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)

We present characterization of the rhodopsin, Gq and phosphatidylinositol-specific phospholipase C (PLC) from the signal transduction pathway of cephalopod photoreceptors. Cephalopod rhodopsins are unique in possessing a C-terminal extension of proline-rich repeats, and they have a strong tendency to form ordered arrays. Two-dimensional arrays of a full-length and C-terminally-truncated cephalopod rhodopsin have been obtained. The C termini appear to cluster the rhodopsins into small groups. An AlF4(-)-activated Gq alpha subunit has been isolated and shown to activate a partially purified PLC beta. This 130 kDa PLC, isolated by absorption on heparin agarose, showed a specific activity of 195 nmol of phosphatidylinositol 4,5-bisphosphate hydrolysed per milligram of protein per minute in the presence of 1.6 microM free calcium.
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PMID:Rhodopsin, Gq and phospholipase C activation in cephalopod photoreceptors. 882 31

The heterotrimeric guanosine 5'-triphosphate (GTP)-binding protein Gq was suggested to couple the light receptor rhodopsin with the effector phospholipase C in visual cells of invertebrates. We indirectly linked Gq from Sepia officinalis to a concanavalin A-sepharose column via rhodopsin. Rhodopsin had been solubilized previously with 10 mM n-dodecyl-beta-maltoside from the purified photosensory membrane under illumination. All three subunits of the Gq were released almost pure by elution with 100 microM GTP. The alpha and beta subunits were identified by specific antipeptide antisera. The alpha subunit has a relative molecular mass of 46 kDa, and the beta subunit of 35 kDa. The gamma subunit corresponds to a 9 kDa polypeptide owing to the molecular mass, which is similar to the G gamma subunit of squid. The use of specific antibodies shows that neither actin nor G-protein related to transducin were in the fractions eluted with GTP or alpha-methyl mannoside. We demonstrate that all three subunits of Gq were associated with rhodopsin of invertebrates. Such use of a lectin column might be useful for further investigations of the interaction of rhodopsin and Gq.
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PMID:Interaction of guanosine 5'-triphosphate binding protein Gq from Sepia officinalis with illuminated rhodopsin bound to concanavalin A. 882 32

We report isolating the Drosophila retinal degeneration E (rdgE) mutation. The hypomorphic rdgE1 allele causes rapid photoreceptor degeneration in light and a slower rate of degeneration when the flies are raised in constant darkness. The rdgE1 flies exhibited an electrophysiological light response that decreased with age, coinciding with the degeneration. This suggests that degeneration caused the loss of the light response. We determined that the ninaE (rhodopsin) mutation, but not norpA [phospholipase C (PLC)], slowed the rdgE-dependent degeneration. This was consistent with the light-enhanced degeneration, but revealed that the degeneration is independent of the PLC-mediated phototransduction cascade. Transmission electron microscopy revealed that rdgE1 photoreceptors exhibited a number of vesicular transport defects including unpacking/vesiculation of rhabdomeres, endocytosis of novel vesicles by photoreceptors, a buildup of very large multivesicular bodies, and an increased amount of rough endoplasmic reticulum. We determined that the rdgE null phenotype is a late embryonic lethality. Therefore, rdgE+ is required in cells outside of the retina, quite possibly in a large number of neurons. Thus, rdgE may define a mutational class that exhibits both light-enhanced retinal degeneration and a recessive null lethality by perturbing neuronal membrane biosynthesis and/or recycling.
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PMID:rdgE: a novel retinal degeneration mutation in Drosophila melanogaster. 887 79

DGq is the alpha subunit of the heterotrimeric GTPase (G alpha), which couples rhodopsin to phospholipase C in Drosophila vision. We have uncovered three duplicated exons in dgq by scanning the GenBank data base for unrecognized coding sequences. These alternative exons encode sites involved in GTPase activity and G beta-binding, NorpA (phospholipase C)-binding, and rhodopsin-binding. We examined the in vivo splicing of dgq in adult flies and find that, in all but the male gonads, only two isoforms are expressed. One, dgqA, is the original visual isoform and is expressed in eyes, ocelli, brain, and male gonads. The other, dgqB, has the three novel exons and is widely expressed. Remarkably, all three nonvisual B exons are highly similar (82% identity at the amino acid level) to the Gq alpha family consensus, from Caenorhabditis elegans to human, but all three visual A exons are divergent (61% identity). Intriguingly, we have found a third isoform, dgqC, which is specifically and abundantly expressed in male gonads, and shares the divergent rhodopsin-binding exon of dgqA. We suggest that DGqC is a candidate for the light-signal transducer of a testes-autonomous photosensory clock. This proposal is supported by the finding that rhodopsin 2 and arrestin 1, two photoreceptor-cell-specific genes, are also expressed in male gonads.
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PMID:Novel Gq alpha isoform is a candidate transducer of rhodopsin signaling in a Drosophila testes-autonomous pacemaker. 890 71

In invertebrate photoreceptors, illuminated rhodopsin activates multiple G proteins, which are assumed to initiate multiple phototransduction cascades. In this paper, we focused on one of the phototransduction cascades, which utilizes rhodopsin, a Gq-like G protein, and phospholipase C (PLC). A Gq-like G protein from octopus photoreceptors was successfully purified to apparent homogeneity as an active form by simple two-step chromatography. The purified G protein had an alpha beta gamma-trimeric structure consisting of 44-kDa alpha, 37-kDa beta, and 9-kDa gamma subunits. The 44-kDa alpha subunit was assigned to the Gq class by western blot with antiserum against mammalian Gq alpha and by partial amino acid sequencing of its proteolytic fragments. Light-dependent binding of GTP gamma S was observed when the purified octopus Gq was reconstituted with octopus rhodopsin that had been integrated into phospholipid vesicles. Octopus Gq activated PLC beta 1 purified from bovine brain dose-dependently in the presence of A1F4-. Finally, light- and GTP-dependent activation of PLC beta 1 was observed in a reconstitution system consisting of octopus rhodopsin, Gq, and bovine PLC beta 1.
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PMID:Simple purification and functional reconstitution of octopus photoreceptor Gq, which couples rhodopsin to phospholipase C. 896 50

In the fly, thorough retinoid deprivation is possible, to optimize investigation of the effects of vitamin A metabolites and retinoic acid (RA) on visual development. Retinoids had been found to control fly opsin gene transcription, though this finding was contested. Northern blots on Drosophila heads showed that mRNA of Rh1 (the predominant rhodopsin) was high in vitamin A replete controls, very low in deprived flies, and increased upon feeding carrot juice to deprived flies as early as 1 hr. Expression of the ribosomal protein 49 [rp49] gene (the control) was equal both in deprivation and in replacement. Recovery of Rh1 protein upon such carotenoid replacement followed, barely detectable on Western blots at 4 hr but conspicuous by 8 hr. Alternative chromophore deprivation with yeast-glucose food yielded flies with opsin mRNA on Northerns but not rhodopsin, as demonstrated by Western blots, spectrophotometry and the electroretinogram (ERG). Rh1's mRNA but not Rh1 protein resulted from rearing flies from egg to adult on the otherwise deprivational medium supplemented with RA or beef brain-heart infusion. By comparing results from these different media it was concluded that: [1] deprivation and replacement affect opsin gene transcription; and [2] contradictory conclusions were from chromophore deprivation which does not eliminate all retinoid dependent factors which could affect the opsin promoter. Preliminary evidence shows that carotenoid deprivation decreases two proteins relevant to visual function: [1] phospholipase C (PLC); and [2] Drosophila retinoid binding protein (DRBP).
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PMID:Control of Drosophila opsin gene expression by carotenoids and retinoic acid: northern and western analyses. 899 52

In Drosophila, the store-operated Ca2+ channel, TRP, is required in photoreceptor cells for a sustained response to light. Here, we show that TRP forms a complex with phospholipase C-beta (NORPA), rhodopsin (RH1), calmodulin, and the PDZ domain containing protein INAD. Proteins with PDZ domains have previously been shown to cluster ion channels in vitro. We show that in InaD mutant flies, TRP is no longer spatially restricted to its normal subcellular compartment, the rhabdomere. These results provide evidence that a PDZ domain protein is required, in vivo, for anchoring of an ion channel to a signaling complex. Furthermore, disruption of this interaction results in retinal degeneration. We propose that the TRP channel is linked to NORPA and RH1 to facilitate feedback regulation of these upstream signaling molecules.
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PMID:Requirement for the PDZ domain protein, INAD, for localization of the TRP store-operated channel to a signaling complex. 901 Feb 8

We have used Drosophila mutants which are deficient in one or both of the arrestins present in photoreceptor cells to critically test the requirements for arrestin in the stabilization of Rh1 metarhodopsin under in vitro and in vivo conditions. Heads from flies illuminated with blue light were homogenized to obtain membranes or micellar extracts, and the amount of metarhodopsin present was quantitated by spectroscopic methods. Compared to wild-type, approximately 64% Rh1 metarhodopsin was recovered in flies deficient in arrestin-1 (arr1(1) mutant), approximately 38% in flies deficient in arrestin-2 (arr2(3) mutant), and approximately 6% in flies deficient in both arrestin-1 and arrestin-2 (arr1(1), arr2(3) double mutant). In contrast, no decrease was observed in the amounts of Rh1 metarhodopsin recovered from illuminated flies which were deficient either in the eye-specific phosphatase (rdgC mutant) or in the eye-specific phospholipase C (norpA(H24) and norpA(H52) mutants). Further, reconstitution experiments in total head homogenates showed that metarhodopsin produced in the arr1(1), arr2(3) double mutant could be stabilized upon the addition of exogenous arrestin-2. These studies provide definitive evidence that arrestin binding stabilizes Rh1 metarhodopsin under in vitro conditions. To test whether arrestin was also required to stabilize metarhodopsin in intact photoreceptor cells, metarhodopsin was generated in arr1(1), arr2(3) double mutant flies by in vivo illumination, and after a wait period of 20 min, converted back into rhodopsin by further illumination with red light. Quantitation of the regenerated rhodopsin in extracts from Drosophila heads showed no significant change in the level of rhodopsin recovered by this illumination protocol. Together, these experiments demonstrate that in disrupted photoreceptor cells, metarhodopsin is not stabilized unless arrestin is present, but in intact photoreceptor cells, significant metarhodopsin stabilization occurs even in the absence of bound arrestin.
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PMID:Studies of Rh1 metarhodopsin stabilization in wild-type Drosophila and in mutants lacking one or both arrestins. 904 19

The phototransduction cascade in invertebrates involves the coupling of rhodopsin activation to the action of the enzyme phospholipase C. This step is performed by G-proteins. An antibody against the alpha-subunit of a mouse Gq type G-protein recognized protein bands in Western blots of lateral eye and ventral nerve photoreceptors of Limulus. The protein bands had an apparent molecular mass of about 42 kDa. The antibody also recognized protein bands of a similar molecular mass in immunoblots of brain and intestine tissue. Immunoreactivity was found in lateral eye frozen sections where it was confined to the rhabdom region. When the antibody was applied to ultrathin sections of ventral nerve photoreceptors, the highest density of labeling was found on the rhabdomeral microvilli, but gold particles were also scattered in the cytoplasm. We conclude that a G-protein of the type Gq participates in the phototransduction of Limulus.
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PMID:Immunological demonstration of Gq-protein in Limulus photoreceptors. 914 81

We have previously demonstrated that the phospholipase C-coupled m3-muscarinic receptor is phosphorylated in an agonist-sensitive manner by a protein kinase of approximately 40 kDa purified from porcine cerebellum (Tobin, A. B., Keys, B., and Nahorski, S. R. (1996) J. Biol Chem. 271, 3907-3916). This kinase, called muscarinic receptor kinase (MRK), is distinct from second messenger-regulated protein kinases and from beta-adrenergic receptor kinase and other members of the G-protein-coupled receptor kinase family. In the present study we propose that MRK is casein kinase 1alpha (CK1alpha) based on the following evidence: 1) the amino acid sequence from two proteolytic peptide fragments derived from purified MRK corresponded exactly to sequences within CK1alpha. 2) Casein kinase activity co-eluted with MRK activity from the final two chromatography steps in the purification of porcine brain MRK. 3) Recombinant CK1alpha expressed in Sf9 cells is able to phosphorylate both casein and the bacterial fusion protein, Ex-m3, that contains a portion of the third intracellular loop of the m3-muscarinic receptor downstream of glutathione S-transferase. 4) Partially purified CK1alpha increased the level of muscarinic receptor phosphorylation in an agonist-sensitive manner when reconstituted with membranes from Chinese hamster ovary-m3 cells expressing the human recombinant m3-muscarinic receptor. 5) Partially-purified CK1alpha phosphorylated rhodopsin, contained in urea-treated bovine rod outer segment membranes, and the extent of phosphorylation was increased in the presence of light. These data demonstrate that the kinase previously called MRK is CK1alpha, and that CK1alpha offers an alternative protein kinase pathway from that of the G-protein-coupled receptor kinase family for the stimulus-dependent phosphorylation of the m3-muscarinic receptor, rhodopsin, and possibly other G-protein-coupled receptors.
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PMID:Stimulus-dependent phosphorylation of G-protein-coupled receptors by casein kinase 1alpha. 925 10


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