Biomedical subjects

Giordano Pula

Publications and source records attributed to Giordano Pula.

11 recordsLinked to original sources

PubMed2006

PKCdelta regulates collagen-induced platelet aggregation through inhibition of VASP-mediated filopodia formation.

Protein kinase Cdelta (PKCdelta) has been shown by pharmacologic approaches to negatively regulate collagen-induced platelet aggregation. Here we addressed the molecular and cellular mechanisms underlying this negative regulation. Using PKCdelta-/- platelets, we show that the mechanism did not involve altered inside-out signaling to integrin alpha(IIb)beta3 and did not affect early signaling events downstream of GPVI, because there was no difference in tyrosine phosphorylation of PLCgamma2 between wild-type and PKCdelta-/- platelets. There was also no increase in secretion of dense granule content, in contrast to studies using rottlerin where secretion was enhanced. Importantly, however, there was marked enhancement of filopodia generation in PKCdelta-/- platelets upon adhesion to collagen compared with wild-type platelets. Filopodia play an essential role regulating adhesive events leading to platelet aggregation by increasing platelet-platelet contact. We show that the critical effector for PKCdelta is vasodilator-stimulated phosphoprotein (VASP), a major regulator of actin cytoskeleton dynamics. PKCdelta physically interacts with VASP constitutively and regulates its phosphorylation on Ser157. In VASP-/- platelets, the enhancement of filopodia generation, actin polymerization, and platelet aggregation by rottlerin is ablated. PKCdelta is therefore a critical negative regulator of filopodia, and hence platelet aggregation, through a functional interaction with the actin organizer VASP.

Acetophenones↗

PubMed2006

Vasodilator-stimulated phosphoprotein (VASP) is phosphorylated on Ser157 by protein kinase C-dependent and -independent mechanisms in thrombin-stimulated human platelets.

VASP (vasodilator-stimulated phosphoprotein) is an actin- and profilin-binding protein that is expressed in platelets at high levels and plays a major role in negatively regulating secretory and adhesive events in these cells. VASP is a major substrate for cAMP- and cGMP-regulated protein kinases and it has been shown to be directly phosphorylated on Ser157 by PKC (protein kinase C). In the present paper, we show that, in human platelets, VASP is phosphorylated by PKC on Ser157, but not Ser239, in response to phorbol ester stimulation, in a manner blocked by the PKC inhibitor BIM I (bisindolylmaleimide I). In response to thrombin, VASP was also phosphorylated on Ser157, but this response was only partially inhibited by BIM I, indicating PKC-dependent and -independent pathways to VASP phosphorylation by thrombin. Using inhibitors, we have ruled out the possibility that the PKC-independent pathway acts through guanylate cyclase generation of cGMP, or through a phosphoinositide 3-kinase-dependent kinase. Inhibition of Rho kinase, however, substantially reduced Ser157 VASP phosphorylation, and its effects were additive with BIM I. This implicates Rho kinase and PKC as the major kinases that phosphorylate VASP Ser157 in response to thrombin in platelets.

Blood Platelets↗

PubMed2005

Analysis of mGluR1a constitutive internalization using a pulse-chase enzyme-linked immuno-sorbant assay (ELISA).

The surface expression of G protein-coupled receptors is regulated by internalization. For many receptors, a constitutive level of internalization in the absence of agonist has been reported. The constitutive internalization of metabotropic glutamate receptor 1a (mGluR1a) has been described, but in general little attention has been dedicated to this important aspect of receptor regulation. Here we describe a pulse-chase ELISA method that allows the investigation of mGluR1a constitutive internalization. When investigated by pulse-chase ELISA, the constitutive internalization of mGluR1a was inhibited by dominant negative mutant constructs of arrestin-2 or Eps-15. This observation, besides indicating the arrestin- and clathrin-dependence of mGluR1a constitutive internalization, also confirmed the physiological relevance of the method described in this article. Confocal microscopy experiments to study receptor localization further validated the pulse-chase labelling procedure. The application of the pulse-chase ELISA to mGluR1b, revealed that this splice variant undergoes marginal constitutive internalization. Two COOH-terminal deletion mutants of mGluR1a, DMI (Arg847stop) and DMII (Arg868stop), were also tested for constitutive internalization. Interestingly, only DMII underwent significant constitutive internalization, suggesting that the region between Arg847 and Arg868 might play a regulatory role in mGluR1a trafficking. Taken together, the pulse-chase ELISA appears to be an efficient tool to analyze the constitutive internalization of different mGluR1 splice variants.

Alanine Transaminase↗

PubMed2004

Functional interaction of protein kinase Calpha with the tyrosine kinases Syk and Src in human platelets.

There is a high degree of cross-talk between tyrosine phosphorylation and the serine/threonine phosphorylation signaling pathways. Here we show a physical and functional interaction between the classical protein kinase C isoform (cPKC), PKCalpha, and two major nonreceptor tyrosine kinases in platelets, Syk and Src. In the presence of the cPKC-selective inhibitor Go6976, platelet 5-hydroxytryptamine release was abolished in response to co-activation of glycoproteins VI and Ib-IX-V by the snake venom alboaggregin A, whereas platelet aggregation was substantially inhibited. Of the two platelet cPKCs, PKCalpha but not PKCbeta was activated, occurring in an Syk- and phospholipase C-dependent manner. Syk and PKCalpha associate in a stimulation-dependent manner, requiring Syk but not PKC activity. PKCalpha and Syk also co-translocate from the cytosol to the plasma membrane upon platelet activation, in a manner dependent upon the activities of both kinases. Although PKCalpha is phosphorylated on tyrosine downstream of Syk, we provide evidence against phosphorylation of Syk by PKCalpha, consistent with a lack of effect of PKCalpha inhibition on Syk activity. PKCalpha also associates with Src; although in contrast to interaction with Syk, PKCalpha activity is required for the association of these kinases but not the stimulation-induced translocation of Src to the cell membrane. Finally, the activity of Src is negatively regulated by PKC, as shown by potentiation of Src activity in the presence of the PKC inhibitors GF109203X or Go6976. Therefore, there is a complex interplay between PKCalpha, Syk, and Src involving physical interaction, phosphorylation, translocation within the cell, and functional activity regulation.

Blood Platelets↗

PubMed2004

Desensitization and internalization of metabotropic glutamate receptor 1a following activation of heterologous Gq/11-coupled receptors.

In this study we characterized the heterologous desensitization and internalization of the metabotropic glutamate receptor 1 (mGluR1) splice variants mGluR1a and mGluR1b following activation of endogenous G(q/11)-coupled receptors in HEK293 cells. Agonist activation of M1 muscarinic acetylcholine or P2Y1 purinergic receptors triggered the PKC- and CaMKII-dependent internalization of mGluR1a. In co-immunoprecipitation studies, both glutamate and carbachol increased the association of GRK2 with mGluR1a. Co-addition of the protein kinase C (PKC) inhibitor GF109203X and the Ca(2+) calmodulin-dependent kinase II (CaMKII) inhibitor KN-93 blocked the ability of glutamate and carbachol to increase the association of GRK2 with mGluR1a. Glutamate also increased the association of GRK2 with mGluR1b, whereas carbachol did not. However, unlike mGluR1a, glutamate-stimulated association of GRK2 with mGluR1b was not reduced by PKC/CaMKII inhibition. Pretreatment of cells expressing mGluR1a or mGluR1b with carbachol rapidly desensitized subsequent glutamate-stimulated inositol phosphate accumulation. The carbachol-induced heterologous desensitization and internalization of mGluR1a was blocked by LY367385, an mGluR1a antagonist with inverse agonist activity. Furthermore, LY367385 blocked the ability of carbachol to increase the association of GRK2 with mGluR1a. On the other hand, LY367385 had no effect on the carbachol-induced desensitization and internalization of the nonconstitutively active mGluR1b splice variant. These results demonstrate that the internalization of mGluR1a, triggered homologously by glutamate or heterologously by carbachol, is PKC/CaMKII-, GRK2-, arrestin-, and clathrin-dependent and that PKC/CaMKII activation appears to be necessary for GRK2 to associate with mGluR1a. Furthermore, the heterologous desensitization of mGluR1a is dependent upon the splice variant being in an active conformation.

Arrestins↗

PubMed2004

PKC-interacting proteins: from function to pharmacology.

Protein kinase C (PKC) is a ubiquitously expressed family of kinases that have key roles in regulating multiple cellular activities. The activity of this family is controlled tightly by several molecular mechanisms, including interaction with binding-partner proteins. These PKC-interacting proteins (C-KIPs) confer specificity for individual PKC isoforms by regulating the activity and cellular localization of PKC isoforms and, subsequently, the ability of these isoforms to specifically regulate cellular functional events. Although many C-KIPs have been identified by genome and proteome-mining approaches, it is important to address the specificity and function of the interactions in greater detail because they might form novel drug targets. In this article, we review recent work on C-KIPs and the implications for pharmacological and therapeutic development.

Animals↗

PubMed2004

Agonist-independent internalization of metabotropic glutamate receptor 1a is arrestin- and clathrin-dependent and is suppressed by receptor inverse agonists.

Three group I mGluR antagonists CPCCOEt, LY367385 and BAY36-7620, were analyzed for their effect on cell surface expression of metabotropic glutamate receptor 1a and 1b. All three antagonists inhibited glutamate-induced internalization of mGluR1a and mGluR1b. However, when added alone, either LY367385 or BAY36-7620 increased the cell surface expression of mGluR1a but not mGluR1b. Both LY367385 and BAY36-7620 displayed inverse agonist activity as judged by their ability to inhibit basal inositol phosphate accumulation in cells expressing the constitutively active mGluR1a. Interestingly, mGluR1a but not mGluR1b was constitutively internalized in HEK293 cells and both LY367385 and BAY36-7620 inhibited the constitutive internalization of this splice variant. Furthermore, coexpression of dominant negative mutant constructs of arrestin-2 [arrestin-2-(319-418)] or Eps15 [Eps15(E Delta 95-295)] increased cell surface expression of mGluR1a and blocked constitutive receptor internalization. In the presence of these dominant negative mutants, incubation of cells with LY367385 and BAY36-7620 produced no further increase in cell surface expression of mGluR1a. Taken together, these results suggest that the constitutive activity of mGluR1a triggers the internalization of the receptor through an arrestin- and clathrin-dependent pathway, and that inverse agonists increase the cell surface expression of mGluR1a by promoting an inactive form of mGluR1a, which does not undergo constitutive internalization.

Animals↗

PubMed2004

Activation of cyclic AMP-dependent protein kinase inhibits the desensitization and internalization of metabotropic glutamate receptors 1a and 1b.

In this study, we characterized the effects of activation of cyclic AMP-dependent protein kinase (PKA) on the internalization and functional coupling of the metabotropic glutamate receptor (mGluR1) splice variants mGluR1a and mGluR1b. Using an enzyme-linked immunosorbent assay technique to assess receptor internalization, we found that the glutamate-induced internalization of mGluR1a or mGluR1b transiently expressed in human embryonic kidney (HEK) 293 cells was inhibited by coactivation of endogenous beta2-adrenoceptors with isoprenaline or by direct activation of adenylyl cyclase with forskolin. The PKA inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H89) blocked the effects of both isoprenaline and forskolin. The heterologous internalization of the mGluR1 splice variants triggered by carbachol was also inhibited by isoprenaline and forskolin in a PKA-sensitive fashion, whereas the constitutive (agonist-independent) internalization of mGluR1a was inhibited only modestly by PKA activation. Using inositol phosphate (IP) accumulation in cells prelabeled with [3H]inositol to assess receptor coupling, PKA activation increased basal IP accumulation in mGluR1a receptor-expressing cells and also increased glutamate-stimulated IP accumulation in both mGluR1a- and mGluR1b-expressing cells, but only at short times of glutamate addition. Furthermore, PKA activation completely blocked the carbachol-induced heterologous desensitization of glutamate-stimulated IP accumulation in both mGluR1a- and mGluR1b-expressing cells. In coimmunoprecipitation experiments, the ability of glutamate to increase association of GRK2 and arrestin-2 with mGluR1a and mGluR1b was inhibited by PKA activation with forskolin. Together, these results indicate that PKA activation inhibits the agonist-induced internalization and desensitization of mGluR1a and mGluR1b, probably by reducing their interaction with GRK2 and nonvisual arrestins.

Arrestins↗

PubMed2003

The antidepressant fluoxetine blocks the human small conductance calcium-activated potassium channels SK1, SK2 and SK3.

The effects of fluoxetine (Prozac) on the activity of human small-conductance calcium-activated potassium (SK) channels were investigated utilizing a functional fluorescence assay with bis-(1,3-dibutylbarbituric acid)trimethine oxonol (DiBAC(4)(3)). Fluoxetine blocked SK channels stably expressed in HEK 293 cells in a concentration-dependent manner displaying half-maximal inhibitory concentrations (IC(50)) of 9 microM for hSK1, 7 microM for hSK2 and 20 microM for hSK3. The block of hSK3 channels was confirmed by whole cell patch-clamp recordings of the recombinant cells and human TE 671 cells. Fluoxetine also inhibited [(125)I]apamin binding in a concentration-dependent manner displaying IC(50) values of 63 microM for hSK1, 148 microM for hSK2 and 295 microM for hSK3. These results provide new information concerning the mechanism of therapeutic and/or side effects of one of the most widely used antidepressant drugs.

Antidepressive Agents, Second-Generation↗

PubMed2003

Agonist-induced internalization of metabotropic glutamate receptor 1A: structural determinants for protein kinase C- and G protein-coupled receptor kinase-mediated internalization.

To investigate the role of the intracellular C-terminal tail of the rat metabotropic glutamate receptor 1a (mGlu1a) in receptor regulation, we constructed three C-terminal tail deletion mutants (Arg847stop, DM-I; Arg868stop, DM-II; Val893stop, DM-III). Quantification of glutamate-induced internalization provided by ELISA indicated that DM-III, like the wild-type mGlu1a, underwent rapid internalization whilst internalization of DM-I and DM-II was impaired. The selective inhibitor of protein kinase C (PKC), GF109203X, which significantly reduced glutamate-induced mGlu1a internalization, had no effect on the internalization of DM-I, DM-II, or DM-III. In addition activation by carbachol of endogenously expressed M1 muscarinic acetylcholine receptors, which induces PKC- and Ca2+-calmodulin-dependent protein kinase II-dependent internalization of mGlu1a, produced negligible internalization of the deletion mutants. Co-expression of a dominant negative mutant form of G protein-coupled receptor kinase 2 (DNM-GRK2; Lys220Arg) significantly attenuated glutamate-induced internalization of mGlu1a and DM-III, whilst internalization of DM-I and DM-II was not significantly affected. The glutamate-induced internalization of mGlu1a and DM-III, but not of DM-I or DM-II, was inhibited by expression of DNM-arrestin [arrestin-2(319-418)]. In addition glutamate-induced rapid translocation of arrestin-2-Green Fluorescent Protein (arr-2-GFP) from cytosol to membrane was only observed in cells expressing mGlu1a or DM-III. Functionally, in cells expressing mGlu1a, glutamate-stimulated inositol phosphate accumulation was increased in the presence of PKC inhibition, but so too was that in cells expressing DM-II and DM-III. Together these results indicate that different PKC mechanisms regulate the desensitization and internalization of mGlu1a. Furthermore, PKC regulation of mGlu1a internalization requires the distal C terminus of the receptor (Ser894-Leu1199), whilst in contrast glutamate-stimulated GRK- and arrestin-dependent regulation of this receptor depends on a region of 25 amino acids (Ser869-Val893) in the proximal C-terminal tail.

Amino Acid Sequence↗

PubMed2002

Metabotropic glutamate receptor 1 internalization induced by muscarinic acetylcholine receptor activation: differential dependency of internalization of splice variants on nonvisual arrestins.

In this study, we characterized the glutamate- or second-messenger kinase-dependent internalization of the rat metabotropic glutamate receptor 1 (mGluR1) splice variants 1a, 1b, and 1c, and assessed the arrestin and dynamin dependence of these processes. To facilitate this we inserted a hemagglutinin epitope tag in the extracellular N-terminal domain of the splice variants. Quantification of glutamate-induced mGluR1 splice variant internalization provided by enzyme-linked immunosorbent assay and confirmed by immunofluorescent microscopy indicated that each splice variant underwent rapid internalization, which was strongly inhibited by coexpression of dominant-negative mutant (DNM) arrestin or dynamin. In addition glutamate-induced rapid translocation of arrestin-2-green fluorescent protein (GFP) or arrestin-3-GFP from cytosol to membrane was observed in cells expressing mGluR1 splice variants. Glutamate-induced internalization of mGluR1a and mGluR1c was partially blocked by a selective inhibitor of protein kinase C (PKC), 2-[1-(3-dimethylamino-propyl)indol-3-yl]-3-(1H-indol-3-yl)maleimide (GF 109203X), whereas mGluR1b internalization was not significantly affected by this inhibitor. Similarly, inositol phosphate production after glutamate-induced activation of mGluR1a and mGluR1c was increased after PKC inhibition, whereas glutamate-induced mGluR1b stimulation was unaffected. Activation by carbachol of endogenously expressed M(1) muscarinic receptors in human embryonic kidney 293 cells, induced the internalization of mGluR1 splice variants, which was partially blocked by pretreatment with inhibitors of either PKC or Ca(2+) calmodulin-dependent kinase II (CaMKII). Expression of DNM-arrestin with mGluR1a or 1c strongly inhibited carbachol-induced internalization. However, coexpression of DNM-arrestin with mGluR1b was less effective in reducing carbachol-induced receptor internalization. In addition, arrestin-2-GFP or arrestin-3-GFP underwent significant carbachol-induced translocation from cytosol to membrane in cells coexpressing mGluR1a or 1c but not in cells coexpressing mGluR1b. This study demonstrates that the internalization of mGluR1 splice variants is subject to PKC and CaMKII regulation. In addition, regulation by these kinases confers differential arrestin dependence.

Alternative Splicing↗