence, the MT1-MMP biosensor provides a powerful tool for characterizing the molecular processes underlying the spatiotemporal regulation of this critical class of enzymes.