Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: 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)

Agonist-dependent phosphorylation of G protein-coupled receptors (GPRs) by G protein-coupled receptor kinases (GRKs) is proposed to be a key event initiating homologous receptor desensitization. A technical limitation hindering identification of GPRs as GRK substrates has been the necessity to use purified and reconstituted receptors in GRK assays. Here, the human m2 and human m3 (hm3) muscarinic cholinergic receptors (mAChRs), which couple to attenuation of adenylyl cyclase and stimulation of phospholipase C, respectively, were expressed in Spodoptera frugiperda insect cells and an in vitro approach to studying GPR phosphorylation by GRKs in crude membranes was developed. The m2 mAChR, a known substrate of certain GRKs, was used to validate the approach. The GRK isoform beta-adrenergic receptor kinase (beta ARK)1 phosphorylated the membrane-bound human m2 mAChRs in an agonist-dependent manner. The results demonstrated that endogenous membrane-bound beta gamma subunits of G proteins stimulated the phosphorylation of the membrane-bound m2 mAChR. To reveal new GRK substrates, we tested the expressed hm3 mAChRs. The membrane-bound hm3 mAChRs were phosphorylated by beta ARK1 in an agonist-dependent, G beta gamma-enhanced manner. This is the first demonstration that hm3 mAChRs can serve as substrates for GRKs. The stoichiometry of receptor phosphorylation was approximately 2 mol of phosphate/mol of receptors in the absence of G beta gamma and approximately 4 mol of phosphate/mol of receptors upon addition of G beta gamma. When the specificity of various GRKs towards mAChRs was assessed, beta ARK2 phosphorylated the agonist-activated hm3 mAChRs as efficiently as did beta ARK1; however, neither GRK5 nor GRK6 significantly phosphorylated the hm3 mAChRs under similar conditions. The approach of studying GRK-mediated phosphorylation of GPRs in their membrane-bound state identified the hm3 mAChRs as new substrates for GRKs. This approach should be valuable in identifying other new substrates of GRKs and should aid in studies that elucidate GRK/GPR pairing.
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PMID:Agonist-dependent phosphorylation of human muscarinic receptors in Spodoptera frugiperda insect cell membranes by G protein-coupled receptor kinases. 787 29

Continuous stimulation of anaphylatoxin receptors C3aR and C5aR with their cognate ligands engenders, within minutes, diminished responsiveness of these receptors. We tested the hypothesis that agonist-induced desensitization involves C3aR and C5aR phosphorylation by G protein-coupled receptor kinases (GRK). When expressed in rat basophilic leukemia cells and exposed to C3a, the C3aR underwent rapid (t(1/2) approximately 15 s), dose-dependent (EC50 approximately 10 nM) and reversible phosphorylation by a kinase refractory to the effects of PKC inhibitors. Phosphoamino acid analysis revealed that the C3aR is phosphorylated on serine and threonine, but not on tyrosine residues. Overexpression of GRK2, GRK3, GRK5 or GRK6 together with C3aR in COS-7 cells enhanced the C3a-induced C3aR phosphorylation 1.5 - 1.9-fold (p < 0.05), but each kinase reduced ligand-stimulated phospholipase C activity differently. Conversely, antibody-mediated inhibition of endogenous GRK2 and GRK3 significantly inhibited C3aR phosphorylation in permeabilized cells. GRK overexpression in cells which co-expressed C5aR and were exposed to C5a resulted in the hyperphosphorylation of the C5aR. These findings are of physiological relevance, since we observed anaphylatoxin-induced phosphorylation of C3aR and C5aR endogenously expressed in human mast cells (HMC-1) which contain significant intracellular levels of GRK2 and GRK3.
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PMID:Ligand-induced phosphorylation of anaphylatoxin receptors C3aR and C5aR is mediated by "G protein-coupled receptor kinases. 1050 78

G protein-coupled receptor kinases (GRKs) are well characterized regulators of G protein-coupled receptors, whereas regulators of G protein signaling (RGS) proteins directly control the activity of G protein alpha subunits. Interestingly, a recent report (Siderovski, D. P., Hessel, A., Chung, S., Mak, T. W., and Tyers, M. (1996) Curr. Biol. 6, 211-212) identified a region within the N terminus of GRKs that contained homology to RGS domains. Given that RGS domains demonstrate AlF(4)(-)-dependent binding to G protein alpha subunits, we tested the ability of G proteins from a crude bovine brain extract to bind to GRK affinity columns in the absence or presence of AlF(4)(-). This revealed the specific ability of bovine brain Galpha(q/11) to bind to both GRK2 and GRK3 in an AlF(4)(-)-dependent manner. In contrast, Galpha(s), Galpha(i), and Galpha(12/13) did not bind to GRK2 or GRK3 despite their presence in the extract. Additional studies revealed that bovine brain Galpha(q/11) could also bind to an N-terminal construct of GRK2, while no binding of Galpha(q/11), Galpha(s), Galpha(i), or Galpha(12/13) to comparable constructs of GRK5 or GRK6 was observed. Experiments using purified Galpha(q) revealed significant binding of both Galpha(q) GDP/AlF(4)(-) and Galpha(q)(GTPgammaS), but not Galpha(q)(GDP), to GRK2. Activation-dependent binding was also observed in both COS-1 and HEK293 cells as GRK2 significantly co-immunoprecipitated constitutively active Galpha(q)(R183C) but not wild type Galpha(q). In vitro analysis revealed that GRK2 possesses weak GAP activity toward Galpha(q) that is dependent on the presence of a G protein-coupled receptor. However, GRK2 effectively inhibited Galpha(q)-mediated activation of phospholipase C-beta both in vitro and in cells, possibly through sequestration of activated Galpha(q). These data suggest that a subfamily of the GRKs may be bifunctional regulators of G protein-coupled receptor signaling operating directly on both receptors and G proteins.
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PMID:Selective regulation of Galpha(q/11) by an RGS domain in the G protein-coupled receptor kinase, GRK2. 1056 30

We have investigated the effects of G protein-coupled receptor kinase (GRK) 3 and GRK6 on the phosphorylation and regulation of the M3 muscarinic acetylcholine receptor (mACh) endogenously expressed in SH-SY5Y cells. Overexpression of GRK3 or GRK6 enhanced M3 mACh receptor phosphorylation after high-concentration methacholine (100 microM, 1 min) addition. However, GRK6 was more potent, increasing receptor phosphorylation even after low (3 microM, 1 min) agonist stimulation. Compared with plasmid-transfected control cells expressing equivalent M3 mACh receptor number, GRK3- or GRK6-overexpressing cells exhibited a reduced phospholipase C activity reflected by a lower accumulation of total [3H]inositol phosphates and Ins(1,4,5)P3 mass. In addition, direct stimulation of G protein activation of phospholipase C (by AlF4(-)) was inhibited in GRK3- but not GRK6-overexpressing cells. Guanosine-5'-O-(3-[35S]thio)triphosphate binding and immunoprecipitation of Galpha(q/11) indicated that acute methacholine-stimulated receptor/Galpha(q/11) coupling was unaffected by GRK overexpression. In contrast, agonist pretreatment of cells for 3 min caused M3 mACh receptor uncoupling from Galpha(q/11), which was markedly enhanced by GRK6 overexpression, particularly at lower agonist pretreatment concentrations. However, the increased M3 mACh receptor phosphorylation seen in clones overexpressing GRK3 was not accompanied by increased receptor-Galpha(q/11) uncoupling. Overall, these data suggest that GRK3 and GRK6 use different pathways to desensitize the M3 mACh receptor. GRK6 seems to act as a classical GRK, inducing increased receptor phosphorylation accompanied by an uncoupling of receptor and Galpha(q/11). Conversely, GRK3 may cause desensitization independently of receptor phosphorylation, possibly via Gbetagamma binding and/or direct Galpha(q) binding via its regulator of G protein signaling domain to inhibit phospholipase C activity.
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PMID:G protein-coupled receptor kinases 3 and 6 use different pathways to desensitize the endogenous M3 muscarinic acetylcholine receptor in human SH-SY5Y cells. 1145 19

We have previously shown that overexpression of G protein-coupled receptor kinase 6 (GRK6) enhanced the phosphorylation and desensitization of the endogenously expressed M(3) muscarinic acetylcholine (mACh) receptor in human SH-SY5Y neuroblastoma cells. In this study we have examined the potential role of endogenous GRK6 in the regulation of M(3) mACh receptor by blocking its action through the introduction of a kinase-dead, dominant-negative GRK6 ((K215R)GRK6). (K215R)GRK6 expression inhibited methacholine-stimulated M(3) mACh receptor phosphorylation by 50% compared with plasmid transfected control cells. Guanosine-5'-O-(3-[(35)S]thio)triphosphate binding and immunoprecipitation studies, conducted after agonist pretreatment (3 min), indicated that M(3) mACh receptor-G alpha(q/11) uncoupling was attenuated by 50% in cells expressing (K215R)GRK6 when compared with control cells. In contrast, expression of the related dominant-negative kinase (K215R)GRK5 had no effect on M(3) mACh receptor phosphorylation or uncoupling. Time course studies also showed that agonist-stimulated [(3)H]inositol phosphate accumulations were more sustained in cells expressing (K215R)GRK6 compared with control and (K215R)GRK5-expressing cells, whereas (K215R)GRK6 expression had no effect on the phospholipase C response to direct stimulation of G proteins with AlF(4)(-). The ability of (K215R)GRK6 to inhibit agonist-mediated M(3) mACh receptor phosphorylation and G protein uncoupling suggests that endogenous GRK6 mediates, at least in part, M(3) mACh receptor desensitization in the SH-SY5Y cell line.
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PMID:Endogenous G protein-coupled receptor kinase 6 Regulates M3 muscarinic acetylcholine receptor phosphorylation and desensitization in human SH-SY5Y neuroblastoma cells. 1185 37

Previously we have shown that G protein-coupled receptor kinase (GRK) 6 plays a major role in the regulation of the human M3 muscarinic acetylcholine receptor (M3 mAChR) in the human neuroblastoma SH-SY5Y. However, 30-fold overexpression of the catalytically inactive, dominant-negative K215RGRK6 produced only a 50% suppression of M3 mAChR phosphorylation and desensitization. Here, we have attempted to determine whether other endogenous kinases play a role in the regulation of M3 mAChR signaling. In contrast to the clear attenuating effect of K215RGRK6 expression on M3 mAChR regulation, dominant-negative forms of GRKs (K220RGRK2, K220RGRK3, K215RGRK5) and casein kinase 1alpha (K46RCK1alpha) were without effect. In addition, inhibition of a variety of second-messenger-regulated kinases and the tyrosine kinase Src also had no effect upon agonist-stimulated M3 mAChR regulation. To investigate further the desensitization process we have followed changes in inositol 1,4,5-trisphosphate in single SHSY5Y cells using the pleckstrin homology domain of PLCdelta1 tagged with green fluorescent protein (eGFP-PHPLCdelta1). Stimulation of cells with approximate EC50 concentrations of agonist before and after a desensitizing period of agonist exposure resulted in a marked attenuation of the latter response. Altered GRK6 activity, through overexpression of wild-type GRK6 or K215RGRK6, enhanced or reduced the degree of M3 mAChR desensitization, respectively. Taken together, our data indicate that M3 mAChR desensitization is mediated by GRK6 in human SH-SY5Y cells, and we show that receptor desensitization of phospholipase C signaling can be monitored in 'real-time' in single, living cells.
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PMID:Specificity of g protein-coupled receptor kinase 6-mediated phosphorylation and regulation of single-cell m3 muscarinic acetylcholine receptor signaling. 1457 54

We used the inositol 1,4,5-trisphosphate (IP3) biosensor, the pleckstrin homology (PH) domain of PLCdelta1 (phospholipase C) tagged with enhanced green fluorescent protein (eGFP-PH(PLCdelta)), to examine muscarinic acetylcholine (mACh) receptor regulation of phospholipase C/IP3 signaling in intact single hippocampal neurons in "real time." Initial experiments produced a pharmacological profile consistent with the presence of a predominant M1 mACh receptor population coupled to the IP3 response. To investigate M1 mACh receptor regulation, neurons were stimulated with approximate EC50 concentrations of the mACh receptor agonist methacholine before (R1) and after (R2) a short (60 sec) exposure to a high concentration of agonist. This resulted in a marked attenuation in the R2 relative to R1 response. Inhibition of endogenous GRK6 (G-protein-coupled receptor kinase) activity, by the introduction of catalytically inactive (K215R)GRK6, partially reversed the attenuation of agonist-induced responsiveness, whereas overexpression of wild-type GRK6 increased receptor desensitization. Manipulation of endogenous GRK2 activity through introduction of either wild-type or catalytically inactive GRK2 ((K220R)GRK2) almost completely inhibited agonist-stimulated IP3 production, implying a phosphorylation-independent regulation of M1 mACh receptor signaling, most probably mediated by a GRK2 N-terminal RGS-like (regulator of G-protein signaling) domain interaction with GTP-bound Galpha(q/11). Together, our data suggest a role for both phosphorylation-dependent and -independent regulation of M1 mACh receptors in hippocampal neurons.
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PMID:Imaging of muscarinic acetylcholine receptor signaling in hippocampal neurons: evidence for phosphorylation-dependent and -independent regulation by G-protein-coupled receptor kinases. 1511 10

Histamine stimulates uterine contraction; however, little is known regarding the mechanism or regulation of uterine histamine receptor signaling. Here we investigated the regulation of Galpha(q/11)-coupled histamine receptor signaling in human myometrial smooth muscle cells using the inositol 1,4,5-trisphosphate biosensor pleckstrin homology domain of phospholipase-delta1 tagged to enhanced green fluorescent protein and the Ca(2+)-sensitive dye Fluo-4. Histamine addition caused concentration-dependent increases in inositol 1,4,5-trisphosphate and [Ca(2+)](i) in the ULTR human uterine smooth muscle cell line and primary human myometrial cells. These effects were completely inhibited by the H(1) histamine receptor antagonist, diphenhydramine, and were unaffected by the H(2) histamine receptor antagonist, cimetidine. ULTR and primary myometrial cells were transfected with either dominant-negative G protein-coupled receptor kinases (GRKs) or small interfering RNAs targeting specific GRKs to assess the roles of this protein kinase family in H(1) histamine receptor desensitization. Dominant-negative GRK2, but not GRK5 or GRK6, prevented H(1) histamine receptor desensitization. Similarly, transfection with short interfering RNAs (that each caused >70% depletion of the targeted GRK) for GRK2, but not GRK3 or GRK6, also prevented H(1) histamine receptor desensitization. Our data suggest that histamine stimulates phospholipase C-signaling in myometrial smooth muscle cells through H(1) histamine receptors and that GRK2 recruitment is a key mechanism in the regulation of H(1) histamine receptor signaling in human uterine smooth muscle. These data provide insights into the in situ regulation of this receptor subtype and may inform pathophysiological functioning in preterm labor and other conditions involving uterine smooth muscle dysregulation.
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PMID:Selective regulation of H1 histamine receptor signaling by G protein-coupled receptor kinase 2 in uterine smooth muscle cells. 1851 96

Oxytocin plays an important role in the progression, timing, and modulation of uterine contraction during labor and is widely used as an uterotonic agent. We investigated the mechanisms regulating oxytocin receptor (OTR) signaling in human primary myometrial smooth muscle cells and the ULTR cell-line. Oxytocin produced concentration-dependent increases in both total [(3)H]inositol phosphate accumulation and intracellular Ca(2+) concentration ([Ca(2+)](i)); however, responses were greater and more reproducible in the ULTR cell line. Assessment of phospholipase C activity in single cells revealed that the OTR desensitizes rapidly (within 5 min) in the presence of oxytocin (100 nm). To characterize OTR desensitization further, cells were stimulated with a maximally effective concentration of oxytocin (100 nm, 30 sec) followed by a variable washout period and a second identical application of oxytocin. This brief exposure to oxytocin caused a marked decrease (>70%) in OTR responsiveness to rechallenge and was fully reversed by increasing the time period between agonist challenges. To assess involvement of G protein-coupled receptor kinases (GRKs) in OTR desensitization, cells were transfected with small interfering RNAs to cause specific > or =75% knockdown of GRKs 2, 3, 5, or 6. In both primary myometrial and ULTR cells, knockdown of GRK6 largely prevented oxytocin-induced OTR desensitization; in contrast, selective depletion of GRKs 2, 3, or 5 was without effect. These data indicate that GRK6 recruitment is a cardinal effector of OTR responsiveness and provide mechanistic insight into the likely in vivo regulation of OTR signaling in uterine smooth muscle.
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PMID:Regulation of oxytocin receptor responsiveness by G protein-coupled receptor kinase 6 in human myometrial smooth muscle. 1942 52

Vasoconstrictor-driven G protein-coupled receptor (GPCR)/phospholipase C (PLC) signaling increases intracellular Ca²⁺ concentration to mediate arterial contraction. To counteract vasoconstrictor-induced contraction, GPCR/PLC signaling can be desensitized by G protein-coupled receptor kinases (GRKs), with GRK2 playing a predominant role in isolated arterial smooth muscle cells. In this study, we use an array of GRK2 inhibitors to assess their effects on the desensitization of UTP and angiotensin II (AngII)-mediated arterial contractions. The effects of GRK2 inhibitors on the desensitization of UTP- or AngII-stimulated mesenteric third-order arterial contractions, and PLC activity in isolated mesenteric smooth muscle cells (MSMC), were determined using wire myography and Ca²⁺ imaging, respectively. Applying a stimulation protocol to cause receptor desensitization resulted in reductions in UTP- and AngII-stimulated arterial contractions. Preincubation with the GRK2 inhibitor paroxetine almost completely prevented desensitization of UTP- and attenuated desensitization of AngII-stimulated arterial contractions. In contrast, fluoxetine was ineffective. Preincubation with alternative GRK2 inhibitors (Takeda compound 101 or CCG224063) also attenuated the desensitization of UTP-mediated arterial contractile responses. In isolated MSMC, paroxetine, Takeda compound 101, and CCG224063 also attenuated the desensitization of UTP- and AngII-stimulated increases in Ca²⁺, whereas fluoxetine did not. In human uterine smooth muscle cells, paroxetine reversed GRK2-mediated histamine H₁ receptor desensitization, but not GRK6-mediated oxytocin receptor desensitization. Utilizing various small-molecule GRK2 inhibitors, we confirm that GRK2 plays a central role in regulating vasoconstrictor-mediated arterial tone, highlighting a potentially novel strategy for blood pressure regulation through targeting GRK2 function.
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PMID:Small-Molecule G Protein-Coupled Receptor Kinase Inhibitors Attenuate G Protein-Coupled Receptor Kinase 2-Mediated Desensitization of Vasoconstrictor-Induced Arterial Contractions. 2998 Jun 59


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