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Richard A Cerione

Publications and source records attributed to Richard A Cerione.

At least 19 recordsLinked to original sources
PubMed2006

Two isoforms of tissue transglutaminase mediate opposing cellular fates.

Opposing cellular responses are typically regulated by distinct sets of genes. However, tissue transglutaminase (TGase) provides an interesting example of a single gene product that has been implicated both in affording protection against cellular insults as well as in promoting cell death. Here, we shed some light on how these conflicting activities might be manifested by demonstrating that alternative transcripts of TGase differentially affect cell viability. We show that although the full-length TGase protein affords strong protection against cell death signals, a shorter version of TGase that is truncated at the 3' end, and thus called TGase-short (TGase-S), is cytotoxic. The apoptotic activity of TGase-S is not dependent on its transamidation activity because the mutation of a cysteine residue that is essential for catalyzing this reaction does not compromise the ability of TGase-S to induce cell death. Intriguingly, TGase-S undergoes inappropriate oligomer formation in cells before cell death, suggesting a novel mechanism for the apoptotic effects of this protein.

Animals↗
PubMed2006

Importance of Ca(2+)-dependent transamidation activity in the protection afforded by tissue transglutaminase against doxorubicin-induced apoptosis.

Tissue transglutaminase II (TGase-II), which is capable of both GTP binding and transamidation activities, has been implicated in a variety of biological disorders ranging from cancer to neurodegenerative diseases. Recent studies have suggested that the transamidation activity of TGase-II is necessary for the survival of cancer cells confronted with different stresses and cellular insults. When assayed in vitro, the transamidation activity of TGase-II is Ca(2+)-dependent. However, at present, little is known with regard to how the regulation by Ca(2+) is manifested or if in fact it is important for the cellular functions of TGase-II. Here, we have set out to further examine the Ca(2+)-mediated regulation of TGase-II's transamidation activity, with our goals being to identify the Ca(2+)-regulatory sites on the protein and determine whether they are essential for TGase-II to confer survival to human breast cancer cells. On the basis of comparisons between the X-ray crystal structures of TGase-II and TGase-III, we identified three putative Ca(2+)-regulatory sites on TGase-II. Site-directed mutagenesis was performed to individually alter key residues at each of the sites. These substitutions did not affect the ability of TGase-II to bind guanine nucleotides, nor did they cause any obvious changes in its cellular localization. While substitutions at the different Ca(2+)-regulatory sites could either slightly enhance or markedly reduce the GTP hydrolytic activity of TGase-II, mutations at each of the three sites inhibited the Ca(2+)-responsive transamidation activity. We further showed that the same substitutions inhibited the ability of TGase-II to protect human breast cancer cells against the apoptotic activity of doxorubicin. Overall, these findings demonstrate that the Ca(2+)-mediated regulation of transamidation activity is essential for the ability of TGase-II to confer cell survival.

Amides↗
PubMed2006

Identification of a DOCK180-related guanine nucleotide exchange factor that is capable of mediating a positive feedback activation of Cdc42.

Cdc42, a member of the Rho subfamily of small GTPases, influences a wide range of activities including the establishment of cell polarity, migration, and the regulation of cell growth and differentiation. Here we describe the identification of an approximately 220-kDa protein that formed a stable complex with activated forms of Cdc42 and thereby showed characteristics of a downstream target/effector for this GTPase. However, molecular cloning of the cDNA encoding this protein (p220) revealed that it was highly related to Zizimin-1 and identical in sequence to a gene product in the data base designated DOCK11, which are members of the DOCK180 family of guanine nucleotide exchange factors (GEFs) for Cdc42 and Rac. Biochemical characterization shows that p220 is a specific GEF for Cdc42, with the GEF activity originating from its DHR2 (for DOCK homology region 2) domain. Nucleotide-depleted Cdc42 forms a stable complex with the DHR2 domain, whereas the binding of activated Cdc42 requires both the DHR2 domain and residues 66-126 within the amino-terminal portion of p220. Moreover, the full-length protein shows markedly higher GEF activity than the isolated DHR2 domain, whereas removal of the amino-terminal 126 amino acids necessary for binding-activated Cdc42 dramatically diminishes the activity. These and other results point to activated Cdc42 providing a positive feedback regulation of the GEF activity of p220. Thus, we refer to p220/DOCK11 as activated Cdc42-associated GEF, befitting its functional activity.

Amino Acid Sequence↗
PubMed2006

Cool-1 functions as an essential regulatory node for EGF receptor- and Src-mediated cell growth.

Cool-1 (cloned-out of library 1) has a key role in regulating epidermal growth factor receptor (EGFR) degradation. Here, we show that Cool-1 performs this function by functioning as both an upstream activator and downstream target for Cdc42. EGF-dependent phosphorylation of Cool-1 enables it to act as a nucleotide exchange factor for Cdc42 and to form a complex with the E3 ligase Cbl, thus regulating Cbl-catalysed EGFR degradation. The EGF-dependent phosphorylation is normally transient; however, Cool-1 phosphorylation is sustained in cells expressing v-Src and is essential for cellular transformation, as well as for v-Src-induced tumour formation in mice. These findings demonstrate that the regulated phosphorylation of Cool-1 is necessary to maintain the balance between normal signalling by EGFR and Src versus aberrant growth and transformation.

Animals↗
PubMed2006

Influencing cellular transformation by modulating the rates of GTP hydrolysis by Cdc42.

The small GTPase Cdc42 has been implicated in a number of cellular responses ranging from the regulation of the actin cytoskeletal architecture to intracellular trafficking and cell cycle progression. Cdc42 mutants that constitutively exchange GDP for GTP but still hydrolyze GTP (called 'fast-cycling' mutants) promote cellular transformation, whereas Cdc42 mutants that are unable to hydrolyze GTP and are irreversibly trapped in the GTP-bound state often inhibit cell growth. In this work, we have set out to further establish that Cdc42 needs to cycle between its 'on' and 'off' states to stimulate cell growth, by examining the consequences of manipulating its GTP-binding/GTP hydrolytic cycle in two different ways. One approach was to examine whether substitutions that act in a manner opposite to the 'fast cyclers', and extend the lifetime of the activated GTP-bound state by slowing the GTP hydrolytic reaction (i.e., 'slow-cycling' mutations), positively influence cell growth. Indeed we show that one such slow-cycling mutant, Cdc42[Y32A], which is insensitive to Cdc42GAP but still exhibits a measurable intrinsic GTP hydrolytic activity, gives rise to increased levels of activated Cdc42 in NIH 3T3 cells. We go on to show that the Y32A mutant stimulates the actin cytoskeletal changes that lead to filopodia formation, confer growth advantages to fibroblasts under low serum conditions, and enable cells to grow to high densities when exposed to normal levels of serum. The second approach was to determine whether the transforming activity of the fast-cycling Cdc42[F28L] mutant can be reversed by compensating for its accelerated nucleotide exchange reaction through the expression of the GTPase-activating protein (Cdc42GAP) and the ensuing stimulation of GTP hydrolytic activity. We showed that expression of the limit functional domain of Cdc42GAP inhibited Cdc42[F28L]-induced transformation, as well as selectively reversed the transformed phenotypes caused by the hyperactivation of wild-type Cdc42 in cells expressing the oncogenic version of Dbl (for Diffuse B cell lymphoma), a guanine nucleotide exchange factor for Cdc42 and the related Rac and Rho GTPases. Overall, the results reported here establish the requirement for Cdc42 to cycle between its signaling-on and -off states in order to positively influence cell growth and highlight how the Cdc42GAP can play an important role in regulating cell proliferation.

Animals↗
PubMed2006

New insights into the role of conserved, essential residues in the GTP binding/GTP hydrolytic cycle of large G proteins.

The GTP hydrolytic (GTPase) reaction terminates signaling by both large (heterotrimeric) and small (Ras-related) GTP-binding proteins (G proteins). Two residues that are necessary for GTPase activity are an arginine (often called the "arginine finger") found either in the Switch I domains of the alpha subunits of large G proteins or contributed by the GTPase-activating proteins of small G proteins, and a glutamine that is highly conserved in the Switch II domains of Galpha subunits and small G proteins. However, questions still exist regarding the mechanism of the GTPase reaction and the exact role played by the Switch II glutamine. Here, we have characterized the GTP binding and GTPase activities of mutants in which the essential arginine or glutamine residue has been changed within the background of a Galpha chimera (designated alpha(T)*), comprised mainly of the alpha subunit of retinal transducin (alpha(T)) and the Switch III region from the alpha subunit of G(i1). As expected, both the alpha(T)*(R174C) and alpha(T)*(Q200L) mutants exhibited severely compromised GTPase activity. Neither mutant was capable of responding to aluminum fluoride when monitoring changes in the fluorescence of Trp-207 in Switch II, although both stimulated effector activity in the absence of rhodopsin and Gbetagamma. Surprisingly, each mutant also showed some capability for being activated by rhodopsin and Gbetagamma to undergo GDP-[(35)S]GTPgammaS exchange. The ability of the mutants to couple to rhodopsin was not consistent with the assumption that they contained only bound GTP, prompting us to examine their nucleotide-bound states following their expression and purification from Escherichia coli. Indeed, both mutants contained bound GDP as well as GTP, with 35-45% of each mutant being isolated as GDP-P(i) complexes. Overall, these findings suggest that the R174C and Q200L mutations reveal Galpha subunit states that occur subsequent to GTP hydrolysis but are still capable of fully stimulating effector activity.

Arginine↗
PubMed2006

Effects of tissue transglutaminase on beta -amyloid1-42-induced apoptosis.

Tissue transglutaminase (TGase) has been implicated in both cell survival and apoptosis. Here we investigate the role of TGase in beta-amyloid-induced neurotoxicity using retinoic acid (RA)-differentiated, neuronal SH-SY5Y cells. We show that beta-amyloid-induced cell death was reduced in RA-differentiated SH-SY5Y cells treated with the TGase inhibitor monodansyl cadaverine. Expression of wild-type TGase enhanced beta-amyloid1-42-induced apoptosis, whereas transamidation-defective TGase did not. These effects were specific for beta-amyloid-treated cells, as TGase reversed the neurotoxic effects caused by hydrogen peroxide treatment. Enhancement of beta-amyloid1-42-induced cell death by TGase was accompanied by marked increases in TGase activity in the membrane fractions and translocation of TGase to the cell surface. Overall, these findings suggest that the ability of TGase to exhibit pro-survival versus pro-apoptotic activity is linked to its cellular localization, with beta-amyloid-induced recruitment of TGase to the cell surface accentuating neuronal toxicity and apoptosis.

Amyloid beta-Peptides↗
PubMed2006

Biochemical characterization of the Cool (Cloned-out-of-Library)/Pix (Pak-interactive exchange factor) proteins.

The Cool (Cloned out of Library)/Pix (Pak interactive exchange factor) proteins have been implicated in a diversity of biological activities, ranging from pathways initiated by growth factors and chemoattractants to X-linked mental retardation. Initially discovered through yeast two-hybrid and biochemical analyses as binding partners for the Cdc42/Rac-target/effector, Pak (p21 activated kinase), the sequences for the Cool/Pix proteins revealed a DH (Dbl homology) domain. Because the DH domain is the limit functional unit for stimulating guanine nucleotide exchange on Rho family GTP-binding proteins, it was assumed that the Cool/Pix proteins would act as guanine nucleotide exchange factors (GEFs) for the Rho proteins. Of the three known isoforms, (p50Cool-1, p85Cool-1/beta-Pix, and 90Cool-2/alpha-Pix), only Cool-2/alpha-Pix has exhibited significant GEF activity. A number of experimental techniques have been used to characterize Cool-2, and in vitro analysis has revealed that its GEF activity is under tight control through intramolecular interactions involving several binding partners. Here we describe the biochemical methods used to study the Cool/Pix proteins and, in particular, the regulation of the GEF activity of Cool-2/alpha-Pix.

Animals↗
PubMed2006

Measurement of epidermal growth factor receptor turnover and effects of Cdc42.

Ligand-induced degradation represents an essential component of the overall regulation of EGF receptor (EGFR)-coupled signal transduction. Following activation, EGFRs are monoubiquitinated, subsequently sorted by ubiquitin-interaction-based sorting machinery, and transported to multivesicular bodies (MVBs) and lysosomes for degradation. The Rho-family small G-protein, Cdc42, has been implicated in the regulation of EGFR degradation. Here we describe routine methods for assaying EGFR endocytosis and degradation. In addition, we have introduced procedures for determining the effects of Cdc42 and its downstream targets, in particular, ACK (Activated Cdc42-associated Kinase) and p85Cool-1 (Cloned out of library)/Pix (for Pak-interactive exchange factor), on EGFR degradation.

Animals↗
PubMed2005

A Cdc42 mutant specifically activated by intersectin.

The Rho family GTPase Cdc42 functions as a molecular switch and controls many fundamental cellular processes such as cytoskeletal regulation, cell polarity, and vesicular trafficking. Guanine nucleotide exchange factors of the Dbl family activate Cdc42 and other Rho GTPases by catalyzing the removal of bound GDP, allowing for GTP loading, and subsequent effector recognition ultimately leading to downstream signaling events. Analysis of existing structural data reveals that the Dbl exchange factor intersectin engages a strictly conserved GTPase residue of Cdc42 (tyrosine 32) in a unique mode with respect to all other visualized exchange factor-Rho GTPase interfaces. To investigate this differential binding architecture, we analyzed the role of tyrosine 32 of Cdc42 in binding, and stimulation by Dbl family exchange factors. Deletion of the hydroxyl side chain of tyrosine 32 substantially increases the affinity of Cdc42 for intersectin, yet severely cripples interaction with Dbs, a normally potent exchange factor of Cdc42. Moreover, Cdc42(Y32F) is exclusively activated by intersectin, while virtually unresponsive to other Cdc42-activating exchange factors in vitro and in vivo. Further, the structural determinants unique to intersectin, which permit selective recognition and concomitant stimulation of Cdc42(Y32F), have been defined. Cdc42 and other individual Rho GTPases receive input stimulatory signals from a multitude of Dbl exchange factors, and therefore, Cdc42(Y32F) could act as a valuable reagent for understanding the specific influence of ITSN on Cdc42-mediated signaling phenomena.

Adaptor Proteins, Vesicular Transport↗
PubMed2005

A switch 3 point mutation in the alpha subunit of transducin yields a unique dominant-negative inhibitor.

The rhodopsin/transducin-coupled vertebrate vision system has served as a paradigm for G protein-coupled signaling. We have taken advantage of this system to identify new types of constitutively active, transducin-alpha (alphaT) subunits. Here we have described a novel dominant-negative mutation, made in the background of a chimera consisting of alphaT and the alpha subunit of G(i1) (designated alphaT*), which involves the substitution of a conserved arginine residue in the conformationally sensitive Switch 3 region. Changing Arg-238 to either lysine or alanine had little or no effect on the ability of alphaT* to undergo rhodopsin-stimulated GDP-GTP exchange, whereas substituting glutamic acid for arginine at this position yielded an alphaT* subunit (alphaT*(R238E)) that was incapable of undergoing rhodopsin-dependent nucleotide exchange and was unable to bind or stimulate the target/effector enzyme (cyclic GMP phosphodiesterase). Moreover, unlike the GDP-bound forms of alphaT*, alphaT*(R238A) and alphaT*(R238K), the alphaT*(R238E) mutant did not respond to aluminum fluoride (AlF4(-)), as read out by changes in Trp-207 fluorescence. However, surprisingly, we found that alphaT*(R238E) effectively blocked rhodopsin-catalyzed GDP-GTP exchange on alphaT*, as well as rhodopsin-stimulated phosphodiesterase activity. Analysis by high pressure liquid chromatography indicated that the alphaT*(R238E) mutant exists in a nucleotide-free state. Nucleotide-free forms of G alpha subunits were typically very sensitive to proteolytic degradation, but alphaT*(R238E) exhibited a resistance to trypsin-proteolysis similar to that observed with activated forms of alphaT*. Overall, these findings indicated that by mutating a single residue in Switch 3, it is possible to generate a unique type of dominant-negative G alpha subunit that can effectively block signaling by G protein-coupled receptors.

Aluminum Compounds↗
PubMed2005

Cdc42 and Ras cooperate to mediate cellular transformation by intersectin-L.

Cdc42, a Ras-related GTP-binding protein, has been implicated in the regulation of the actin cytoskeleton, membrane trafficking, cell-cycle progression, and malignant transformation. We have shown previously that a Cdc42 mutant (Cdc42(F28L)), capable of spontaneously exchanging GDP for GTP (referred to as "fast-cycling"), transformed NIH 3T3 cells because of its ability to interfere with epidermal growth factor receptor (EGFR)-Cbl interactions and EGFR down-regulation. To further examine the link between the hyperactivation of Cdc42 and its ability to alter EGFR signaling and thereby cause cellular transformation, we examined the effects of expressing different forms of the Cdc42-specific guanine nucleotide exchange factor, intersectin-L, in fibroblasts. Full-length intersectin-L exhibited little ability to stimulate nucleotide exchange on Cdc42, whereas a truncated version that contained five Src homology 3 (SH3) domains, the Dbl and pleckstrin homology domains (DH and PH domains, respectively), and a C2 domain (designated as SH3A-C2) showed modest guanine nucleotide exchange factor activity, whereas a form containing just the DH, PH, and C2 domains (DH-C2) strongly activated Cdc42. However, DH-C2 showed little ability to stimulate growth in low serum or colony formation in soft agar, whereas SH3A-C2 gave rise to a much stronger stimulation of cell growth in low serum and was highly effective in stimulating colony formation. Moreover, although SH3A-C2 strongly transformed fibroblasts, it differed from the actions of the Cdc42(F28L) mutant, as SH3A-C2 showed little ability to alter EGFR levels or the lifetime of EGF-coupled signaling through ERK. Rather, we found that SH3A-C2 exhibited strong transforming activity through its ability to mediate cooperation between Ras and Cdc42.

Actins↗
PubMed2005

The Cool-2/alpha-Pix protein mediates a Cdc42-Rac signaling cascade.

BACKGROUND: Cloned-out of library-2 (Cool-2)/PAK-interactive exchange factor (alpha-Pix) was identified through its ability to bind the Cdc42/Rac target p21-activated kinase (PAK) and has been implicated in certain forms of X-linked mental retardation as well as in growth factor- and chemoattractant-coupled signaling pathways. We recently found that the dimeric form of Cool-2 is a specific guanine nucleotide exchange factor (GEF) for Rac, whereas monomeric Cool-2 is a GEF for Cdc42 as well as Rac. However, unlike many GEFs, Cool-2 binds to activated forms of Cdc42 and Rac. Thus, we have investigated the functional consequences of these interactions. RESULTS: We show that the binding of activated Cdc42 to the Cool-2 dimer markedly enhances its ability to associate with GDP bound Rac1, resulting in a significant activation of Rac-GEF activity. While the Rac-specific GEF activity of Cool-2 is mediated through the Dbl homology (DH) domain from one monomer and the Pleckstrin homology domain from the other, activated Cdc42 interacts with the DH domain, most likely opposite the DH domain binding site for GDP bound Rac. Activated Rac also binds to Cool-2; however, it strongly inhibits the GEF activity of dimeric Cool-2. CONCLUSIONS: We provide evidence for novel mechanisms of allosteric regulation of the Rac-GEF activity of the Cool-2 dimer, involving stimulatory effects by Cdc42 and feedback inhibition by Rac. These findings demonstrate that by serving as a target for GTP bound Cdc42 and a GEF for Rac, Cool-2 mediates a GTPase cascade where the activation of Cdc42 is translated into the activation of Rac.

Animals↗
PubMed2004

Interaction of activated Cdc42-associated tyrosine kinase ACK2 with HSP90.

ACK2 (activated Cdc42-associated tyrosine kinase 2) is a specific downstream effector for Cdc42, a member of the Rho family of small G-proteins. ACK2 interacts with clathrin, an endocytic vesicle coating protein, and SH3PX1, a sorting nexin, and is involved in clathrin-mediated endocytosis. While searching for proteins that interact with ACK2, we found that HSP90 (heat-shock protein 90) binds to ACK2. Analysis of a series of truncation mutants of ACK2 has defined the regions within the kinase domain of ACK2 that are required for binding to HSP90. The binding of HSP90 to ACK2 is blocked upon treatment with geldanamycin, an HSP90-specific ATPase inhibitor, and is required for the in vivo kinase activity of ACK2 and its association with Cdc42. Overall, our data suggest a novel mechanism of regulation in which HSP90 serves as a regulatory component in an ACK2 functional complex and plays a role in sustaining its kinase activity.

Adenosine Triphosphatases↗
PubMed2004

Novel regulatory mechanisms for the Dbl family guanine nucleotide exchange factor Cool-2/alpha-Pix.

The Cool-2 (cloned-out of library-2) protein (identical to alpha-Pix for Pak-interactive exchange factor) has been implicated in various biological responses including chemoattractant signaling and in certain forms of mental retardation. We show that when Cool-2 exists as a dimer, it functions as a Rac-specific guanine nucleotide exchange factor (GEF). Dimerization of Cool-2 enables its Dbl (diffuse B-cell lymphoma) and pleckstrin homology domains to work together (in trans) to bind specifically to Rac-GDP. Dissociation of dimeric Cool-2 into its monomeric form allows it to act as a GEF for Cdc42 as well as for Rac. The binding of either PAK (p21-activated kinase) or Cbl (Casitas B-lymphoma) to the SH3 domain of monomeric Cool-2 is necessary for the functional interactions between GDP-bound Cdc42 or Rac and the Cool-2 monomer. The betagamma subunit complex of large GTP-binding proteins, by interacting with PAK, stimulates the dissociation of the Cool-2 dimer and activates its GEF activity for Cdc42. Overall, these findings highlight novel mechanisms by which extracellular signals can direct the specific activation of Rac versus Cdc42 by Cool-2/alpha-Pix.

Animals↗
PubMed2004

Augmentation of tissue transglutaminase expression and activation by epidermal growth factor inhibit doxorubicin-induced apoptosis in human breast cancer cells.

Tissue transglutaminase (TGase) exhibits both a GTP binding/hydrolytic capability and an enzymatic transamidation activity. Increases in TGase expression and activation often occur in response to stimuli that promote cellular differentiation and apoptosis, yet the signaling mechanisms used by these stimuli to regulate TGase expression and activation and the role of TGase in these cellular processes are not well understood. Retinoic acid (RA) consistently induces TGase expression and activation, and it was shown recently that RA-induced TGase expression was inhibited in NIH3T3 mouse fibroblasts co-stimulated with epidermal growth factor (EGF). Here we investigate whether EGF also antagonized RA-induced TGase expression in breast cancer cells. We found that EGF stimulation affected TGase expression and activation very differently in these cancer cells. Not only did EGF fail to block RA-induced TGase expression, but also EGF alone was sufficient to potently up-regulate TGase expression and activation in SKBR3 cells, as well as MDAMB468 and BT-20 cells. Inhibiting phosphoinositide 3-kinase activity severely diminished the ability of EGF and RA to increase TGase protein levels, whereas a constitutively active form of phosphoinositide 3-kinase potentiated the induction of TGase expression by EGF in SKBR3 cells. Because EGF is an established antiapoptotic factor, we examined whether the protection afforded by EGF was dependent on its ability to up-regulate TGase activity in SKBR3 and BT-20 cells. Exposure of cells to a TGase inhibitor or expression of a dominant-negative form of TGase potently inhibited EGF-mediated protection from doxorubicin-induced apoptosis. Moreover, expression of exogenous TGase in SKBR3 cells mimicked the survival advantage of EGF, suggesting that TGase activation is necessary and sufficient for the antiapoptotic properties of EGF. These findings indicate for the first time that EGF can induce TGase expression and activation in human breast cancer cells and that this contributes to their oncogenic potential by promoting chemoresistance.

Animals↗
PubMed2004

Perturbing the linker regions of the alpha-subunit of transducin: a new class of constitutively active GTP-binding proteins.

The GDP-GTP exchange activity of the retinal G protein, transducin, is markedly accelerated by the photoreceptor rhodopsin in the first step of visual transduction. The x-ray structures for the alpha subunits of transducin (alpha(T)) and other G proteins suggest that the nucleotide-binding (Ras-like) domain and a large helical domain form a "clam shell" that buries the GDP molecule. Thus, receptor-promoted G protein activation may involve "opening the clam shell" to facilitate GDP dissociation. In this study, we have examined whether perturbing the linker regions connecting the Ras-like and helical domains of Galpha subunits gives rise to a more readily exchangeable state. The sole glycine residues in linkers 1 and 2 were individually changed to proline residues within an alpha(T)/alpha(i1) chimera (designated alpha(T)(*)). Both alpha(T)(*) linker mutants showed significant increases in their basal rates of GDP-GTP exchange when compared either to retinal alpha(T) or recombinant alpha(T)(*). The alpha(T)(*) linker mutants were responsive to aluminum fluoride, which binds to alpha-GDP complexes and induces changes in Switch 2. Although both linker mutants were further activated by light-activated rhodopsin together with the betagamma complex, their activation was not influenced by betagamma alone, arguing against the idea that the betagamma complex helps to pry apart the helical and Ras-like domains of Galpha subunits. Once activated, the alpha(T)(*) linker mutants were able to stimulate the cyclic GMP phosphodiesterase. Overall, these findings highlight a new class of activated Galpha mutants that constitutively exchange GDP for GTP and should prove valuable in studying different G protein-signaling systems.

3',5'-Cyclic-GMP Phosphodiesterases↗