Src Homology Domains of Phospholipase C γ1 Inhibit Nerve Growth Factor-Induced Differentiation of PC12 Cells

Abstract: Phospholipase C γ1 (PLC-γ1) is phosphorylated on treatment of cells with nerve growth factor (NGF). To assess the role of PLC-γ1 in mediating the neuronal differentiation induced by NGF treatment, we established PC12 cells that overexpress whole PLC-γ1 (PLC-γ1PC12), the SH2-SH2-SH3 domain (PLC-γ1SH223PC12), SH2-SH2-deleted mutants (PLC-γ1ΔSH22PC12), and SH3-deleted mutants (PLC-γ1ΔSH3PC12). Overexpressed whole PLC-γ1 or the SH2-SH2-SH3 domain of PLC-γ1 stimulated cell growth and inhibited NGF-induced neurite outgrowth of PC12 cells. However, cells expressing PLC-γ1 lacking the SH2-SH2 domain or the SH3 domain had no effect on NGF-induced neuronal differentiation. Overexpression of intact PLC-γ1 resulted in a threefold increase in total inositol phosphate accumulation on treatment with NGF. However, overexpression of the SH2-SH2-SH3 domain of PLC-γ1 did not alter total inositol phosphate accumulation. To investigate whether the SH2-SH2-SH3 domain of PLC-γ1 can mediate the NGF-induced signal, tyrosine phosphorylation of the SH2-SH2-SH3 domain of PLC-γ1 on NGF treatment was examined. The SH2-SH2-SH3 domain of PLC-γ1 as well as intact PLC-γ1 could be tyrosine-phosphorylated on NGF treatment. These results indicate that the overexpressed SH2-SH2-SH3 domain of PLC-γ1 can block the differentiation of PC12 cells induced by NGF and that the inhibition appears not to be related to the lipase activity of PLC-γ1 but to the SH2-SH2-SH3 domain of PLC-γ1.     

Transient Ischemia Differentially Increases Tyrosine Phosphorylation of NMDA Receptor Subunits 2A and 2B

Abstract: Activation of the N -methyl- d -aspartate (NMDA) receptor has been implicated in the events leading to ischemia-induced neuronal cell death. Recent studies have indicated that the properties of the NMDA receptor channel may be regulated by tyrosine phosphorylation. We have therefore examined the effects of transient cerebral ischemia on the tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B in different regions of the rat brain. Transient (15 min) global ischemia was produced by the four-vessel occlusion procedure. The tyrosine phosphorylation of NR2A and NR2B subunits was examined by immunoprecipitation with anti-tyrosine phosphate antibodies followed by immunoblotting with antibodies specific for NR2A or NR2B, and by immunoprecipitation with subunit-specific antibodies followed by immunoblotting with anti-phosphotyrosine antibodies. Transient ischemia followed by reperfusion induced large (23–29-fold relative to sham-operated controls), rapid (within 15 min of reperfusion), and sustained (for at least 24 h) increases in the tyrosine phosphorylation of NR2A and smaller increases in that of NR2B in the hippocampus. Ischemia-induced tyrosine phosphorylation of NR2 subunits in the hippocampus was higher than that of cortical and striatal NR2 subunits. The enhanced tyrosine phosphorylation of NR2A or NR2B may contribute to alterations in NMDA receptor function or in signaling pathways in the postischemic brain and may be related to pathogenic events leading to neuronal death.     

Regulation of Phospholipase Cγ in the Mesolimbic Dopamine System by Chronic Morphine Administration

Neurotrophic signaling pathways have been implicated in the maintenance of the mesolimbic dopamine system, as well as in changes in this system induced by chronic morphine exposure. We found that many of these signaling pathway proteins are expressed at appreciable levels within the ventral tegmental area (VTA) and related regions, although with substantial regional variation. Moreover, phospholipase Cgamma1 (PLCgamma1) was significantly and specifically up-regulated within the VTA by 30% following chronic exposure to morphine. PLCgamma1 mRNA expression is enriched in dopaminergic neurons within the VTA; however, the up-regulation of PLCgamma1 in this region was not seen at the mRNA level. In contrast to PLCgamma1, insulin receptor substrate (IRS)-2, a protein involved in phosphatidylinositol 3-kinase signaling, and another putative IRS-like protein were significantly down-regulated within the VTA by 49 and 45%, respectively. Levels of several proteins within the Ras-ERK pathway were not altered. Regulation of neurotrophic factor signaling proteins may play a role in morphine-induced plasticity within the mesolimbic dopamine system.     

An Examination of the Involvement of Phospholipases A2 and C in the α-Adrenergic and γ-Aminobutyric Acid Receptor Modulation of Cyclic AMP Accumulation in Rat Brain Slices

Experiments were undertaken to define the role of two calcium-associated enzyme systems in modulating transmitter-stimulated production of cyclic nucleotides in rat brain. Cyclic AMP (cAMP) accumulation was examined in cerebral cortical slices using a prelabeling technique. The enhancement of isoproterenol-stimulated cAMP production by alpha-adrenergic and gamma-aminobutyric acid-B (GABAB) agonists was reduced by exposing the tissue to EGTA, a chelator of divalent cations, or quinacrine, a nonselective inhibitor of phospholipase A2. Likewise, chronic (2 weeks) administration of corticosterone decreased the alpha-adrenergic and GABAB receptor modulation of second messenger production. Neither cyclooxygenase nor lipoxygenase inhibitors selectively influenced the facilitating response of alpha-adrenergic and GABAB agonists. Other experiments revealed that although norepinephrine and 6-fluoronorepinephrine stimulated inositol phosphate (IP) production in cerebral cortical slices with potencies equal to those displayed in the cyclic nucleotide assay, selective alpha 1-adrenergic agonists were less efficacious on IP formation and were without effect in the cAMP assay. Conversely, a selective alpha 2-adrenergic receptor agonist facilitated the cAMP response to a beta-adrenergic agonist without affecting IP formation. The rank orders of potency of a series of alpha-adrenergic antagonists suggest that IP accumulation is mediated solely by alpha 1-adrenergic receptors, whereas the augmentation of cAMP accumulation is regulated by a mixed population of alpha-adrenergic sites. The results suggest that the alpha-adrenergic and GABAB receptor-mediated enhancement of isoproterenol-stimulated cAMP formation appears to be more closely associated with phospholipase A2 than phospholipase C and may be mediated by arachidonate or some other fatty acid.     

Short and Long Form γ2 Subunits of the GABAA/Benzodiazepine Receptors

Abstract: Three novel antisera to the γ₂ subunit of the γ-aminobutyric acid_A (GABA_A) receptor/benzodiazepine receptor (GABA_AR/BZDR) complex have been made. Anti-γ_2S and anti-γ_2L are specific antibodies to synthetic peptides that recognize the γ_2S (short) and γ_2L (long) forms, respectively, of the γ₂ subunit. An antibody (anti-γ₂IL2) to staphylococcal protein A fusion protein of the large intracellular loop (γ₂IL) located between the putative transmembrane segments M3 and M4 of γ_2S recognizes both γ_2S and γ_2L subunits. The antibodies immunoprecipitated both the solubilized and affinity-purified GABA_AR/BZDR from rat and bovine brain. Immunoblots with membranes from rat brain cerebral cortex as well as with affinity-purified receptor from bovine cortex show that anti-γ_2S and anti-γ_2L recognize peptides of 45,000 and 47,000 M_r, respectively. Immunoprecipitation experiments indicate that γ_2S is more prevalent in hippocampus, whereas γ_2L is more abundant in cerebellum. Intermediate values for each form are found in the cerebral cortex. The results suggest that in the rat brain there is a considerable amount of colocalization of γ_2S and γ_2L in the same receptor complex. In the cerebral cortex, 15% of the BZDRs contain both γ_2S and γ_2L subunits and 41–48% of the γ_2L subunit coexists with γ_2S in the same receptor complex. In cerebellum, in 27% of the clonazepam-sensitive and 39% of the clonazepam-insensitive BZDRs the γ_2S and γ_2L coexist in the same receptor complex. The latter are presumably localized in granule cells and also contain α₆. In addition, almost all (93%) the clonazepam-insensitive BZDRs that contain γ_2L also contain a γ_2S subunit in the same receptor complex. The most likely interpretation of the results is that there is an important population of granule cell receptors that contain α₆, γ_2S, and γ_2L coexisting in the same receptor complex. Nevertheless, 31% of the cerebellar receptors that contain α₆ subunit(s) have neither γ_2S nor γ_2L subunits. There are also species differences with respect to the relative abundance of γ_2S and γ_2L. These results might be relevant for understanding the molecular mechanisms underlying some of the GABA_AR/BZDR-mediated effects of ethanol intoxication involving cerebellar granule cells.     

Calcium/Calmodulin-Dependent Protein Kinase II Is Associated with NR2A/B Subunits of NMDA Receptor in Postsynaptic Densities

Abstract: NMDA receptors and Ca²⁺/calmodulin-dependent kinase II (CaMKII) have been reported to be highly concentrated in the postsynaptic density (PSD). Although the possibility that CaMKII in PSD might be associated with specific proteins has been put forward, the protein or proteins determining the targeting of the kinase in PSD have not yet been identified. Here we report that CaMKII binds to NR2A and NR2B subunits of NMDA receptors in PSD isolated from cortex and hippocampus. The association of NMDA receptor subunits and CaMKII was assessed by immunoprecipitating PSD proteins with antibodies specific for NR2A/B and CaMKII: CaMKII coprecipitated with NR2A/B and NR1 but not with other glutamate ionotropic receptor subunits, such as GluR1 and GluR2-3. A direct association between CaMKII and NR2A/B subunits was further confirmed by overlay experiments using either ³²P-autophosphorylated CaMKII or ³²P-NR2A/B and by evaluating the formation of a CaMKII-NR2A/B complex by means of the cross-linker disuccimidyl suberate. These data demonstrate an association between the NMDA receptor complex and CaMKII in the postsynaptic compartment, suggesting that this colocalization may be relevant for synaptic plasticity.     

Transient ischemia enhances tyrosine phosphorylation and binding of the NMDA receptor to the Src homology 2 domain of phosphatidylinositol 3-kinase in the rat hippocampus

Tyrosine phosphorylation of the NMDA receptor has been implicated in the regulation of the receptor channel. We investigated the effects of transient (15 min) global ischemia on tyrosine phosphorylation of NMDA receptor subunits NR2A and NR2B, and the interaction of NR2 subunits with the SH2 domain of phosphatidylinositol 3-kinase (PI3-kinase) in vulnerable CA1 and resistant CA3/dentate gyrus of the hippocampus. Transient ischemia induced a marked increase in the tyrosine phosphorylation of NR2A in both regions. The tyrosine phosphorylation of NR2B in CA3/dentate gyrus after transient ischemia was sustained and greater than that in CA1. PI3-kinase p85 was co-precipitated with NR2B after transient global ischemia. The SH2 domain of the p85 subunit of PI3-kinase bound to NR2B, but not to NR2A. Binding to NR2B was increased following ischemia and the increase in binding in CA3/dentate gyrus (4.5-fold relative to sham) was greater than in CA1 (1.7-fold relative to sham) at 10 min of reperfusion. Prior incubation of proteins with an exogenous protein tyrosine phosphatase or with a phosphorylated peptide (pYAHM) prevented binding. The results suggest that sustained increases in tyrosine phosphorylation and increased interaction of NR2B with the SH2 domain of PI3-kinase may contribute to altered signal transduction in the CA3/dentate gyrus after transient ischemia.     

Generation of Two Forms of the γ-Aminobutyric AcidA Receptor γ-2-Subunit in Mice by Alternative Splicing

gamma-Aminobutyric acidA (GABAA) receptors are multisubunit ligand-gated ion channels which mediate neuronal inhibition by GABA and are composed of at least four subunit types (alpha, beta, gamma, and delta). The gamma 2-subunit appears to be essential for benzodiazepine modulation of GABAA receptor function. In cloning murine gamma 2-subunits, we isolated cDNAs encoding forms of the subunit that differ by the insertion of eight amino acids. LLRMFSFK, in the major intracellular loop between proposed transmembrane domains M3 and M4. The two forms of the gamma 2-subunit are generated by alternative splicing, as demonstrated by cloning and partial sequencing of the corresponding gene. The eight-amino-acid insertion encodes a potential consensus serine phosphorylation site for protein kinase C. These results suggest a novel mechanism for the regulation of the GABAA receptor by protein phosphorylation.     

Molecular Dissection of Native Mammalian Forebrain NMDA Receptors Containing the NR1 C2 Exon: Direct Demonstration of NMDA Receptors Comprising NR1, NR2A, and NR2B Subunits Within the Same Complex

Abstract: The subunit compositions of the NR1 C2 exon-containing N -methyl- d -aspartate (NMDA) receptors of adult mammalian forebrain were determined by using a combination of immunoaffinity chromatography and immunoprecipitation studies with NMDA receptor subunit-specific antibodies. NMDA receptors were solubilised by sodium deoxycholate, pH 9, and purified by anti-NR1 C2 antibody affinity chromatography. The purified receptor subpopulation showed immunoreactivity with anti-NR1 C2, anti-NR1 N1, anti-NR1 C2', anti-NR2A, and anti-NR2B NMDA receptor antibodies. The NR1 C2-receptor subpopulation was subjected to immunoprecipitation using anti-NR2B antibodies and the resultant immune pellets analysed by immunoblotting where anti-NR1 C2, anti-NR1 C2', anti-NR2A, and anti-NR2B immunoreactivities were all found. Quantification of the immunoblots showed that 46% of the NR1 C2 immunoreactivity was associated with the NR2B subunit. Of this, 87% (i.e., 40% of total) were NR1 C2/NR2B receptors and 13% (6% of total) were NR1 C2/NR2A/NR2B, thus identifying the triple combination as a minor receptor subset. These results demonstrate directly, for the first time, the coexistence of the NR2A and NR2B subunits in native NMDA receptors. They show the coexistence of two splice forms of the NR1 subunit, i.e., NR1 C2 and NR1 C2', in native receptors and, in addition, they imply an NMDA receptor subpopulation containing four types of NMDA receptor subunit, NR1 C2, NR1 C2', NR2A, and NR2B, which, in accord with molecular size determinations, predicts that the NMDA receptor is at least tetrameric. These results are the first quantitative study of NMDA receptor subtypes and demonstrate molecular heterogeneity for both the NR1 and the NR2 subunits in native forebrain NMDA receptors.     

Binding of SH2 containing proteins to the insulin receptor: A new way for modulating insulin signalling

Prior studies have established a role in insulin action for the tyrosine phosphorylation of substrates and their subsequent complexing with SH2 containing proteins. More recently, SH2 proteins have been identified which can tightly bind to the tyrosine phosphorylated insulin receptor. The major protein identified so far (called Grb-IR or Grb10) of this type appears to be present in at least 3 isoforms, varying in the presence of a pleckstrin homology domain and in the sequence of its amino terminus. The binding of this protein to the insulin receptor appears to inhibit signalling by the receptor. The present review will discuss the current knowledge of the structure and function of this protein.     

Protein kinase C activation induces tyrosine phosphorylation of the NR2A and NR2B subunits of the NMDA receptor

The N-methyl-D-aspartate receptor (NMDAR) is an ionotropic glutamate receptor, which plays crucial roles in synaptic plasticity and development. We have recently shown that potentiation of NMDA receptor function by protein kinase C (PKC) appears to be mediated via activation of non-receptor tyrosine kinases. The aim of this study was to test whether this effect could be mediated by direct tyrosine phosphorylation of the NR2A or NR2B subunits of the receptor. Following treatment of rat hippocampal CA1 mini-slices with 500 nM phorbol 12-myristate 13-acetate (PMA) for 15 min, samples were homogenized, immunoprecipitated with anti-NR2A or NR2B antibodies and the resulting pellets subjected to Western blotting with antiphosphotyrosine antibody. An increase in tyrosine phosphorylation of both NR2A (76 +/- 11% above control) and NR2B (41 +/- 11%) was observed. This increase was blocked by pretreatment with the selective PKC inhibitor chelerythrine, with the tyrosine kinase inhibitor Lavendustin A or with the Src family tyrosine kinase inhibitor PP2. PMA treatment also produced an increase in the phosphorylation of serine 890 on the NR1 subunit, a known PKC site, at 5 min with phosphorylation returning to near basal levels by 10 min while tyrosine phosphorylation of NR2A and NR2B was sustained for up to 15 min. These results suggest that the modulation of NMDA receptor function seen with PKC activation may be the result of tyrosine phosphorylation of NR2A and/or NR2B.     

Regulation of EGF-induced phospholipase C-gamma1 translocation and activation by its SH2 and PH domains

Translocation of phospholipase C-γ1 is essential for its function in response to growth factors. However, in spite of recent progress, the phospholipase C-γ1 translocation pattern and the molecular mechanism of the translocation are far from fully understood. Contradictory results were reported as to which domain, PH or SH2, controls the epidermal growth factor-induced translocation of phospholipase C-γ1. In this communication, we studied epidermal growth factor-induced translocation of phospholipase C-γ1 by using comprehensive approaches including biochemistry, indirect fluorescence and live fluorescence imaging. We provided original evidence demonstrating that: (i) endogenous phospholipase C-γ1, similar to YFP-tagged phospholipase C-γ1, translocated to endosomes following its initial translocation from cytosol to the plasma membrane in response to epidermal growth factor; (ii) phospholipase C-γ1 remained phosphorylated in endosomes, but phospholipase C-γ1 activity is not required for its translocation, which suggests a signaling role for phospholipase C-γ1 in endosomes; (iii) the PH domain was not required for the initial translocation of phospholipase C-γ1 from cytosol to the plasma membrane, but it stabilizes phospholipase C-γ1 in the membrane at a later time; (iv) the function of the phospholipase C-γ1 PH domain in stabilizing phospholipase C-γ1 membrane association is very important in maintaining the activity of phospholipase C-γ1; and (v) the role of the PH domain in phospholipase C-γ1 membrane association and activation is dependent on PI3K activity. We conclude that the phospholipase C-γ1 SH2 and PH domains coordinate to determine epidermal growth factor-induced translocation and activation of phospholipase C-γ1.     

Immunological Identification of Multiple α-Like Subunits of the γ-Aminobutyric AcidA Receptor Complex Purified from Neonatal Rat Cortex

Antibodies were prepared against a synthetic peptide corresponding to amino acid sequences 174-203 of the bovine gamma-aminobutyric acidA (GABAA) receptor alpha 1-subunit. The antibodies recognized this synthetic alpha 1-peptide, but failed to react with the homologous peptide sequence, 170-199, of the bovine beta 1-subunit. On Western blots, anti-alpha 1-subunit antibody recognized a 50-kilodalton (kDa) protein in affinity-purified receptor preparations from adult rat cortex and cerebellum. In receptor purified from neonatal cortex, the anti-alpha 1-antibody reacted with 50-kDa, 53-54-kDa, and 59-kDa proteins. After digestion with endoglycosidase F, these three protein bands retained differing electrophoretic mobilities. The 50-kDa and 59-kDa subunits of affinity-purified neonatal receptor, which were photoaffinity-labeled with [3H]flunitrazepam, were immunoprecipitated to different extents by alpha-subunit antibody. These data suggest the existence in GABAA receptor from neonatal cortex of three proteins (50 kDa, 53 kDa, and 59 kDa) which have immunological homology to alpha 1-subunit of bovine GABAA receptor. The presence of an alpha- and a beta-like subunit with similar mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis may account for the relatively high concentration of protein in the 53-54-kDa band which has been observed in receptor purified from neonatal cortex. The presence of multiple alpha-like subunits may be related to the presence of a relatively high concentration of type II GABA receptor in this tissue.     

Antibodies to the Human γ2 Subunit of the γ-Aminobutyric AcidA/Benzodiazepine Receptor

Abstract: A γ-aminobutyric acid_A (GABA_A) receptor (GABA_AR) γ₂ subunit (short form) was cloned from an adult human cerebral cortex cDNA library in bacteriophage λgt11. The 261-bp intracellular loop (IL) located between M3 and M4 was amplified using the polymerase chain reaction and inserted into the expression vectors λgt11 and pGEX-3X. Both γ-galactosidase (LacZ) and glutathione-S-transferase (GST) fusion proteins containing the γ₂IL were purified, and a rabbit antibody to the LacZ–γ₂IL was made. The antibody reacted with the γ₂IL of both LacZ and GST fusion proteins and immunoprecipitated the GABA_AR/ benzodiazepine receptor (GABA_AR/BZDR) from bovine and rat brain. The antibody reacted in affinity-purified GABA_AR/BZDR immunoblots with a wide peptide band of 44,000–49,000 M_r. Immunoprecipitation studies with the anti-γ₂IL antibody suggest that in the cerebral cortex, 87% of the GABA_ARs with high affinity for benzodiazepines and 70% of the GABA_ARs with high affinity for muscimol contain at least a γ subunit, probably a γ₂. These results indicate that there are [³H]muscimol binding GABA_ARs that do not bind [³H]flunitrazepam with high affinity. Immunoprecipitations with this and other anti-GABA_AR/BZDR antibodies indicate that the most abundant combination of GABA_AR subunits in the cerebral cortex involves α₁, γ₂ (or other γ), and β₂ and/or β₃ subunits. These subunits coexist in >60% of the GABA_AR/BZDRs in the cerebral cortex. The results also show that a considerable proportion (20–25%) of the cerebellar GABA_AR/BZDRs is clonazepam insensitive. At least 74% of these cerebellar receptors, which likely contain α₆, also contain γ₂ (or other γ) subunit(s). The α₁ and β₂ or β₃ subunits are also frequently associated with γ₂ (or other γ) and α₆ in these cerebellar receptors.     

Characterization of NMDA Receptor Subunit-Specific Antibodies: Distribution of NR2A and NR2B Receptor Subunits in Rat Brain and Ontogenic Profile in the Cerebellum

Abstract: Selective antisera for NMDA receptor subunits NR2A and NR2B have been developed. Each antiserum identifies a single band on an immunoblot at ∼175 kDa that appears to be the appropriate subunit of the NMDA receptor. Using these antisera the relative densities of the subunits in eight areas of adult rat brain have been determined. The NR2A subunit was found to be at its highest level in hippocampus and cerebral cortex, to be at intermediate levels in striatum, olfactory tubercle, midbrain, olfactory bulb, and cerebellum, and to be at lowest levels in the pons-medulla. The NR2B subunit was found to be expressed at its highest levels in the olfactory tubercle, hippocampus, olfactory bulb, and cerebral cortex. Intermediate levels were expressed in striatum and midbrain, and low levels were detected in the pons-medulla. No signal for NR2B was found in the cerebellum. These regional distributions were compared with that for [³H]MK-801 binding sites. It was found that although the distribution of the NR2A subunit corresponds well with radioligand binding, the distribution of the NR2B subunit does not. The ontogenic profiles of NR2A and NR2B subunits in the rat cerebellum were also determined. Just following birth [postnatal day (P) 2] NR2A subunits are undetectable, whereas NR2B subunits are expressed at amounts easily measurable. Beginning at about P12 the levels of NR2A rise rapidly to reach adult levels by P22. At the same time (P12), levels of NR2B protein begin to decline rapidly to reach undetectable levels by 22 days after birth. The results suggest that NMDA receptors are likely to be composed of different subunits in different parts of the brain and that even in the same tissue the receptors are likely to show different properties at various times during development due to alterations in the subunit composition of the receptor.     

Interaction of Axl receptor tyrosine kinase with C1-TEN,a novel C1 domain-containing protein with homology to tensin

Axl receptor tyrosine kinase is implicated in several malignancies and is the receptor for the vitamin K-dependent growth factor Gas6. From a yeast two-hybrid screen of protein-protein interactions with the Axl cytoplasmic domain, we detected a previously uncharacterised SH2 domain-containing protein. We cloned two novel splice variants of this protein that give rise to 1409- and 1419-amino acid proteins, differing only in their N-terminal residues and yielding a 150-kDa protein product by in vitro translation. The Axl-interacting C-terminus contains a tandem SH2 and PTB domain combination homologous to the focal adhesion protein tensin. We detected interaction of Axl with both domains in mammalian cells by co-immunoprecipitation and two-hybrid analyses. In addition, the protein possesses an N-terminal putative phorbol ester-binding C1 domain as well as a central tyrosine phosphatase motif. Thus, we have named the protein C1 domain-containing phosphatase and TENsin homologue (C1-TEN). Northern blot analysis of C1-TEN in human tissues revealed highest expression in heart, kidney, and liver. In summary, we have identified a novel multi-domain intracellular protein that interacts with Axl and which may furthermore be involved in other signal transduction pathways.     

HIV-1 Tat binds to SH3 domains: Cellular and viral outcome of Tat/Grb2 interaction

The Src-homology 3 (SH3) domain is one of the most frequent protein recognition modules (PRMs), being represented in signal transduction pathways and in several pathologies such as cancer and AIDS. Grb2 (growth factor receptor-bound protein 2) is an adaptor protein that contains two SH3 domains and is involved in receptor tyrosine kinase (RTK) signal transduction pathways. The HIV-1 transactivator factor Tat is required for viral replication and it has been shown to bind directly or indirectly to several host proteins, deregulating their functions. In this study, we show interaction between the cellular factor Grb2 and the HIV-1 trans-activating protein Tat. The binding is mediated by the proline-rich sequence of Tat and the SH3 domain of Grb2. As the adaptor protein Grb2 participates in a wide variety of signaling pathways, we characterized at least one of the possible downstream effects of the Tat/Grb2 interaction on the well-known IGF-1R/Raf/MAPK cascade. We show that the binding of Tat to Grb2 impairs activation of the Raf/MAPK pathway, while potentiating the PKA/Raf inhibitory pathway. The Tat/Grb2 interaction affects also viral function by inhibiting the Tat-mediated transactivation of HIV-1 LTR and viral replication in infected primary microglia.     

P2Y Receptor Linked to Phospholipase C: Stimulation of Neuro 2A Cells by UTP and ATP and Possible Regulation by Protein Kinase C Subtype ε

Abstract: Incubation of Neuro 2A mouse neuroblastoma cells with UTP and UDP results in a concentration-dependent increase in the accumulation of inositol phosphates with equal potency and maximal effect; ATP, ADP, and 2-methylthioadenosine 5′-triphosphate were much less potent, indicating the expression of P2Y receptor in these cells. The effects of UTP and ATP were not affected by pretreatment of cells with pertussis toxin, indicating that the P2Y receptor in Neuro 2A cells is coupled to pertussis toxin-insensitive G_q protein. Short-term (10 min) treatment of cells with 1 µM 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in the inhibition of the UTP and ATP effects; this inhibitory effect was gradually attenuated with increased length of TPA treatment (1.5–6 h) and was not seen after long-term (24 h) treatment. Western blot analysis showed the expression of protein kinase C (PKC) α, ε, θ, and ζ in Neuro 2A cells. Translocation of PKCα, ε, and θ from the cytosol to the membrane was seen after 10 min or 1.5 h of treatment with TPA. However, partial and complete down-regulation of both membrane PKCα and θ were seen after 3 and 6 h of treatment, respectively. In contrast, the TPA-induced translocation of PKCε was maintained after 3–6 h of treatment, and almost complete down-regulation occurred only after a 24-h treatment. The observed TPA-induced inhibition of UTP- or ATP-stimulated phosphoinositide hydrolysis, therefore, correlated well with the extent of translocation of PKCε. Phosphoinositide hydrolysis induced by AlF₄^?, but not Ca²⁺ ionophores, was inhibited by a 10-min treatment with TPA. This was not seen after a 24-h treatment, indicating that the site of action of PKCε in the P2Y receptor/G_q protein/phospholipase Cβ pathway might be the G_q protein. This is the first study to show the existence of the P2Y receptor in Neuro 2A cells and the possible involvement of neuronal PKCε in the regulation of the receptor-mediated phosphoinositide turnover.     

Effects of C-Terminal Truncation of the Recombinant δ-Opioid Receptor on Phospholipase C and Adenylyl Cyclase Coupling

Abstract: Opioid receptors belong to the superfamily of guanine nucleotide binding (G) protein-coupled receptors. There is now growing evidence in support of a stimulatory coupling of opioid receptors to phospholipase C (PLC), via a pertussis toxin-sensitive G protein, leading to the generation of the second messenger inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃]. We have generated two C-terminal truncation mutants of the δ-opioid receptor lacking the final 15 or 37 amino acids and examined their coupling to PLC and adenylyl cyclase. d -[Pen^2,5]-enkephalin (DPDPE) mediated Ins(1,4,5)P₃ formation and cyclic AMP inhibition was measured in whole cells and assayed using radioreceptor mass assays. DPDPE produced a time- and dose-dependent increase in Ins(1,4,5)P₃ mass formation in Chinese hamster ovary (CHO) cells expressing the δ_wt, δ₁₅, and δ₃₇ receptors. As the C terminus was truncated, the time to maximum stimulation (15 s in CHOδ_wt, 60 s in CHOδ₁₅, and 120 s in CHOδ₃₇) increased and removal of the C terminus resulted in a prompt return to basal Ins(1,4,5)P₃ levels. Whereas the dose-response curves to Ins(1,4,5)P₃ formation and cyclic AMP inhibition remained largely unaffected by C-terminal truncation, there were large differences in the pEC/IC₅₀ values, with cyclic AMP inhibition being the more potent, perhaps indicating G_iα coupling to adenylyl cyclase and G_iβ/γ coupling to PLC. Collectively, these data indicate that the C terminus of the δ-opioid receptor is unimportant in the acute coupling to adenylyl cyclase but may have a role to play in PLC coupling. We hypothesize that an intact C terminus is required to allow normal “strong” coupling of receptor to G_i and that truncation weakens this link as reflected in an increased time to peak. In addition, if the coupling is weak, the acute response to agonist stimulation rapidly uncouples.     

Regulation of Multiple Effectors by the Cloned δ-Opioid Receptor: Stimulation of Phospholipase C and Type II Adenylyl Cyclase

Abstract: The δ-opioid receptor is known to regulate multiple effectors in various tissues. When expressed in human embryonic kidney 293 cells, the cloned δ-opioid receptor inhibited cyclic AMP (cAMP) accumulation in response to the δ-selective agonist [ d -Pen², d -Pen⁵]enkephalin. The inhibitory response of [ d -Pen², d -Pen⁵]enkephalin was dependent on the expression of the δ-opioid receptor and exhibited an EC₅₀ of 1 n M . The receptor showed ligand selectivity and a pharmacological profile that is appropriate for the δ-opioid subtype. The inhibition was blocked by the opiate antagonist naloxone or by pretreatment of the cells with pertussis toxin. Cotransfection of the δ-opioid receptor with type II adenylyl cyclase and an activated mutant of α_s converted the δ-opioid signal from inhibition to stimulation of cAMP accumulation. It is interesting that when transfected into Ltk⁻ fibroblasts, the cloned δ-opioid receptor was able to stimulate the formation of inositol phosphates (EC₅₀ = 8 n M ). This response was sensitive to pertussis toxin. The opioid-mediated formation of inositol phosphates exhibited the same ligand selectivity as seen with the inhibition of cAMP accumulation. The ability of the δ-opioid receptor to couple to G proteins other than G_i was also examined. Cotransfection studies revealed that the δ-opioid receptor can utilize G_z to regulate cAMP accumulation and to stimulate the formation of inositol phosphates.