Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.1.4.3 (phospholipase C)
 18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Gq mediates hormonal stimulation of phosphoinositide-specific phospholipase C (PI-PLC). We mutated the alpha subunit of Gq (alpha q) to replace arginine 183 with cysteine. Mutations that substitute cysteine for the corresponding arginine residues of alpha s and alpha i2 constitutively activate their respective effector pathways, creating the gsp and gip2 oncogenes. Transient expression of alpha q-R183C in COS-7 and HEK-293 cells constitutively activates PI-PLC, but wild type (WT) alpha q does not. This suggests that the mutated arginines in alpha s, alpha i2, and alpha q share a common function in regulating the active state of these proteins and that the alpha q gene may serve as a target for oncogenic mutations in human tumors. In an attempt to develop an assay for receptor stimulation of recombinant alpha q, we co-expressed receptors with alpha q-WT. We found that the alpha 2-adrenoceptor stimulates PI-PLC activation in HEK-293 cells in a fashion that depends completely on co-expression of alpha q-WT. These findings create an experimental model, similar to that provided for alpha s by S49 cyc- cells, that should make it possible to analyze receptor and effector coupling by mutant alpha q against a null background.
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PMID:Recombinant Gq alpha. Mutational activation and coupling to receptors and phospholipase C. 130 40

The gonadotropin-releasing hormone receptor (GnRH-R) desensitizes following chronic exposure to GnRH or its agonists. However, it is not certain whether the GnRH-R undergoes rapid homologous desensitization analogous to other members of the G-protein coupled receptor (GPCR) superfamily. This study investigated rapid desensitization events in two cell lines expressing the GnRH-R; (the pituitary gonadotrope alpha T3-1 cell line and the stably transfected human embryonal kidney cells, HEK-293). In both cell types, total inositol phosphate (IP) production did not desensitize, increasing linearly over 10 min. Short-term GnRH pretreatment also did not desensitize the rapid phase ( < or = sec) of the early Ins1,4,5P3 response despite a partial desensitization of the plateau phase ( > 1 min). It is likely that Ins1,4,5P3 metabolism rather than desensitization is responsible for this partial effect. In contrast, GnRH-stimulated calcium responses did desensitize in a dose-dependent fashion in both alpha T3-1 and HEK 293 cells expressing the GnRH-R. These results suggest that rapid GnRH-R desensitization occurs at a level beyond both the receptor and phospholipase C (PLC) activation. These events were receptor specific and not related to cell type, since similar rapid desensitization profiles were observed in both GnRH-R expressing pituitary and nonpituitary cell types. In contrast, profiles of GnRH-stimulated calcium responses were cell type specific.
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PMID:Rapid desensitization of GnRH-stimulated intracellular signalling events in alpha T3-1 and HEK-293 cells expressing the GnRH receptor. 758 62

1. Diacylglycerols (DAGs) are common intracellular second messengers produced as a result of activation of phospholipase C. We have examined the direct effects of DAG on currents from cloned voltage-dependent potassium channels. Potassium channels were studied by overexpression of their cRNAs in Xenopus oocytes or of their cDNAs in HEK 293 cells, and macroscopic currents were recorded from inside-out membrane patches. 2. When applied to the intracellular side of the patch, 1,2-dioctanoyl-sn-glycerol (C8:0) (DOG) blocks Shaker IR, Kv1.3, and Kv1.6 channels. This block appears macroscopically as a large speeding of the inactivation rate. Longer carbon chain length DAGs (10 and 12 carbons) are less effective in producing this response. 3. DOG is effective at low concentrations, doubling the apparent inactivation rate at 162 nM, and has a fast time course, with a wash-in and reversal to control each within approximately 30 s. 4. Voltage steps delivered with a two pulse protocol in the presence of DOG indicate that recovery from DOG block is voltage dependent. Recovery occurs quickly (tau = 507 ms) when channels are closed quickly by hyperpolarized (-90 mV) potentials, and occurs slowly (tau = 1.3 s) when channels are closed incompletely by depolarized (-60 mV) potentials. 5. The action of DOG is independent of protein kinase C (PKC) activation, because it does not require ATP, nor is it blocked by staurosporin or by the PKC inhibitor peptide 19-36.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Block of cloned voltage-gated potassium channels by the second messenger diacylglycerol independent of protein kinase C. 766 34

Pleckstrin is a 40-kDa protein present in platelets and leukocytes that contains two PH domains separated by a 150-residue intervening sequence. Pleckstrin is a major substrate for protein kinase C, but its function is unknown. The present studies examine the effects of pleckstrin on second messenger generation. When expressed in cos-1 or HEK-293 cells, pleckstrin inhibited 1) the G alpha-mediated activation of phospholipase C beta initiated by thrombin, M1-muscarinic acetylcholine, and angiotensin II receptors, 2) the stimulation of phospholipase C beta by constitutively active Gq alpha, 3) the G beta gamma-mediated activation of phospholipase C beta caused by alpha 2A-adrenergic receptors, and 4) the tyrosine phosphorylation-mediated activation of phospholipase C gamma caused by Trk A. However, pleckstrin had no effect on either the stimulation or inhibition of adenylyl cyclase. The inhibition of phosphoinositide hydrolysis caused by pleckstrin was similar in magnitude to that caused by activating protein kinase C with phorbol 12-myristate 13-acetate (PMA). When combined, pleckstrin and PMA had an additive effect, inhibiting phosphoinositide hydrolysis by as much as 90%. Structure-function analysis highlighted the role of pleckstrin's N-terminal PH domain in these events. Although deleting the C-terminal PH domain had no effect, deleting the N-terminal PH domain abolished activity (but not expression) and mutating a highly conserved tryptophan residue within the N-terminal PH domain decreased activity by one-third. Notably, however, a pleckstrin variant in which the N-terminal PH domain was replaced with a second copy of the C-terminal PH domain was nearly as active as native pleckstrin. These results show that: 1) pleckstrin can inhibit pathways leading to both phospholipase C beta- and phospholipase C gamma-mediated phosphoinositide hydrolysis, 2) this inhibition affects activation of phospholipase C beta mediated by either G alpha or G beta gamma, but does not affect the regulation of adenylyl cyclase activity by G alpha or G beta gamma, 3) although pleckstrin is a substrate for protein kinase C, the effects of pleckstrin and PMA are at least partially independent, 4) the inhibition caused by pleckstrin appears to be mediated by the PH domain at the N terminus, rather than the C terminus of the molecule, and 5) location of the two PH domains within the molecule clearly contributes to their individual activity.2+1
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PMID:Pleckstrin inhibits phosphoinositide hydrolysis initiated by G-protein-coupled and growth factor receptors. A role for pleckstrin's PH domains. 778 10

It is now widely appreciated that G-protein-coupled cell-surface receptors can modulate distinct signal transduction pathways via coupling to different GTP-binding proteins. In the present study, we have used a transient co-expression approach to study the coupling of a single alpha 2-adrenergic receptor (alpha 2AAR) population to three different G protein subtypes (Gi, Gq, and Gs) acting on two different cellular effectors in HEK 293 cells. In all cases, the affinity of the receptor for the alpha 2A-adrenergic agonist, UK14304, is unchanged (KD approximately equal to 670 nM). However, there is a dramatic difference in the EC50 of UK14304 in eliciting inhibition of endogenous adenylyl cyclase via endogenous Gi (0.09 nM) versus activation of phospholipase C via co-transfected Gq (50 nM) or stimulation of endogenous adenylyl cyclase via co-transfected Gs (70 nM) in HEK 293 cells. These findings are consistent with the interpretations that the alpha 2AAR preferentially interacts with Gi rather than Gs or Gq. When the alpha 2AAR was mutated at Asp79, a residue highly conserved among G-protein-coupled receptors, the mutant D79N alpha 2AAR lost the ability to couple to Gq and Gs and, although it was able to couple to inhibition of cyclase via pertussis toxin-sensitive pathways (Gi), it did so with a lower potency than observed for the wild-type alpha 2AAR (EC50 = 7.2 nM). The most straightforward interpretation of these data is that the D79N mutation in the alpha 2AAR reduces the efficiency of coupling of the alpha 2AAR to all G-proteins, thus eliminating signal transduction through those pathways less efficiently coupled to the alpha 2AAR. Since the transient expression assays described permit manipulation of the structure of both the receptor or the G-protein, the present strategies could be exploited to delineate the complementary domains specifying the affinity and/or efficacy of receptor coupling to distinct GTP-binding proteins.
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PMID:Coupling of the alpha 2A-adrenergic receptor to multiple G-proteins. A simple approach for estimating receptor-G-protein coupling efficiency in a transient expression system. 790 86

Gq alpha is palmitoylated at residues Cys9 and Cys10. Removal of palmitate from purified Gq alpha with palmitoylthioesterase in vitro failed to alter interactions of Gq alpha with phospholipase C-beta 1, the G protein beta gamma subunit complex, or m1 muscarinic cholinergic receptors. Mutants C9A, C10A, C9A/C10A, C9S/C10S, and truncated Gq alpha (removal of residues 1-6) were synthesized in Sf9 cells and purified. Loss of both Cys residues or truncation prevented palmitoylation of Gq alpha. However, truncated Gq alpha and the single Cys mutants activated phospholipase C-beta 1 normally, while the double Cys mutants were poor activators. Loss of both Cys residues impaired but did not abolish interaction of Gq alpha with m1 receptors. These Cys residues are thus important regardless of their state of palmitoylation. When expressed in HEK-293 or Sf9 cells, all of the proteins studied associated entirely or predominantly with membranes, although a minor fraction of nonpalmitoylated Gq alpha proteins accumulated in the cytosol of HEK-293 cells. When subjected to TX-114 phase partitioning, a significant fraction of all of the proteins, including those with no palmitate, was found in the detergent-rich phase. Removal of residues 1-34 of Gq alpha caused a loss of surface hydrophobicity as evidenced by complete partitioning into the aqueous phase. The Cys residues at the amino terminus of Gq alpha are thus important for its interactions with effector and receptor, and the amino terminus conveys a hydrophobic character to the protein distinct from that contributed by palmitate.
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PMID:Functional importance of the amino terminus of Gq alpha. 855 Jun 9

A series of chimeras between a constitutively active mutant of the alpha-subunit of Gq and the alpha-subunit of Gs was constructed to identify the domains in alphaq specifically involved in interaction with its effector phosphoinositide phospholipase C (PLC). Transient expression of the chimeric proteins and measurement of the production of inositol phosphates and cAMP in HEK-293 cells revealed that the Ile217-Lys276 sequence of alphaq contained the PLC interaction sites, whereas the residues for activation of adenylyl cyclase were in the Ile235-Leu294 sequence of alphas. Alanine scanning mutagenesis of the Ile217-Lys276 region of alphaq further identified two clusters of amino acids (Asp243,Asn244,Glu245 and Arg256,Thr257) that were specifically required for interaction with PLC. Comparison of the sequences of alphaq, alphas, and alphat showed that the PLC-interacting residues identified in alphaq are different from the corresponding residues in alphas and alphat that are involved in effector activation. Alignment of the sequences of alphaq and alphat, based on the crystal structure of alphat (Noel, J. P., Hamm, H. E., and Sigler, P. D. (1993) Nature 366, 654-663), indicated that the PLC-activating residues of alphaq are located in alpha-helix 3 and its linker to beta-sheet 4, which are adjacent to a switch region whose conformation changes with activation. It is proposed that the selectivity of alphaq for PLC involves relatively few amino acids, but that the effector may interact with other nonselective sequences in the alpha-subunit.
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PMID:Identification of determinants in the alpha-subunit of Gq required for phospholipase C activation. 861 84

The metabotropic glutamate receptors (mGluRs) share no sequence homology and show different structural features compared with most other G protein-coupled receptors (GPCRs). In particular, some isoforms of the phospholipase C (PLC)-coupled mGluRs (mGluR1a, mGluR5a, and mGluR5b) have a surprisingly long carboxyl-terminal intracellular domain of more than 350 residues, whereas the splice variants mGluR1b and mGluR1c have a much shorter carboxyl terminus. In the current study, the different splice variants of mGluR1 were expressed in porcine kidney epithelial (LLC-PK1) or the human embryonic kidney (HEK 293) cells, and their levels of expression were examined with the use of Western blot analysis. Expression of the short isoforms mGluR1b and mGluR1c did not modify the basal inositol phosphate production. In contrast, expression to similar levels of mGluR1a resulted in a 2-fold increase in the basal inositol phosphate formation. This increase in basal PLC activity was due to neither the presence of a low concentration of glutamate in the incubation medium nor a modification of the PLC pathway, resulting, for example, from the constant activation of mGluR1a++ by glutamate during the culture. Surprisingly none of the known competitive antagonists of mGluR1 inhibited the basal PLC activity, indicating that none of these molecules act as inverse agonists. Taken together, these results indicate that the long carboxyl-terminal domain confers a small agonist-independent activity to mGluR1. This indicates that, as already observed for other GPCRs, little constitutive activity of wild-type mGluRs can be detected. Our results also add to the splice variants and further suggest that the long carboxyl-terminal domain of mGluR1a confers better coupling efficiency to the G proteins.
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PMID:Changes in the carboxyl-terminal domain of metabotropic glutamate receptor 1 by alternative splicing generate receptors with differing agonist-independent activity. 864 81

Calcium (Ca2+) ions serve multiple roles both intra- and extracellularly. We recently cloned a cell surface, Cao(2+)-sensing receptor (CaR) that plays a central role in Cao2+ homeostasis by enabling direct regulation by Cao2+ of parathyroid hormone (PTH) secretion and the function of other tissues involved in mineral ion homeostasis. In parathyroid cells, the CaR activates phospholipase C, thereby raising the levels of inositol trisphosphate (IP3) and releasing Ca2+ from intracellular stores. High Cao2+ also activates Ca2+ influx into parathyroid cells through poorly defined mechanisms that may involve Ca(2+)-permeable, nonselective cation channels (NCC). We now show that human embryonic kidney (HEK293) cells also have NCC and, furthermore, that these channels are regulated by the CaR. We have utilized the cell-attached configuration of the patch clamp technique to characterize the properties of these channels as well as their regulation by various CaR agonists added to the external bath solution. The polycationic CaR agonist, neomycin (100 microM), as well as an elevated concentration of Cao2+ (3 mM), both of which activate the cloned CaR, significantly increased the probability of channel opening (Po) in HEK cells stably transfected with the CaR but not in nontransfected HEK cells which do not contain the receptor. Thus, the activation of the CaR enhances the activity of Ca(2+)-permeable NCC in these cells, which could contribute to the sustained increase in Cai2+ in parathyroid cells which is observed in response to elevated Cao2+. The CaR may also regulate the membrane functions of other CaR-expressing cells (e.g., those in the brain), at least in part, by modulating similar channels.
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PMID:Agonists of the Ca(2+)-sensing receptor (CaR) activate nonselective cation channels in HEK293 cells stably transfected with the human CaR. 880 75

Parathyroid hormone (PTH) regulates calcium metabolism through a specific G protein-coupled, seven-transmembrane helix-containing receptor. This receptor also binds and is activated by PTH-related protein (PTHrP). The human (h) PTH/PTHrP receptor is a membrane glycoprotein with an apparent molecular weight of approximately 85000 which contains four putative N-glycosylation sites. To elucidate the functional role of receptor glycosylation, if any, we studied hormone binding and signal transduction in human embryonic kidney cells transfected with hPTH/PTHrP receptor (HEK-293/C-21). These cells stably express 300000-400000 receptors per cell. Inhibition of N-glycosylation with an optimized concentration of tunicamycin yielded completely nonglycosylated hPTH/PTHrP receptor (approximately 60 kDa). This receptor form is fully functional; it maintains nanomolar binding affinity for PTH- and PTHrP-derived agonists and antagonists. PTH and PTHrP agonists stimulate cyclic AMP accumulation and increases in cytosolic calcium levels. In addition, the highly potent benzophenone (pBz2)-containing PTH-derived radioligand [Nle8,18,Lys13(epsilon-pBz2),L-2-Nal23,Tyr34 3-125I)]bPTH(1-34)NH2 can photoaffinity cross-link specifically to the nonglycosylated receptor. The molecular weight (approximately 60000) of the band representing the photo-cross-linked, nonglycosylated receptor (obtained from the tunicamycin-treated HEK-293/C-21 cells) was similar to that of the deglycosylated photo-cross-linked receptor (obtained by enzymatic treatment with Endoglycosidase-F/N-glycosidase-F). Our findings indicate that glycosylation of the hPTH/PTHrP receptor is not essential for its effective expression on the plasma membrane or for the binding of ligands known to interact with the native receptor. The nonglycosylated hPTH/PTHrP receptor remains fully functional with regard to both of its known signal transduction pathways: cAMP-protein kinase A and phospholipase C-cytosolic calcium.
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PMID:Role of glycosylation in expression and function of the human parathyroid hormone/parathyroid hormone-related protein receptor. 896 54


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