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

Phosducin is a soluble phosphoprotein found in retinal photoreceptor cells and in the pineal gland. It binds to the beta gamma subunits of guanine nucleotide-binding proteins (G proteins) (G beta gamma) and may regulate G-protein function. In this study, the ability of specific regions of phosducin to bind G beta gamma was characterized. A series of deletion mutants were made in bovine phosducin. They were tested in cotransfection assays for their ability to inhibit G beta gamma-mediated phospholipase C beta 2 isoform activation. Overexpression of the N-terminal half of phosducin showed inhibition, whereas overexpression of the C-terminal half did not. The first 63 amino acid residues were required for inhibition. A tryptophan-to-valine substitution at residue 29, which is part of a well conserved 11-amino acid sequence, severely impaired phosducin inhibitory function. Glutathione S-transferase-phosducin fusion proteins were expressed in Escherichia coli to study phosducin-G beta gamma interaction in vitro. The N-terminal 63-amino acid fragment was able to bind to G beta gamma. In contrast, the C-terminal half failed to bind to G beta gamma. The substitution mutants showed little or no binding. Furthermore, direct measurements of interaction between G beta gamma and fragments of phosducin, using surface plasmon resonance technology, confirmed the assignment of binding activity to the 63-amino acid fragment and the importance of the tryptophan residue.
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PMID:The N terminus of phosducin is involved in binding of beta gamma subunits of G protein. 753 10

HIKE is a highly conserved sequence motif that selectively occurs in proteins candidate to bind PH domains, e.g., the beta subunit of heterotrimeric G proteins, kinases, ankyrin and kinesin. Thus, the HIKE region has been predicted to be a protein docking site for PH domains. This work evidentiates recent experimental evidence that unambiguously defines the functional role of HIKE in Gbeta as a multiple effector docking site and as a major regulatory region of G protein's function. Indeed, the Gbeta HIKE interacts with the beta-adrenergic receptor kinase, Galpha, Ggamma, adenylyl cyclase 2, phospholipase C beta2, inward rectifier K channels, calcium channel alpha1B, calmodulin, phosducin, ste20. Quite interestingly, HIKE is located in the Gbeta region that faces the cell membrane. Thus, HIKE also interacts with the cell membrane and may dynamically regulate membrane vs effector binding of the Galphabetagamma trimer. These findings fulfill a major prediction of the HIKE model, i.e., that HIKE is a regulatory region for protein-protein interactions. A role of HIKE as a proteic binding site for PH domains is supported by the profound influence of HIKE mutations on the largely PH-mediated binding of beta-ARK to Gbeta.
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PMID:HIKE, a candidate protein binding site for PH domains, is a major regulatory region of Gbeta proteins. 1032 71

Over the past 20 years, the general mechanism for signaling through 7-transmembrane helix receptors coupled to GTP hydrolysis has been worked out. Although similar in overall organization, subtype variability and subcellular localization of components have built in considerable signaling specificity. Atomic resolution structures for many of the components have delineated the domain organization of these complex proteins and have given physical form to the idea of subtype specificity. This review describes what is known about the physical structures of the 7-transmembrane helix receptors, the heterotrimeric GTP binding coupling proteins, the adenylate cyclase and phospholipase C effector proteins, and signaling modulatory proteins, such as arrestin, phosducin, recoverin-type myristoyl switch proteins, and the pleckstrin homology domain of G-protein receptor kinase-2. These images allow experimenters to contemplate the details of the supramolecular organization of the multiprotein complexes involved in the transmission of signals across the cellular lipid bilayer.
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PMID:Structural features of heterotrimeric G-protein-coupled receptors and their modulatory proteins. 1037 66

The prenyl group on the G protein gamma subunit is an important determinant of protein-protein interactions between the betagamma dimer and its targets, such as alpha subunits, receptors, and effectors. In an effort to identify domains of the beta subunit important for the activation of effectors, we have prepared two types of mutants, one set in the domain suggested to form a hydrophobic prenyl-binding pocket for the gamma subunit's prenyl group (prenyl pocket mutants) and the other set in a domain between Gly(306) and Gly(319) in the beta propeller, which undergoes a conformational change when the dimer binds to phosducin (conformational change mutants). Recombinant baculoviruses for each set of mutants were prepared, and the nine mutant beta subunits were overexpressed with either the gamma(2) subunit (modified with geranylgeranyl) or the gamma(2-L71S) subunit (gamma(2) with altered CAAX sequence and modified with farnesyl). The purified dimers were tested for their ability to couple Galpha(i1) to the A1 adenosine receptor and to activate phospholipase C-beta or type II adenylyl cyclase. All dimers containing mutant beta subunits were indistinguishable from wild-type beta(1)gamma(2) or beta(1)gamma(2-L71S) in coupling the receptor to Galpha(i1). The prenyl pocket mutants expressed with gamma(2) were 10-fold less potent in activating phospholipase C-beta and adenylyl cyclase than beta(1)gamma(2) and had similar activities to beta(1)gamma(2-L71S). The conformational change mutants caused a 15- to 23-fold decrease in EC(50) values for activation of these two effectors. Overall, the results suggest that the sites in Gbeta identified by these mutants are very important in the activation of effectors. Furthermore, the nature of the prenyl group on Ggamma may play an important role in the conformational change leading to the activity of Gbetagamma on effectors.
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PMID:Role of C-terminal domains of the G protein beta subunit in the activation of effectors. 1092 79

To understand the requirements for binding to G protein betagamma subunits, phage-displayed random peptide libraries were screened using immobilized biotinylated betagamma as the target. Selected peptides were grouped into four different families based on their sequence characteristics. One group (group I) had a clear conserved motif that has significant homology to peptides derived from phospholipase C beta (PLC beta) and to a short motif in phosducin that binds to G protein beta subunits. The other groups had weaker sequence homologies or no homology to the group I sequences. A synthetic peptide from the strongest consensus group blocked activation of PLC by G protein betagamma subunits. The peptide did not block betagamma-mediated inhibition of voltage-gated calcium channels and had little effect on betagamma-mediated inhibition of Gs-stimulated type I adenylate cyclase. Competition experiments indicated that peptides from all four families bound to a single site on betagamma. These peptides may bind to a protein-protein interaction 'hot spot' on the surface of betagamma subunits that is used by a subclass of effectors.
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PMID:Evidence that a protein-protein interaction 'hot spot' on heterotrimeric G protein betagamma subunits is used for recognition of a subclass of effectors. 1117 21

Our laboratory has previously demonstrated the ability of kappa- and delta-opioid agonists to decrease aqueous flow rates and intraocular pressure of rabbits. The mechanisms by which these agents act in the ciliary body of the rabbit could involve inhibition of cAMP production, as well as increased generation of inositol phosphates (IPs). With regard to enhanced production of IPs, it has been suggested that their levels can be augmented by stimulation of phospholipase C via opioid receptors linked to either Galpha-subunits derived from Gq proteins or Gbetagamma-subunits derived from G(i/o)-proteins. The aim of the current study is to investigate the role of pertussis toxin (PTX)-sensitive G-proteins and Gbetagamma-subunits in delta-opioid agonist-mediated changes in IP production in the isolated, rabbit iris-ciliary body (ICB). In one set of experiments, ICB segments were treated with the delta agonist, SNC80 (10(-12)-10(-7) mol/l) alone. Other experiments were conducted utilizing SNC80 following pretreatment with either phosducin (Gbetagamma-subunit scavenger), PTX, or the delta antagonist, naltrindole. IP production was measured by ion exchange chromatography. Basal levels of IPs in the rabbit ICB were 58,287 +/- 2162, 15,218 +/- 969 and 2083 +/- 367 dpm/mg protein for IP1, IP2 and IP3, respectively. The highly selective delta-opioid receptor agonist, SNC80, produced concentration-dependent increases in the levels of the IPs in the ICB, which were diminished in the presence of the delta antagonist, naltrindole, indicating the effect was mediated via activation of delta-opioid receptors. Pretreatment of tissues with PTX (75 ng/ml), completely abolished the concentration-dependent production of IPs generated by SNC80 (10(-11)-10(-7) mol/l). In addition, pretreatment with phosducin (1 nmol/l) ablated the SNC80 (1 nmol/l)-induced increase in the formation of all three inositol phosphates. Results from this study indicate that the delta-opioid receptor-mediated increase in IP production is a PTX-sensitive G(i/o) response that involves participation of Gbetagamma-subunits. Thus, delta-opioid receptor activation by SNC80 in the ICB could be responsible, in part, for suppression of aqueous humor dynamics.
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PMID:Delta-opioid agonist-stimulated inositol phosphate formation in isolated, rabbit iris-ciliary bodies: role of G(i/o) proteins and Gbetagamma-subunits. 1460 52