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

---

Query: EC:3.1.4.3 (phospholipase C)
18,461 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The cellular and molecular mechanisms of gonadotrope desensitization are unknown but transduction of the GnRH signal is known to involve sequentially the GnRH receptor, Gq alpha protein, phospholipase C beta-1, inositol-1,4,5-trisphosphate (IP3), and intracellular Ca+2 release. Here, we report the results of studies of a new family of proteins known as regulators of G protein signaling (RGS) that recently have been implicated in desensitization of several ligand induced processes. Using DNA-mediated transfection, we co-expressed the GnRH receptor and RGS1,2,3, or 4 in COS-1 cells. Control cells and those expressing RGS1,2, and 4 produced five fold increases in IP3 levels during the 30 sec after treatment with GnRH. In contrast, RGS3 expression suppressed by 75% the GnRH-induced IP3 responses. RGS3 was shown to bind Gq alpha protein in a model in vitro system: recombinant RGS3-glutathione-S-transferase (GST) fusion protein bound five-fold more 35S-met labeled Gq alpha protein than did with GST alone, suggesting that the mechanism of RGS3 action is attenuation of Gq alpha protein activation of phospholipase C. RGS3 mRNA and protein were observed to be expressed endogenously in the gonadotropic alpha T3-1 cell line. These results suggest a potential role for RGS3 in modulating the LH secretory responsiveness of the pituitary gonadotrope to GnRH.
...
PMID:Potential role for a regulator of G protein signaling (RGS3) in gonadotropin-releasing hormone (GnRH) stimulated desensitization. 900 25

To define the structural requirements of the parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptor necessary for activation of phospholipase C (PLC), receptors with random mutations in their second cytoplasmic loop were synthesized, and their properties were assessed. A mutant in which the wild type (WT) rat PTH/PTHrP receptor sequence EKKY (amino acids 317-320) was replaced with DSEL had little or no PTH-stimulated PLC activity when expressed transiently in COS-7 cells, but it retained full capacity to bind ligand and to generate cAMP. This phenotype was confirmed in LLC-PK1 cells stably expressing the DSEL mutant receptor, where both PTH-stimulated PLC activity and sodium-dependent phosphate co-transport were essentially abolished. Individual mutations of these four residues point to a critical role for Lys-319 in receptor-G protein coupling. PTH-generated IPs were reduced to 27 +/- 13% when K319E, compared with the WT receptor, and PLC activation was fully recovered in a receptor revertant in which Glu-319 in the DSEL mutant cassette was restored to the WT residue, Lys. Moreover, the WT receptor and a mutant receptor in which K319R had indistinguishable properties, thus suggesting that a basic amino acid at this position may be important for PLC activation. All of these receptors had unimpaired capacity to bind ligand and to generate cAMP. To ensure adequacy of Galphaq-subunits for transducing the receptor signal, Galphaq was expressed in HEK293 and in LLC-PK1 cells together with either WT receptors or receptors with the DSEL mutant cassette. PTH generated no inositol phosphates (IPs) in either HEK293 or LLC-PK1 cells, when they expressed DSEL mutant receptors together with Galphaq. In contrast, PTH generated 2- and 2. 5-fold increases in IPs, respectively, when these cells co-expressed both the WT receptor and Galphaq. Thus, generation of IPs by the activated PTH/PTHrP receptor can be selectively abolished without affecting its capacity to generate cAMP, and Lys-319 in the second intracellular loop is critical for activating the PLC pathway. Moreover, alpha-subunits of the Gq family, rather than betagamma-subunits, transduce the signal from the activated receptor to PLC, and the PLC, rather than the adenylyl cyclase, pathway mediates sodium-dependent phosphate co-transport in LLC-PK1 cells.
...
PMID:Mutations in the second cytoplasmic loop of the rat parathyroid hormone (PTH)/PTH-related protein receptor result in selective loss of PTH-stimulated phospholipase C activity. 905 74

In common with other Gq protein-coupled receptors, the third intracellular loop of the cholecystokinin-B (CCK-B) receptor contains three basic amino acids (K333/K334/R335) at the C-terminal segment. To determine the importance of these conserved basic residues in Gq-protein activation and stimulation of phospholipase C, these basic amino acids were mutated. Subsequently, the ability of resulting mutant receptors to activate phospholipase C was investigated by measuring inositol phosphate formation in COS-7 cells and recording Ca(2+)-activated Cl- currents from Xenopus oocytes. Site-directed mutagenesis was performed to mutate the three basic amino acids, K333/K334/R335, to neutral amino acids, M333/T334/L335. When the resulting mutant CCK-B receptors were expressed in COS-7 cells and Xenopus oocytes, sulfated cholecystokinin octapeptide (CCK-8) failed to induce inositol phosphate formation in COS-7 cells and evoke Ca(2+)-activated Cl- currents from oocytes. Each basic amino acid was also mutated (K333M, K334T, and R335L). All three single-point mutations resulted in a significant reduction in CCK-8-induced inositol phosphate formation and CCK-8-activated Ca(2+)-dependent Cl- currents. It is interesting that substituting the basic amino acids, K333/K334/R335, with three other basic residues, R333/R334/K335, did not change the maximal CCK-8-simulated inositol phosphate formation and the amplitude of CCK-8-evoked Ca(2+)-dependent Cl- currents. Radioligand-binding studies showed that the above-mentioned mutations did not affect the affinity for CCK-8 and receptor expression level in COS-7 cells. These findings suggest that basic amino acids at the C-terminus of the third cytoplasmic loop are required for the signal transduction by CCK-B receptors.
...
PMID:Basic amino acids at the C-terminus of the third intracellular loop are required for the activation of phospholipase C by cholecystokinin-B receptors. 908 47

Stimulation of high affinity IgE Fc receptors (FcepsilonRI) in basophils and mast cells activates the tyrosine kinases Lyn and Syk and causes the tyrosine phosphorylation of phospholipase C-gamma, resulting in the Ca2+- and protein kinase C-dependent secretion of inflammatory mediators. Concomitantly, FcepsilonRI stimulation initiates a number of signaling events resulting in the activation of mitogen-activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK), which, in turn, regulate nuclear responses, including cytokine gene expression. To dissect the signaling pathway(s) linking FcepsilonRI to MAPK and JNK, we reconstructed their respective biochemical routes by expression of a chimeric interleukin-2 receptor alpha subunit (Tac)-FcepsilonRI gamma chain (Tacgamma) in COS-7 cells. Cross-linking of Tacgamma did not affect MAPK in COS-7 cells, but when coexpressed with the tyrosine kinase Syk, Tacgamma stimulation potently induced Syk and Shc tyrosine phosphorylation and MAPK activation. In contrast, Tacgamma did not signal JNK activation, even when coexpressed with Syk. Ectopic expression of a hematopoietic-specific guanine nucleotide exchange factor (GEF), Vav, reconstituted the Tacgamma-induced, Syk- and Rac1-dependent JNK activation; and tyrosine-phosphorylation of Vav by Syk stimulated its GEF activity for Rac1. Thus, these data strongly suggest that Vav plays a critical role linking FcepsilonRI and Syk to the Rac1-JNK pathway. Furthermore, these findings define a novel signal transduction pathway involving a multimeric cell surface receptor acting on a cytosolic tyrosine kinase, which, in turn, phosphorylates a GEF, thereby regulating its activity toward a small GTP-binding protein and promoting the activation of a kinase cascade.
...
PMID:Tyrosine phosphorylation of the vav proto-oncogene product links FcepsilonRI to the Rac1-JNK pathway. 909 26

Thromboxane A2 (TXA2) is a major arachidonic acid metabolite of platelets and induces platelet functions by binding to specific receptors on the membrane. We have found patients with hemostatic defects due to impaired platelet responses to TXA2, and molecular characterization of the patients has been carried out. Platelets from these two unrelated patients showed impaired aggregation responses to TXA2 and its analogues despite the normal response to thrombin. Although the patients' platelets exhibited normal binding activities to TXA2 analogues, they showed decreased GTPase activity and second messenger formation when stimulated by STA2, a stable TXA2 agonist. To understand the molecular basis of this abnormality, we determined the cDNA sequence of the TXA2 receptor by reverse transcription-polymerase chain reaction (RT-PCR) from the patient's platelet RNA, and identified a single amino acid substitution (Arg60 for Leu) in the first cytoplasmic loop of the receptor. This mutation was found in both isoforms of the platelet TXA2 receptor which we have recently found: TXR alpha with the same structure as the placental TXA2 receptor and TXR beta with the same structure as the endothelial TXA2 receptor, and was detected exclusively in affected members of two unrelated families with the disorder. The mutant TXR alpha and TXR beta expressed in COS-m6 cells showed decreased agonist-induced phospholipase C activation despite their normal ligand binding affinities. These results suggest that the Arg60 for Leu mutation is responsible for the disorder and imply a critical role for the first cytoplasmic loop in the interaction of the TXA2 receptor with the G protein.
...
PMID:Molecular characterization of a dominantly inherited bleeding disorder with impaired platelet responses to thromboxane A2. 911 32

Recombinant regulators of G protein-signaling (RGS) proteins stimulate hydrolysis of GTP by alpha subunits of the Gi family but have not been reported to regulate other G protein alpha subunits. Expression of recombinant RGS proteins in cultured cells inhibits Gi-mediated hormonal signals probably by acting as GTPase-activating proteins for Galphai subunits. To ask whether an RGS protein can also regulate cellular responses mediated by G proteins in the Gq/11 family, we compared activation of mitogen-activated protein kinase (MAPK) by a Gq/11-coupled receptor, the bombesin receptor (BR), and a Gi-coupled receptor, the D2 dopamine receptor, transiently co-expressed with or without recombinant RGS4 in COS-7 cells. Pertussis toxin, which uncouples Gi from receptors, blocked MAPK activation by the D2 dopamine receptor but not by the BR. Co-expression of RGS4, however, inhibited activation of MAPK by both receptors causing a rightward shift of the concentration-effect curve for both receptor agonists. RGS4 also inhibited BR-stimulated synthesis of inositol phosphates by an effector target of Gq/11, phospholipase C. Moreover, RGS4 inhibited inositol phosphate synthesis activated by addition of AlF4- to cells overexpressing recombinant alphaq, probably by binding to alphaq.GDP.AlF4-. These results demonstrate that RGS4 can regulate Gq/11-mediated cellular signals by competing for effector binding as well as by acting as a GTPase-activating protein.
...
PMID:RGS4 inhibits Gq-mediated activation of mitogen-activated protein kinase and phosphoinositide synthesis. 911 54

The molecular mechanism by which cell surface receptors stimulate the serine/threonine kinase activity of c-Jun N-terminal kinases (JNKs) was investigated using a transient cotransfection experiments in COS-7 cells. Our data demonstrate that JNK activity is potently induced by platelet derived growth factor (PDGF) upon expression of beta PDGFR wild type (beta RWT). However, PDGF failed to mediate JNK activation in cells expressing beta PDGFR mutant lacking the binding site for phosphatidylinositol-3 (PI-3) kinase but not for phospholipase C gamma (PLC gamma) or Syp. Consistent with this result, a PI-3 kinase inhibitor, wortmannin inhibited activation of JNK by PDGF. Furthermore, overexpression of P110 the catalytic domain of PI-3 kinase was sufficient for activation of JNKs which could be efficiently inhibited by dominant negative forms of Ras, Rac but not of RhoA or Cdc42. Taken together all of these findings suggest that activation of JNK by PDGF involves receptor association with PI-3 kinase activity, which in turn acts on a ras- and rac-dependent pathway.
...
PMID:Requirement of phosphatidylinositol-3 kinase for activation of JNK/SAPKs by PDGF. 912 62

Isolated central hypothyroidism, characterized by insufficient TSH secretion resulting in low levels of thyroid hormones, is a rare disorder. We report a boy in whom isolated central hypothyroidism was diagnosed at 9 yr of age. Complete absence of TSH and PRL responses to TRH led us to speculate that he had an inactivating mutation of the TRH receptor gene. The patients' genomic DNA was isolated, and the entire coding region of the TRH receptor was amplified by the PCR and sequenced directly. Confirmation of the mutations and haplotyping of the family was performed using restriction enzymes. The biological activity of the wild-type and mutated TRH receptors was verified by evaluating the binding of labeled TRH and stimulation by TRH of total inositol phosphate accumulation in transfected HEK-293 and COS-1 cells. The patient was found to be a compound heterozygote, having inherited a different mutated allele from each of the parents; both mutations were in the 5'-part of the gene. Mutated receptors were unable to bind TRH and to activate total inositol phosphate accumulation. Our report is the first description of naturally occurring inactivating mutations of a G protein-coupled receptor linked to the phospholipase C second messenger pathway. The prevalence and phenotypic spectrum of TRH receptor mutations in isolated central hypothyroidism remain to be established.
...
PMID:A novel mechanism for isolated central hypothyroidism: inactivating mutations in the thyrotropin-releasing hormone receptor gene. 914 50

1. An alanine residue at the C-terminal tail of the third intracellular loop is highly conserved among various Gq protein-coupled receptors including rat cholecystokininB (CCKB) and neurotensin receptors. To investigate the functional significance of the conserved alanine in the activation of Gq proteins and phospholipase C (PLC) by CCKB and neurotensin receptors, the alanine residue was mutated in the present study. Subsequently, the ability of resulting mutant receptors to activate PLC was investigated by measuring the formation of inositol phosphates (IP) in COS-7 cells and recording Ca(2+)-activated chloride currents from Xenopus oocytes. 2. Site-directed mutagenesis was performed to mutate alanine at position 332 of rat CCKB receptor to glutamate. When the (A332E) mutant receptor was expressed in COS-7 cells and Xenopus oocytes, the efficacy and the potency of sulphated cholecystokinin octapeptide (CCK-8) to stimulate polyphosphoinositide hydrolysis in COS-7 cells and evoke calcium-dependent Cl- currents in oocytes were not significantly affected. 3. Alanine residue at position 302 of rat neurotensin receptor was also mutated to glutamate. When expressed in COS-7 cells and Xenopus oocytes, the resulting (A302E) mutant receptor was strongly defective in stimulating phosphatidylinositol turnover in COS-7 cells and evoking Ca(2+)-dependent chloride currents in oocytes. 4. In summary, the present study demonstrates that alanine residue at the C-terminus of third cytoplasmic domain is required for the full activation of Gq proteins and PLC by neurotensin receptors. However, in contrast to other Gq protein-coupled receptors, alanine at the distal third intracellular loop does not play a significant role in CCKB receptor activation of PLC.
...
PMID:A site-directed mutagenesis study on the conserved alanine residue in the distal third intracellular loops of cholecystokininB and neurotensin receptors. 915 42

Proposed mechanisms by which alpha 2-adrenergic receptors (alpha 2AR) regulate intracellular calcium ([Ca2+]i) include stimulation and inhibition of cell surface calcium channels, stimulation of calcium release via receptor coupling to Gq with subsequent activation of phospholipase C and release of IP3, or stimulation of calcium release via coupling to Gi in an IP3-independent manner. These potential mechanisms were explored in cells that expressed alpha(2A)AR endogenously (HEL cells), permanently transfected CHO cells, and transiently transfected COS-7 cells. Each cell type displayed agonist (UK14304)-dependent increases in [Ca2+]i that were blocked by yohimbine, ablated by pertussis toxin, and largely unaffected by chelation of extracellular calcium. Furthermore, calcium release was associated with IP3 accumulation and was blocked by an inhibitor of phospholipase C (PLC). When expressed in CHO cells, a mutated alpha(2A)AR which has the amino and carboxyl termini of the third intracellular loop substituted with beta 2AR sequence poorly coupled to Gi in adenylyl cyclase assays, and likewise displayed virtually no coupling to increased [Ca2+]i. These results all point toward a Gi- versus a Gq-mediated coupling pathway triggering release of intracellular calcium stores. The possibility that G(beta gamma) subunits released from alpha(2A)AR-Gi coupling is the mechanism of PLC activation was explored in COS-7 cells by coexpressing alpha(2A)AR with the G(beta gamma) inhibitors transducin or a carboxy-terminal portion of the beta AR kinase. Both beta gamma inhibitors markedly inhibited alpha(2A)AR modulation of [Ca2+]i while not affecting thromboxane A2 receptor mediated stimulation of [Ca2+]i via Gq coupling. Thus, alpha(2A)AR couple to calcium release via Gi-associated G(beta gamma) subunits. This coupling is present in multiple cell types and should be considered a major signal transduction pathway of this receptor.
...
PMID:Alpha 2A-adrenergic receptor stimulated calcium release is transduced by Gi-associated G(beta gamma)-mediated activation of phospholipase C. 917 58


<< Previous 1 2 3 4 5 6 7 8 9 10

---