The yeast and mammalian isoforms of phosphatidylinositol transfer protein can all restore phospholipase C-mediated inositol lipid signaling in cytosol-depleted RBL-2H3 and HL-60 cells.

The mammalian phosphatidylinositol transfer proteins (PITP) and the yeast Saccharomyces cerevisiae PITP (SEC14p) that show no sequence homology both catalyze exchange of phosphatidylinositol (PI) between membranes compartments in vitro. In HL-60 cells where the cytosolic proteins are depleted by permeabilization, exogenously added PITPalpha is required to restore G protein-mediated phospholipase Cbeta (PLCbeta) signaling. Recently, a second mammalian PITPbeta form has been described that shows 77% identity to rat PITPalpha. We have examined the ability of the two mammalian PITPs and SEC14p to restore PLC-mediated signaling in cytosol-depleted HL-60 and RBL-2H3 cells. Both PITPalpha and PITPbeta isoforms as well as SEC14p restore G protein-mediated PLCbeta signaling with a similar potency. In RBL-2H3 cells, crosslinking of the IgE receptor by antigen stimulates inositol lipid hydrolysis by tyrosine phosphorylation of PLCgamma1. Permeabilization of RBL cells leads to loss of PLCgamma1 as well as PITP into the extracellular medium and this coincides with loss of antigen-stimulated lipid hydrolysis. Both PLCgamma1 and PITP were required to restore inositol lipid signaling. We conclude that (i) because the PI binding/transfer activities of PITP/SEC14p is the common feature shared by all three transfer proteins, it must be the relevant activity that determines their abilities to restore inositol lipid-mediated signaling and (ii) PITP is a general requirement for inositol lipid hydrolysis regardless of how and which isoform of PLC is activated by the appropriate agonist.

G protein subunits and the stimulation of phospholipase C by Gs-and Gi-coupled receptors: Lack of receptor selectivity of Galpha(16) and evidence for a synergic interaction between Gbeta gamma and the alpha subunit of a receptor activated G protein.

Stimulatory guanine nucleotide binding protein (Gs)-coupled receptors activated by luteinizing hormone, vasopressin, and the catecholamine isoproterenol (luteinizing hormone receptor, type 2 vasopressin receptor, and types 1 and 2 beta-adrenergic receptors) and the Gi-coupled M2 muscarinic receptor (M2R) were expressed transiently in COS cells, alone and in combination with Gbeta gamma dimers, their corresponding Galphas (Galpha(s), or Galpha(i3)) and either Galpha(q) or Galpha(16). Phospholipase C (PLC) activity, assessed by inositol phosphate production from preincorporated myo[3H]inositol, was then determined to gain insight into differential coupling preferences among receptors and G proteins. The following were observed: (i) All receptors tested were able to stimulate PLC activity in response to agonist occupation. The effect of the M2R was pertussis toxin sensitive. (ii) While, as expected, expression of Galpha(q) facilitated an agonist-induced activation of PLC that varied widely from receptor to receptor (400% with type 2 vasopressin receptor and only 30% with M2R), expression of Galpha(16) facilitated about equally well the activation of PLC by any of the tested receptors and thus showed little if any discrimination for one receptor over another. (iii) Gbeta gamma elevated basal (agonist independent) PLC activity between 2- and 4-fold, confirming the proven ability of Gbeta gamma to stimulate PLCbeta. (iv) Activation of expressed receptors by their respective ligands in cells coexpressing excess Gbeta gamma elicited agonist stimulated PLC activities, which, in the case of the M2R, was not blocked by pertussis toxin (PTX), suggesting mediation by a PTX-insensitive PLC-stimulating Galpha subunit, presumably, but not necessarily, of the Gq family. (v) The effects of Gbeta gamma and the PTX-insensitive Galpha elicited by M2R were synergistic, suggesting the possibility that one or more forms of PLC are under conditional or dual regulation of G protein subunits such that stimulation by one sensitizes to the stimulation by the other.

Identification of a phospholipase C beta2 region that interacts with Gbeta-gamma.

To delineate the phospholipase C (PLC; EC 3.1.4.3) beta2 sequences involved in interactions with the beta-gamma subunits of G proteins, we prepared a number of mammalian expression plasmids encoding a series of PLC beta2 segments that span the region from the beginning of the X box to the end of the Y box. We found the sequence extending from residue Glu-435 to residue Val-641 inhibited Gbeta-gamma-mediated activation of PLC beta2 in transfected COS-7 cells. This PLC beta2 sequence also inhibited ligand-induced activation of PLC in COS-7 cells cotransfected with cDNAs encoding the complement component C5a receptor and PLC beta2 but not in cells transfected with the alpha1B-adrenergic receptor, suggesting that the PLC beta2 residues (Glu-435 to Val-641) inhibit the Gbeta-gamma-mediated but not the Galpha-mediated effect. The inhibitory effect on Gbeta-gamma-mediated activation of PLC beta2 may be the result of the interaction between Gbeta-gamma and the PLC beta2 fragment. This idea was confirmed by the observation that a fusion protein comprising these residues (Glu-435 to Val-641) of PLC beta2 and glutathione S-transferase (GST) bound to Gbeta-gamma in an in vitro binding assay. The Gbeta-gamma-binding region was further narrowed down to 62 amino acids (residues Leu-580 to Val-641) by testing fusion proteins comprising various PLC beta2 sequences and GST in the in vitro binding assay.

Platelet-derived growth factor activates protein kinase C epsilon through redundant and independent signaling pathways involving phospholipase C gamma or phosphatidylinositol 3-kinase.

Protein kinase C (PKC), a major cellular receptor for tumor-promoting phorbol esters and diacylglycerols (DGs), appears to be involved in a variety of cellular functions, although its activation mechanism in vivo is not yet fully understood. To evaluate the signaling pathways involved in the activation of PKC epsilon upon stimulation by platelet-derived growth factor (PDGF) receptor (PDGFR), we used a series of PDGFR "add-back" mutants. Activation of a PDGFR mutant (Y40/51) that binds and activates phosphatidylinositol 3-kinase (PI 3-kinase) caused translocation of PKC epsilon from the cytosol to the membrane in response to PDGF. A PDGFR mutant (Y1021) that binds and activates phospholipase C gamma (PLC gamma), but not PI 3-kinase, also caused the PDGF-dependent translocation of PKC epsilon. The translocation of PKC epsilon upon stimulation of PDGFR (Y40/51) was inhibited by wortmannin, an inhibitor of PI 3-kinase. Activation of PKC epsilon was further confirmed in terms of PKC epsilon-dependent expression of a phorbol 12-tetradecanoate 13-acetate response element (TRE)-luciferase reporter. Further, purified PKC epsilon was activated in vitro by either DG or synthetic phosphatidylinositol 3,4,5-trisphosphate. These results clearly demonstrate that PKC epsilon is activated through redundant and independent signaling pathways which most likely involve PLC gamma or PI 3-kinase in vivo and that PKC epsilon is one of the downstream mediators of PI 3-kinase whose downstream targets remain to be identified.

Specific and high-affinity binding of inositol phosphates to an isolated pleckstrin homology domain.

Pleckstrin homology (PH) domains are found in many signaling molecules and are thought to be involved in specific intermolecular interactions. Their binding to several proteins and to membranes containing 1-alpha-phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] has been reported. A region that includes the PH domain has also been implicated in binding of phospholipase C-delta 1 (PLC-delta 1) to both PtdIns(4,5)P2 and D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] [Cifuentes, M. E., Delaney, T. & Rebecchi, M. J. (1994) J. Biol. Chem. 269, 1945-1948]. We report herein that the isolated PH domain from PLC-delta 1 binds to both PtdIns(4,5)P2 and Ins(1,4,5)P3 with high affinity and shows the same binding specificity seen by others with whole PLC-delta 1. Thus the PH domain is functionally and structurally modular. These results demonstrate stereo-specific high-affinity binding by an isolated PH domain and further support a functional role for PH domains in the regulation of PLC isoforms. Other PH domains did not bind strongly to the compounds tested, suggesting that inositol phosphates and phospholipids are not likely physiological ligands for all PH domains. Nonetheless, since all PH-domain-containing proteins are associated with membrane surfaces, several PH domains bind to specific sites on membranes, and PH domains appear to be electrostatically polarized, a possible general role for PH domains in membrane association is suggested.

A gene encoding a phosphatidylinositol-specific phospholipase C is induced by dehydration and salt stress in Arabidopsis thaliana.

A cDNA corresponding to a putative phosphatidylinositol-specific phospholipase C (PI-PLC) in the higher plant Arabidopsis thaliana was cloned by use of the polymerase chain reaction. The cDNA, designated cAtPLC1, encodes a putative polypeptide of 561 aa with a calculated molecular mass of 64 kDa. The putative product includes so-called X and Y domains found in all PI-PLCs identified to date. In mammalian cells, there are three types of PI-PLC, PLC-beta, -gamma, and -delta. The overall structure of the putative AtPLC1 protein is most similar to that of PLC-delta, although the AtPLC1 protein is much smaller than PLCs from other organisms. The recombinant AtPLC1 protein synthesized in Escherichia coli was able to hydrolyze phosphatidylinositol 4,5-bisphosphate and this activity was completely dependent on Ca2+, as observed also for mammalian PI-PLCs. These results suggest that the AtPLC1 gene encodes a genuine PI-PLC of a higher plant. Northern blot analysis showed that the AtPLC1 gene is expressed at very low levels in the plant under normal conditions but is induced to a significant extent under various environmental stresses, such as dehydration, salinity, and low temperature. These observations suggest that AtPLC1 might be involved in the signal-transduction pathways of environmental stresses and that an increase in the level of AtPLC1 might amplify the signal, in a manner that contributes to the adaptation of the plant to these stresses.