P2Y2 purinergic and M3 muscarinic acetylcholine receptors activate different phospholipase C-beta isoforms that are uniquely susceptible to protein kinase C-dependent phosphorylation and inactivation

Activation of phospholipase C-beta (PLC-beta) by G protein-coupled receptors typically results in rapid but transient second messenger generation. Although PLC-beta deactivation may contribute to the transient nature of this response, the mechanisms governing PLC-beta deactivation are poorly characterized. We investigated the involvement of protein kinase C (PKC) in the termination of PLC-beta activation induced by endogenous P2Y(2) purinergic receptors and transfected M(3) muscarinic acetylcholine receptors (mAChR) in Chinese hamster ovary cells. Activation of P2Y(2) receptors causes Galpha(q/11) to associate with PLC-beta3, whereas M(3) mAChR activation causes Galpha(q/11) to associate with both PLC-beta1 and PLC-beta3 in these cells. Phosphorylation of PLC-beta3, but not PLC-beta1, is induced by activating either P2Y(2) receptors or M(3) mAChR. We demonstrate that PKC rather than protein kinase A mediates the G protein-coupled receptor-induced phosphorylation of PLC-beta3. The PKC-mediated phosphorylation of PLC-beta3 diminishes the interaction of Galpha(q/11) with PLC-beta3, thereby contributing to the termination PLC-beta3 activity. These findings indicate that the distinct temporal profiles of PLC activation by P2Y(2) receptors and mAChR may arise from the differential activation of PLC-beta1 and PLC-beta3 by the receptors, coupled with a selective PKC-mediated negative feedback mechanism that targets PLC-beta3 but not PLC-beta1.     

Phospholipase C-beta3 mediates the thrombin-induced Ca2+ response in glial cells

Phospholipase C-beta (PLC-beta) hydrolyses phosphatidylinositol 4,5-bisphosphate and generates inositol 1,4,5-trisphosphate in response to activation of various G protein-coupled receptors (GPCRs). Using glial cells from knock-out mice lacking either PLC-beta1 [PLC-beta1 (-/-)] or PLC-beta3 [PLC-beta3 (-/-)], we examined which isotype of PLC-beta participated in the cellular signaling events triggered by thrombin. Generation of inositol phosphates (IPs) was enhanced by thrombin in PLC-beta1 (-/-) cells, but was negligible in PLC-beta3 (-/-) cells. Expression of PLC-beta3 in PLC-beta3 (-/-) cells resulted in an increase in pertussis toxin (PTx)-sensitive IPs in response to thrombin as well as to PAR1-specific peptide, while expression of PLC-beta1 in PLC-beta1 (-/-) cells did not have any effect on IP generation. The thrombin-induced [Ca2+]i increase was delayed and attenuated in PLC-beta3 (-/-) cells, but normal in PLC-beta1 (-/-) cells. Pertussis toxin evoked a delayed [Ca2+]i increase in PLC-beta3 (-/-) cells as well as in PLC-beta1 (-/-) cells. These results suggest that activation of PLC-beta3 by pertussis toxin-sensitive G proteins is responsible for the transient [Ca2+]i increase in response to thrombin, whereas the delayed [Ca2+]i increase may be due to activation of some other PLC, such as PLC-beta4, acting via PTx-insensitive G proteins.     

Membrane signaling and progesterone in female and male osteoblasts. II. Direct involvement of G alpha q/11 coupled to PLC-beta 1 and PLC-beta 3

We have shown that progesterone (10 pM-10 nM) and progesterone covalently bound to bovine serum albumin (P-CMO BSA; 100 pM-1 microM) rapidly increased (within 5 s) the cytosolic free Ca(2+) concentration and inositol 1,4,5 trisphosphate (InsP(3)) formation in confluent female and male rat osteoblasts via a pertussis toxin-insensitive G-protein. The activation of G-proteins coupled to effectors such as phospholipase C (PLC) is an early event in the signal transduction pathway leading to InsP(3) formation. We used antibodies against the various PLC isoforms to show that only PLC-beta1 and PLC-beta 3 were involved in the Ca(2+) mobilization and InsP(3) formation induced by both progestins in female and male osteoblasts, whereas PLC-beta 2, PLC-gamma 1, and PLC-gamma 2 were not. We also used antibodies against the subunits of heterotrimeric G-proteins to show that the activation of PLC-beta 1 and PLC-beta 3 by both progestins involved the G alpha q/11 subunit, which was insensitive to pertussis toxin, whereas G alpha i, G alpha s, and G beta gamma subunits were not. The membrane effects were independent of the concentration of nuclear progesterone receptor, because the concentration of nuclear progesterone receptors was lower in male than in female osteoblasts. These data suggest that progesterone and P-CMO BSA, which does not enter the cell, directly activate G-protein leading to the very rapid formation of second messengers without involving the nuclear receptor.     

Stimulation of phospholipase C-epsilon by the M3 muscarinic acetylcholine receptor mediated by cyclic AMP and the GTPase Rap2B

Stimulation of phospholipase C (PLC) by G(q)-coupled receptors such as the M(3) muscarinic acetylcholine receptor (mAChR) is caused by direct activation of PLC-beta enzymes by Galpha(q) proteins. We have recently shown that G(s)-coupled receptors can stimulate PLC-epsilon, apparently via formation of cyclic AMP and activation of the Ras-related GTPase Rap2B. Here we report that PLC stimulation by the M(3) mAChR expressed in HEK-293 cells also involves, in part, similar mechanisms. M(3) mAChR-mediated PLC stimulation and [Ca(2+)](i) increase were reduced by 2',5'-dideoxyadenosine (dd-Ado), a direct adenylyl cyclase inhibitor. On the other hand, overexpression of Galpha(s) or Epac1, a cyclic AMP-regulated guanine nucleotide exchange factor for Rap GTPases, enhanced M(3) mAChR-mediated PLC stimulation. Inactivation of Ras-related GTPases with clostridial toxins suppressed the M(3) mAChR responses. The inhibitory toxin effects were mimicked by expression of inactive Rap2B, but not of other inactive GTPases (Rac1, Ras, RalA, Rap1A, and Rap2A). Activation of the M(3) mAChR induced GTP loading of Rap2B, an effect strongly enhanced by overexpression of Galpha(s) and inhibited by dd-Ado. Overexpression of PLC-epsilon and PLC-beta1, but not PLC-gamma1 or PLC-delta1, enhanced M(3) mAChR-mediated PLC stimulation and [Ca(2+)](i) increase. In contrast, expression of a catalytically inactive PLC-epsilon mutant reduced PLC stimulation by the M(3) mAChR and abrogated the potentiating effect of Galpha(s). In conclusion, our findings suggest that PLC stimulation by the M(3) mAChR is a composite action of PLC-beta1 stimulation by Galpha(q) and stimulation of PLC-epsilon apparently mediated by G(s)-dependent cyclic AMP formation and subsequent activation of Rap2B.     

Increased expression of Galpha(q/11) and of phospholipase-Cbeta1/4 in differentiated human NT2-N neurons: enhancement of phosphoinositide hydrolysis

The CNS is enriched in phosphoinositide-specific phospholipase C (PLC) and in the G proteins linked to its activation. Although the regional distributions of these signaling components within the brain have been determined, neither their cell type-specific localizations (i.e., neuronal versus glial) nor the functional significance of their high expression has been definitively established. In this study, we have examined the expression of phosphoinositide signaling proteins in human NT2-N cells, a well characterized model system for CNS neurons. Retinoic acid-mediated differentiation of NT2 precursor cells to the neuronal phenotype resulted in five- to 15-fold increases in the expression of PLC-beta1, PLC-beta4, and Galpha(q/11) (the prime G protein activator of these isozymes). In contrast, the expression of PLC-beta3 and PLC-gamma1 was markedly reduced following neuronal differentiation. Similar alterations in cell morphology and in the expression of PLC-beta1, PLC-beta3, and Galpha(q/11) expression were observed when NT2 cells were differentiated with berberine, a compound structurally unrelated to retinoic acid. NT2-N neurons exhibited a significantly higher rate of phosphoinositide hydrolysis than NT2 precursor cells in response to direct activation of either G proteins or PLC. These results indicate that neuronal differentiation of NT2 cells is associated with dramatic changes in the expression of proteins of the phosphoinositide signaling system and that, accordingly, differentiated NT2-N neurons possess an increased ability to hydrolyze inositol lipids.     

Phospholipase C-beta 2 interacts with mitogen-activated protein kinase kinase 3

Phospholipase C (PLC)-beta enzymes (isoenzymes beta 1-beta 4) are activated by G protein subunits, leading to the generation of intracellular messengers which mobilize calcium and activate protein kinase C. It has recently been recognized that these enzymes interact with and are regulated by proteins other than G proteins. Using the yeast two-hybrid technique to screen a leukocyte library we identified mitogen-activated protein kinase kinase 3 (MKK3) as a partner of PLC-beta 2. The interaction was confirmed by co-immunoprecipitation assays which indicated that MKK3 interacts with PLC-beta 2, but not with other PLC-betas. PLC-beta 2 interacted weakly with MKK6, which is related to MKK3, but not with the other MKK3 tested. The region of PLC-beta 2 involved in the interaction with MKK3 was mapped to the C-terminus of PLC-beta 2. p38MAPK also co-immunoprecipitated with PLC-beta 2. The data suggest that PLC-beta 2 serves an unappreciated role assembling components of the p38MAPK signaling module.     

Reassembly of phospholipase C-beta2 from separated domains: analysis of basal and G protein-stimulated activities

Phosphatidylinositol-specific phospholipase C-betas (PLC-betas) are the only PLC isoforms that are regulated by G protein subunits. To further understand the regulation of PLC-beta(2) by G proteins and the functional roles of PLC-beta(2) structural domains, we tested whether the separately expressed amino and carboxyl halves of PLC-beta(2) could associate to form catalytically active enzymes as two polypeptides, and we explored how the complexes thus formed would be regulated by G protein betagamma subunits (Gbetagamma). We expressed cDNA constructs encoding PLC-beta(2) fragments of different lengths in COS-7 cells and demonstrated by coimmunoprecipitation that the coexpressed fragments could assemble and functionally reconstitute an active PLC-beta(2). The pleckstrin homology domain of PLC-beta(2) was required for its targeting to the membrane and for substrate hydrolysis. Reconstituted enzymes that contained the linker region that joins the two catalytic domains were as active or more active than the wild-type PLC-beta(2). When the linker region was removed, basal PLC-beta(2) enzymatic activity was increased further, suggesting that the linker region exerts an inhibitory effect on basal PLC-beta(2) activity. The reconstituted enzymes, like wild-type PLC-beta(2), were activated by Gbetagamma; when the C-terminal region was present in these constructs, they were also activated by Galpha(q). Gbetagamma and Galpha(q) activated these PLC-beta(2) constructs equally in the presence or absence of the linker region. We conclude that the linker region is an inhibitory element in PLC-beta(2) and that Gbetagamma and Galpha(q) do not stimulate PLC-beta(2) through easing the inhibition of enzymatic activity by the linker region.     

Identification of a region at the N-terminus of phospholipase C-beta 3 that interacts with G protein beta gamma subunits

Members of the phospholipase C-beta (PLC-beta) family of proteins are activated either by G alpha or G beta gamma subunits of heterotrimeric G proteins. To define specific regions of PLC-beta 3 that are involved in binding and activation by G beta gamma, a series of fragments of PLC-beta 3 as glutathione-S-transferase (GST) fusion proteins were produced. A fragment encompassing the N-terminal pleckstrin homology (PH) domain and downstream sequence (GST-N) bound to G protein beta 1 gamma 2 in an in vitro binding assay, and binding was inhibited by G protein alpha subunit, G alpha i1. This PLC-beta 3 fragment also inhibited G beta gamma-stimulated PLC-beta activity in a reconstitution system, while having no significant effect on G alpha q-stimulated PLC-beta 3 activity. The N-terminal G beta gamma binding region was delineated further to the first 180 amino acids, and the sequence Asn150-Ser180, just distal to the PH domain, was found to be required for the interaction. Mutation of basic residues 154Arg, 155Lys, 159Lys, and 161Lys to Glu within this region reduced G beta gamma binding affinity and specifically reduced the EC50 for G beta gamma-dependent activation of the mutant enzyme 3-fold. Basal activity and G alpha q-dependent activation of the enzyme were unaffected by the mutations. While these basic residues may not directly mediate the interaction with G beta gamma, the data provide evidence for an N-terminal G beta gamma binding region of PLC-beta 3 that is involved in activation of the enzyme.     

Identification in bovine liver plasma membranes of a Gq-activatable phosphoinositide phospholipase C.

Phosphoinositide phospholipase C (PLC) activity extracted from bovine liver plasma membranes with sodium cholate was stimulated by GTP gamma S-activated G alpha q/G alpha 11, whereas the enzyme from liver cytosol was not. The membrane-associated PLC was subjected to chromatography on heparin-Sepharose, Q Sepharose, and S300HR, enabling the isolation of the G-protein stimulated activity and its resolution from PLC-gamma and PLC-delta. Following gel filtration, two proteins of 150 and 140 kDa were found to correspond to the activatable enzyme. These proteins were identified immunologically as members of the PLC-beta family and were completely resolved by chromatography on TSK Phenyl 5PW. The 150-kDa enzyme was markedly responsive to GTP gamma S-activated alpha-subunits of G alpha q/G alpha 11 or to purified Gq/G11 in the presence of GTP gamma S. The response of this PLC was of much greater magnitude than that of the 140-kDa enzyme. The partially purified 150-kDa enzyme showed specificity for PtdIns(4,5)P2 and PtdIns4P as compared to PtdIns and had an absolute dependence upon Ca2+. These characteristics were similar to those of the brain PLC-beta 1. The immunological and biochemical properties of the 150-kDa membrane-associated enzyme are consistent with its being the PLC-beta isozyme that is involved in receptor-G-protein-mediated generation of inositol 1,4,5-triphosphate in liver.     

PLC-epsilon: a shared effector protein in Ras-, Rho-, and G alpha beta gamma-mediated signaling

The conceptual segregation of G protein-stimulated cell signaling responses into those mediated by heterotrimeric G proteins versus those promoted by small GTPases of the Ras superfamily is no longer vogue. PLC-epsilon, an isozyme of the phospholipase C (PLC) family, has been identified recently and dramatically extends our understanding of the crosstalk that occurs between heterotrimeric and small monomeric GTPases. Like the widely studied PLC-beta isozymes, PLC-epsilon is activated by Gbetagamma released upon activation of heterotrimeric G proteins. However, PLC-epsilon markedly differs from the PLC-beta isozymes in its capacity for activation by Galpha(12/13) - but not Galpha(q) -coupled receptors. PLC-epsilon contains two Ras-associating domains located near the C terminus, and H-Ras regulates PLC-epsilon as a downstream effector. Rho also activates PLC-epsilon, but in a mechanism independent of the C-terminal Ras-associating domains. Therefore, Ca(2+) mobilization and activation of protein kinase C are signaling responses associated with activation of both H-Ras and Rho. A guanine nucleotide exchange domain conserved in the N terminus of PLC-epsilon potentially confers a capacity for activators of this isozyme to cast signals into additional signaling pathways mediated by GTPases of the Ras superfamily. Thus, PLC-epsilon is a multifunctional nexus protein that senses and mediates crosstalk between heterotrimeric and small GTPase signaling pathways.     

Feedback regulation of phospholipase C-beta by protein kinase C

Treatment of a variety of cells and tissues with 12-O-tetradecanoylphorbol-13-acetate (TPA), an activator of protein kinase C (PKC) results in the inhibition of receptor-coupled inositol phospholipid-specific phospholipase C (PLC) activity. To determine whether or not the targets of TPA-activated PKC include one or more isozymes of PLC, studies were carried out with PC12, C6Bu1, and NIH 3T3 cells, which contain at least three PLC isozymes, PLC-beta, PLC-gamma, and PLC-delta. Treatment of the cells with TPA stimulated the phosphorylation of serine residues in PLC-beta, but the phosphorylation state of PLC-gamma and PLC-delta was not changed significantly. Phosphorylation of bovine brain PLC-beta by PKC in vitro resulted in a stoichiometric incorporation of phosphate at serine 887, without any concomitant effect on PLC-beta activity. We propose, therefore, that rather than having a direct effect on enzyme activity, the phosphorylation of PLC-beta by PKC may alter its interaction with a putative guanine nucleotide-binding regulatory protein and thereby prevent its activation.     

The pleckstrin homology domain of phospholipase C-beta2 as an effector site for Rac

Increasing evidence links the activation of Rho family GTPases to the stimulation of lipid hydrolysis catalyzed by phospholipase C (PLC)-beta isozymes. To better define this relationship, members of a library of recombinant Rho GTPases were screened for their capacity to directly engage various purified PLC-beta isozymes. Of the 17 tested members of the Rho family, only the active isoforms of Rac (Rac1, Rac2, and Rac3) both stimulate PLC-beta activity in vivo and bind PLC-beta2 and PLC-beta3, but not PLC-beta1, in vitro. Furthermore, the recognition site for Rac GTPases was localized to the pleckstrin homology (PH) domain of PLC-beta2, and this PH domain is fully sufficient to selectively interact with the active versions of the Rac GTPases, but not with other similar Rho GTPases. Together, these findings present a quantitative evaluation of the direct interactions between Rac GTPases and PLC-beta isozymes and define a novel role for the PH domain of PLC-beta2 as a putative effector site for Rac GTPases.     

Regulation of phospholipase C-beta activity by phosphatidic acid: isoform dependence,role of protein kinase C,and G protein subunits

Phosphatidic acid (PA) stimulates phospholipase C-beta(1) (PLC-beta(1)) activity and promotes G protein stimulation of PLC-beta(1) activity. The isoform dependence for PA regulation of PLC-beta activity as well as the role of PA in modulating regulation of PLC-beta activity by protein kinase C (PKC) and G protein subunits was determined. As compared to PLC-beta(1), the phospholipase C-beta(3) (PLC-beta(3)) isoform was less sensitive to PA, requiring greater than 15 mol % PA for stimulation. PLC-beta(3) bound weakly to PA. PKC had little effect on PA stimulation of PLC-beta(3) activity. PKC, however, inhibited PA stimulation of PLC-beta(1) activity through a mechanism dependent on the mol % PA. Stimulation by 7.5 mol % PA was completely inhibited by PKC. Increasing the PA and Ca(2+) concentration attenuated PKC inhibition. The binding of PLC-beta(1) to PA containing phospholipid vesicles was also reduced by PKC, in a manner dependent on the mol % PA. PA increased the stimulation of PLC-beta(1) activity by G alpha q but had little effect on the stimulation by beta gamma subunits. These results demonstrate that PA stimulation of PLC-beta activity is tightly regulated, suggesting the existence of a distinct PA binding region in PLC-beta(1). PA may be an important component of a receptor mediated signaling mechanism that determines PLC-beta(1) activation.     

Galpha(q/11) and gbetagamma proteins and membrane signaling of calcitriol and estradiol.

17beta-estradiol and 1,25-dihydroxyvitamin D(3)()(calcitriol) rapidly increase (< 5 sec) the concentration of intracellular calcium by mobilizing Ca(2+) from the endoplasmic reticulum and forming inositol 1,4,5-trisphosphate (InsP(3)) and diacylglycerol. Calcitriol increases InsP(3) formation via activation of phospholipase C (PLC)-beta1 linked to a pertussis toxin (PTX)-insensitive G-protein, and estradiol via activation of PLC-beta2 linked to a PTX-sensitive G-protein. Since PLC are effectors of different subunits of various G-proteins, we looked for and identified several G-subunits (Galpha(q/11), Galphas, Galphai, Gbeta and Ggamma) in female rat osteoblasts using Western immunoblotting. The action of calcitriol on InsP(3) formation and Ca(2+) mobilization in Fura-2-loaded confluent osteoblasts involved Galpha(q/11). The membrane effects of estradiol involved Gbetagamma; subunits, and principally Gbeta subunits, but not alpha-subunits. These results may provide additional evidence for membrane receptors of steroid hormones. Since PLC-beta1 is the target effector of Galpha(q/11), whereas PLC-beta2 is only activated by betagamma subunits, this specificity may help to generate membrane receptor-specific responses in vivo.     

Novel mechanisms for feedback regulation of phospholipase C-beta activity

The receptor-regulated phospholipase C-beta (PLC-beta) signaling pathway is an important component in a network of signaling cascades that regulate cell function. PLC-beta signaling has been implicated in the regulation of cardiovascular function and neuronal plasticity. The Gq family of G proteins mediate receptor stimulation of PLC-beta activity at the plasma membrane. Mitogens stimulate the activity of a nuclear pool of PLC-beta. Stimulation of PLC-beta activity results in the rapid hydrolysis of phosphatidylinositol-4,5-bisphosphate, with production of inositol-1,4,5-trisphosphate and diacylglycerol, intracellular mediators that increase intracellular Ca2+ levels and activate protein kinase C activity, respectively. Diacylglycerol kinase converts diacylglycerol to phosphatidic acid, a newly emerging intracellular mediator of hormone action that targets a number of signaling proteins. Activation of the Gq linked PLC-beta signaling pathway can also generate additional signaling lipids, including phosphatidylinositol-3-phosphate and phosphatidylinositol-3,4,5-trisphosphate, which regulate the activity and/or localization of a number of proteins. Novel feedback mechanisms, directed at the level of Gq and PLC-beta, have been identified. PLC-beta and regulators of G protein signaling (RGS) function as GTPase-activating proteins on Gq to control the amplitude and duration of stimulation. Protein kinases phosphorylate and regulate the activation of specific PLC-beta isoforms. Phosphatidic acid regulates PLC-beta1 activity and stimulation of PLC-beta1 activity by G proteins. These feedback mechanisms coordinate receptor signaling and cell activation. Feedback mechanisms constitute possible targets for pharmacological intervention in the treatment of disease.     

Expression of functional receptor-coupled TRPC3 channels in DT40 triple receptor InsP3 knockout cells

The TRPC3 channel, an intensively studied member of the widely expressed transient receptor potential (TRP) family, is a Ca(2+)-conducting channel activated in response to phospholipase C-coupled receptors. Despite scrutiny, the receptor-induced mechanism to activate TRPC3 channels remains unclear. Evidence indicates TRPC3 channels interact directly with intracellular inositol 1,4,5-trisphosphate receptors (InsP(3)Rs) and that channel activation is mediated through coupling to InsP(3)Rs. TRPC3 channels were expressed in DT40 chicken B lymphocytes in which all three InsP(3)R genes were deleted (DT40InsP(3)R-k/o). Endogenous B-cell receptors (BCR) coupled through Syk kinase to phospholipase C-gamma (PLC-gamma) activated the expressed TRPC3 channels in both DT40w/t and DT40InsP(3)R-k/o cells. The diacylglycerol (DAG) analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) also activated TRPC3 channels independently of InsP(3)Rs. BCR-induced TRPC3 activation was blocked by the PLC enzymic inhibitor, U-73122, and also blocked by wortmannin-induced PLC substrate depletion. Neither U-73122 nor wortmannin modified either OAG-induced TRPC3 activation or store-operated channel activation in DT40 cells. Cotransfection of cells with both G protein-coupled M5 muscarinic receptors and TRPC3 channels resulted in successful M5 coupling to open TRPC3 channels mediated by PLC-beta. We conclude that TRPC3 channels are activated independently of InsP(3)Rs through DAG production resulting from receptor-mediated activation of either PLC-gamma or PLC-beta.     

Evidence for selective coupling of alpha 1-adrenergic receptors to phospholipase C-beta 1 in rat neonatal cardiomyocytes

Activation of phospholipase C (PLC) in neonatal rat cardiomyocytes (NCM) generates primarily inositol 1,4,5-trisphosphate (Ins(1,4,5)P(3)) in response to rises in intracellular Ca(2+), or inositol 1,4-bisphosphate (Ins(1,4)P(2)) in response to norepinephrine (NE) (Matkovich, S. J. and Woodcock, E. A. (2000) J. Biol. Chem. 275, 10845-10850). To examine the PLC subtype mediating the alpha(1)-adrenergic receptor response, PLC-beta(1) and PLC-beta(3) were overexpressed in NCM using adenoviral infection (Ad-PLC-beta(1) NCM and Ad-PLC-beta(3) NCM, respectively) and PLC responses assessed from [(3)H]inositol phosphate (InsP) generation in the presence of 10 mm LiCl. The [(3)H]InsP response to NE (100 microm) was enhanced in Ad-PLC-beta(1) NCM relative to cells infected with blank virus (Ad-MX NCM), but was reduced in Ad-PLC-beta(3) NCM. In contrast, the [(3)H]InsP response to ATP (100 microm) was not elevated in Ad-PLC-beta(1) NCM, and was enhanced rather than diminished in Ad-PLC-beta(3) NCM, showing that effects of the two PLC-beta isoforms were specific for particular receptor types. PLC-delta(1) overexpression selectively reduced NE-induced [(3)H]InsP responses, without affecting the ATP stimulation. The reduced NE response was associated with a selective loss of PLC-beta(1) expression in Ad-PLC-delta(1) NCM. alpha(1)-Adrenergic receptor activation caused phosphorylation of PLC-beta(1) but not PLC-beta(3), whereas stimulation by ATP induced phosphorylation of PLC-beta(3) but not PLC-beta(1.) Taken together, these studies provide evidence that NE-stimulated InsP generation in NCM is primarily mediated by PLC-beta(1), despite the presence of both PLC-beta(1) and PLC-beta(3) isoforms.     

Development of an Assay for Phospholipase C Using Column-Reconstituted, Extruded Phospholipid Vesicles

The reconstitution of heterotrimeric G proteins into phospholipid vesicles has been widely used for the measurement of PLC-beta activity in vitro. We have developed an improved and sensitive method for the assay of PLC-beta activity. This approach involves reconstitution of purified betagamma dimers into extruded phospholipid vesicles containing phosphatidylinositol 4, 5-bisphosphate and using a gel-filtration technique to separate the reconstituted vesicles from monodispersed betagamma dimers and the detergent used to solubilize G proteins. The method provides physical information about the partitioning of betagamma dimers into phospholipid vesicles and was used to examine the effect of different prenyl groups on the gamma subunits in the activation of PLC-beta. The beta1gamma1 dimer (containing the farnesyl group) and the beta1gamma2 dimer (containing the geranylgeranyl group) were purified from baculovirus-infected Sf9 insect cells and were found to partition equally into phospholipid vesicles. The beta1gamma2 dimer is more potent and effective in stimulating PLC-beta activity than the beta1gamma1 dimer. The EC50 values of betagamma dimers for the activation of PLC-beta determined with this method were lower than those determined by previous methodology, showing that betagamma subunits have a subnanomolar affinity for PLC-beta.     

Crystal structure of Rac1 bound to its effector phospholipase C-beta2

Although diverse signaling cascades require the coordinated regulation of heterotrimeric G proteins and small GTPases, these connections remain poorly understood. We present the crystal structure of the GTPase Rac1 bound to phospholipase C-beta2 (PLC-beta2), a classic effector of heterotrimeric G proteins. Rac1 engages the pleckstrin-homology (PH) domain of PLC-beta2 to optimize its orientation for substrate membranes. Gbetagamma also engages the PH domain to activate PLC-beta2, and these two activation events are compatible, leading to additive stimulation of phospholipase activity. In contrast to PLC-delta, the PH domain of PLC-beta2 cannot bind phosphoinositides, eliminating this mode of regulation. The structure of the Rac1-PLC-beta2 complex reveals determinants that dictate selectivity of PLC-beta isozymes for Rac GTPases over other Rho-family GTPases, and substitutions within PLC-beta2 abrogate its stimulation by Rac1 but not by Gbetagamma, allowing for functional dissection of this integral signaling node.     

Phosphorylation and regulation of G-protein-activated phospholipase C-beta 3 by cGMP-dependent protein kinases

Among the drugs that are known to relax the vascular smooth muscle and regulate other cellular functions, beta-adrenergic agonists and nitric oxide-containing compounds are some of the most effective ones. The mechanisms of these drugs are thought to lower agonist-induced intracellular [Ca(2+)] by increasing intracellular cAMP and cGMP, activating their respective protein kinases. However, the physiological targets of cyclic nucleotide-dependent protein kinases are not clear. The molecular basis for the regulation of intracellular Ca(2+) by signaling pathways coupled to cyclic nucleotides is not well defined. G-protein-activated phospholipase C (PLC-beta) catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphates to generate diacylglycerol and inositol 1,4,5-triphosphate, leading to the activation of protein kinase C and the mobilization of intracellular Ca(2+). In this study, we shown that G-protein-activated PLC enzymes are the potential targets of cGMP-dependent protein kinases (PKG). PKG can directly phosphorylate PLC-beta2 and PLC-beta3 in vitro with purified proteins and in vivo with metabolic labeling. Phosphorylation of PLC-beta leads to the inhibition of G-protein-activated PLC-beta3 activity by 50-70% in COS-7 cell transfection assays. By using phosphopeptide mapping and site-directed mutagenesis, we further identified two key phosphorylation sites for the regulation of PLC-beta3 by PKG (Ser(26) and Ser(1105)). Mutation at these two sites (S26A and S1105A) of PLC-beta3 completely blocked the phosphorylation of PLC-beta3 protein catalyzed by PKG. Furthermore, mutation of these serine residues removed the inhibitory effect of PKG on the activation of the mutant PLC-beta3 proteins by G-protein subunits. Our results suggest a molecular mechanism for the regulation of G-protein-mediated intracellular [Ca(2+)] by the NO-cGMP-dependent signaling pathway.