UMLS:C0043167 - FACTA Search

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Query: UMLS:C0043167 (pertussis)
19,595 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Freshly prepared proteolyzed (deprenylated) T beta gamma and material isolated from retina are inert with respect to activating T alpha in the presence of R* in detergent and in disk membranes. In addition, proteolyzed T beta gamma is also incapable of supporting the pertussis toxin-catalyzed ADP ribosylation of T alpha-GDP. These experiments show that isoprenylation/methylation is essential for the fruitful interactions between T alpha and T beta gamma at the membrane. When tested for its ability to support GTP-for-GDP exchange catalyzed by R*, demethylated T beta gamma proved to be approximately 50% as active as methylated T beta gamma in photoreceptor disk membranes (Fig. 3) and in reconstituted liposomes containing rhodopsin. In detergent, no difference was observed between methylated and demethylated T beta gamma, suggesting no role at all for the methyl group in functional interactions between T alpha, T beta gamma, and R*. The twofold activity difference observed in membranes can be accounted for by the twofold lessened affinity of the demethylated T beta gamma, compared with its methylated counterpart, for membranes in the presence of R* and T alpha. It is interesting to note that a substantially larger difference (> 10-fold) in the relative binding of methylated versus demethylated T beta gamma to membranes is observed in the absence of R* and T alpha. However, R* has a substantial affinity for T alpha beta gamma, and the influence of R* and T alpha greatly reduces any differences resulting from the presence or absence of a methyl group on T beta gamma. The results from studies of demethylated T beta gamma demonstrate that specific lipid-receptor interactions are unlikely to play a critical role in the rhodopsin-transducin system, and further show that the effect of methylation is probably due to the increased hydrophobicity of methylated T beta gamma versus its unmethylated counterpart. These studies are, of course, relevant to heterotrimeric G proteins, and specifically to the interactions of receptor (R*) with T alpha and T beta gamma. If a hydrophobic lipid-lipid mechanism is operative, the state of methylation would be expected to have a more profound effect on the membrane-associative properties of farnesylated proteins, but not on those of geranylgeranylated proteins. The increased hydrophobicity of the C20 geranylgeranyl group relative to the C15 farnesyl group will compensate for the loss of the methyl substituent. The results obtained in the transducin-rhodopsin system can be contrasted with the effect of gamma-subunit methylation on effector enzyme activation. In the case of the geranylgeranylated beta 1 gamma 2, methylation proved to have only a small effect on PIPLC beta activation (Fig. 4B). An approximately 25% diminution in efficacy, but not potency, was observed for the demethylated geranylgeranylated beta 1 gamma 2 versus its methylated counterpart. This again shows that specific lipid-protein interactions are unimportant. The effect of methylation on membrane binding would be expected to be small, given that beta 1 gamma 2 is geranylgeranylated. It is of interest to compare these results with those found with methylated and unmethylated T beta gamma as activators of PIPLC beta. In this instance there was a large effect noted, with methylated T beta gamma being at least 10-fold more potent than its unmethylated counterpart with respect to activating either enzyme (Fig. 4A). This result is readily understandable in light of the role of methylation in selectively enhancing hydrophobicity of farnesylated proteins as opposed to geranyl-geranylated proteins. Similar results were obtained for the activation of PI3K, further strengthening the conclusion that it is lipid-lipid interactions that direct beta gamma subunit membrane association. (ABSTRACT TRUNCATED)
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PMID:Isoprenylation/methylation and transducin function. 1080 Jun 94

Light induces retinal synthesis via photoactivation of a small amount of Chlamydomonas rhodopsin pigment (Foster et al., Proc. Natl. Acad. Sci. USA 85, 6379-6383). A reducing agent [dithiothreitol (DDT) or mercaptoacetic acid (MAA)] also induces retinal synthesis in the dark via a rhodopsin with a chromophore. If the opsin is saturated with retinal and is bleached with light in the presence of a thiol trapping agent, the bleaching becomes irreversible. We conclude that the reducing agent as well as light break a disulfide bond resulting in activation of the rhodopsin and induction of carotenogenesis. Both the chemical and light induction is inhibited by GDPbetaS and pertussis toxin. Breaking the bridge between the 3rd and 5th helix may lead to increased proton accessibility of Asp134 leading to the rolling of the 3rd helix and MetaIIb formation.
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PMID:Reducing agents and light break an S-S bond activating rhodopsin in vivo in Chlamydomonas. 1096 59

The retinal receptor rhodopsin undergoes a conformational change upon light excitation to form metarhodopsin II (Meta II), which allows interaction and activation of its cognate G protein, transducin (G(t)). A C-terminal 11-amino acid peptide from transducin, G(talpha)-(340-350), has been shown to both bind and stabilize the Meta II conformation, mimicking heterotrimeric G(t). Using a combinatorial library we identified analogs of G(talpha)-(340-350) that bound light-activated rhodopsin with high affinity (Martin, E. L., Rens-Domiano, S., Schatz, P. J., and Hamm, H. E. (1996) J. Biol. Chem. 271, 361-366). We have made peptides with key substitutions either on the background of the native G(talpha)-(340-350) sequence or on the high affinity sequences and used the stabilization of Meta II as a tool to determine which amino acids are critical in G protein-rhodopsin interaction. Removal of the positive charge at the N termini by acylation or delocalization of the charge by K to R substitution enhances the affinity of the G(talpha)-(340-350) peptides for Meta II, whereas a decrease was observed following C-terminal amidation. Cys-347, a residue conserved in pertussis toxin-sensitive G proteins, was shown to interact with a hydrophobic site in Meta II. These studies provide further insight into the mechanism of interaction between the G(talpha) C terminus and light-activated rhodopsin.
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PMID:Structural requirements for the stabilization of metarhodopsin II by the C terminus of the alpha subunit of transducin. 1101 24

Mutations within the "X1BBX2X3B" motif or its variants in the junction of the third intracellular (i3) loop and the sixth transmembrane domain (TM6) have been shown to lead to constitutive activation of several G protein-coupled receptors (GPCRs). In this study, T6.34(279) at the X3 locus of the rat mu opioid receptor was mutated to Lys and Asp, and the mutants were examined for binding and signaling properties. The T6.34(279)K mutant was poorly expressed, and pretreatment with naloxone greatly enhanced its expression. This construct exhibited properties identified previously with constitutive activation: (1) compared with the wild type, it produced much higher agonist-independent [35S]GTPgammaS binding, which was abolished by pertussis toxin treatment; (2) it displayed an enhanced affinity for the agonist DAMGO similar to that of the high-affinity state of the wild type, which was not altered by GTPgammaS, while having unchanged affinity for the antagonist diprenorphine. The T6.34(279)K mutant displayed a higher intracellular receptor pool than the wild type. Naloxone inhibited the basal [35S]GTPgammaS binding of the T6.34(279)K mutant, demonstrating inverse agonist activity at this mutant receptor. In contrast, the T6.34(279)D substitution did not increase basal [35S]GTPgammaS binding, greatly reduced agonist-promoted [35S]GTPgammaS binding, and markedly decreased affinity for DAMGO. Thus, the T6.34(279)D mutant adopts conformations corresponding to inactive states of the receptor. The results were interpreted in the structural context of a model for the mu opioid receptor that incorporates the information from the crystal structure of rhodopsin. The interaction of T6.34(279) with R3.50(165) in the mu opioid receptor is considered to stabilize the inactive conformations. The T6.34(279)K substitution would then disrupt this interaction and support agonist-free activation, while T6.34(279)D mutation should strengthen this interaction which keeps the receptor in inactive states. T6.34(279) may, in addition, interact with the neighboring R6.35(280) to help constrain the receptor in inactive states, and T6.34(279)K and T6.34(279)D mutations would affect this interaction by disrupting or strengthening it, respectively. To the best of our knowledge, the results presented here represent the first structurally rationalized demonstration that mutations of this locus can lead to dramatically different properties of a GPCR.
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PMID:Functional role of a conserved motif in TM6 of the rat mu opioid receptor: constitutively active and inactive receptors result from substitutions of Thr6.34(279) with Lys and Asp. 1169 97

Many lines of evidence show that membranes contain microdomains, "lipid rafts", that are different from the rest of the membrane in specific lipid and protein composition. In several biological systems, they were shown to be necessary for trafficking and signal transduction. Here, we investigate if lipid rafts have a role in the regulation of the G protein-mediated pathway underlying vertebrate phototransduction. Photoreceptor membranes contain detergent-resistant membrane (DRM) rafts. Rhodopsin and cGMP phosphodiesterase are found in raft and nonraft portions of the membrane; guanylate cyclase is found exclusively in the raft. Distribution of these proteins does not change in the light or dark. In contrast, the G protein transducin, the RGS9-1-Gbeta5L complex, and the p44 isoform of arrestin undergo dramatic translocation to the raft upon illumination. Phosphorylation of RGS9-1 occurs exclusively in the raft. GTPgammaS or pertussis toxin prevent the light-mediated translocation of transducin and RGS9-1, whereas AlF(minus sign)(4) causes both proteins to move to the raft in the dark. This shows that the Galphat-RGS9-1-Gbeta5L complex has the highest affinity to rafts in the transition state of the GTPase. GTPgammaS binds to transducin at a significantly slower rate in the raft, indicating that this translocation results in a reduced rhodopsin-transducin coupling. Thus, an external signal can rearrange components of a G protein pathway in specific domains of the cell membrane, changing its signaling properties. These findings could reveal a novel mechanism utilized by the cells for regulation of G protein-mediated signal transduction.
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PMID:Signal-dependent translocation of transducin, RGS9-1-Gbeta5L complex, and arrestin to detergent-resistant membrane rafts in photoreceptors. 1188 95

Rod photoreceptors are highly compartmentalized sensory neurons that maintain strict ultrastructural and molecular polarity. Structural subdivisions include the outer segment, inner segment, cell body, and synaptic terminal. The visual pigment rhodopsin is found predominantly in membranes of the rod cell outer segment but becomes mislocalized, appearing throughout the plasma membrane of the cell in many retinal diseases and injuries. Currently, there is no known link between rhodopsin redistribution and rod cell death. We propose that activation of mislocalized rhodopsin kills rod cells by stimulating normally inaccessible signaling pathways. This hypothesis was tested in primary retinal cell cultures, which contain photoreceptors. In rod photoreceptors, opsin immunofluorescence occurred throughout the rod cell plasma membrane. Activation of this mislocalized opsin by photostimulation after formation of isorhodopsin or by incubation with beta-ionone (opsin agonist) killed 19-30% of rod cells. Rod cell death was apoptotic, as indicated by marked chromatin condensation and the requirement for caspase-3 activation. Rod cell death could be induced by forskolin (adenylate cyclase agonist), and conversely, beta-ionone-induced cell death could be blocked by cotreatment with SQ22536 (an adenylate cyclase inhibitor). Pertussis toxin (a G protein inhibitor) also blocked beta-ionone-induced cell death. The data support a mechanism by which activation of mislocalized opsin initiates apoptotic rod cell death through G protein stimulation of adenylate cyclase.
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PMID:Activation of mislocalized opsin kills rod cells: a novel mechanism for rod cell death in retinal disease. 1194 54

1. The adrenoceptors (AR) are an important subfamily of rhodopsin-like G-protein-coupled receptors that couple to an increasingly large number of signalling mechanisms. Two important factors that determine the pathways that are used are the C-terminal region of the receptor and the agonist used to activate the receptor. 2. Studies of splice variants of the mouse beta3-AR showed that the C-terminus is a factor controlling the signalling characteristics. Although these receptors differ only at the C-terminus, the beta3b-AR coupled to both Gs and Gi, whereas the beta3a-AR coupled solely to Gs. 3. Examination of four splice variants of the human alpha1A-AR showed that all were able to couple to pertussis toxin-sensitive G-proteins, even though they have radically different C-terminal regions. 4. Comparison of the effects of the beta3-AR ligands CL316243 and SR59230A showed that both can activate the mouse beta3-AR but that SR59230A uses pathways other than cAMP accumulation in 3T3-F442A cells. 5. Examination of a series of alpha1-AR agonists for their ability to activate a number of signalling pathways revealed that A61603 acted as a full agonist in all assays, whereas oxymetazoline was unable to cause cAMP accumulation, suggesting agonist-selective signalling at the human alpha1A-AR.
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PMID:The Janus faces of adrenoceptors: factors controlling the coupling of adrenoceptors to multiple signal transduction pathways. 1556 1

Neovascularization of the retina and choroids is the pathological hallmark of many retinopathies, but its molecular mechanisms remain unclear. Vascular endothelial growth factor (VEGF), which is induced by hypoxia or cytokines, plays a critical role in the abnormal growth of blood vessels. In this study, we report that visible light exposure induces VEGF gene expression in retinoblastoma Y79 cells. Fluorescent light exposure (700 lux, wavelength 400 approximately 740 nm) caused a significant increase in VEGF transcripts and protein levels. Such an induction seemed to be specific to certain cells, including photoreceptor cells, because light-induced VEGF expression was not observed in either nontransformed cells, such as retinal pigment epithelium cells, and bovine aortic endothelial cells or transformed cells, such as CV-1 and HepG2 cells. Pertussis toxin and guanosine 5'-[beta-thio]diphosphate, specific inhibitors for rhodopsin-associated G protein, blunted this induction. Progressive deletion and site-specific mutation analyses indicate that light stimulation increases VEGF promoter activity through G+C-rich sequence, which is proven by Sp1 binding sites by supershift assays. Electrophoretic mobility shift assays show that light stimulation increases Sp1 binding. Synthetic retinoic acid receptor-alpha (RARalpha) antagonist completely abrogated light-mediated increase in VEGF expression. Transfection of Y79 cells with dominant negative mutant of RARalpha significantly attenuated the light-mediated induction of VEGF promoter activity. In conclusion, our data indicate that light exposure increases VEGF expression through the mechanisms involving activation of Sp1 and RARalpha signaling in Y79 cells. This study provides new insight into the role of visible light in the transcription and induction of VEGF gene expression.
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PMID:Visible light exposure induces VEGF gene expression through activation of retinoic acid receptor-alpha in retinoblastoma Y79 cells. 1561 98

The highly conserved Arg in the so-called DRY motif (Asp-Arg-Tyr) at the intracellular end of transmembrane helix 3 is in general considered as an essential residue for G protein coupling in rhodopsin-like seven transmembrane (7TM) receptors. In the open reading frame 74 (ORF74) receptor encoded by equine herpesvirus 2 (EHV2), the DRY motif is substituted with a DTW motif. Nevertheless, this receptor signaled with high constitutive activity through Gi as determined by a receptor-mediated inhibition of forskolin-induced cAMP-production and by an induction of the serum response element-driven transcriptional activity through a pertussis toxin-sensitive manner. Gs and Gq were not activated constitutively as determined by the lack of inositol phosphate turnover and activities of the three transcription factors: cAMP response element-binding protein (CREB), nuclear factor-kappaB, and nuclear factor of activated T cells. Coexpression of the ORF74-EHV2 receptor with the promiscuous G protein Gqi4myr supported the constitutive Gi activation as determined by inositol phosphate turnover and CREB activation. The constitutive activity was inhibited by nonpeptide inverse agonists with micromolar potencies, and the chemokine CXCL6 acted as a high-affinity agonist. It is noteworthy that reconstitution of the DRY motif resulted in a 4- to 5-fold decrease of the constitutive activity. Both the wild type and the receptor with the reconstituted DRY motif were expressed at the cell surface as indicated by immunohistochemistry and enzyme-linked immunosorbent assay analysis. It is concluded that the Arg of the DRY motif in transmembrane helix 3 is not essential for G protein coupling based on the constitutive as well as the ligand-mediated activity observed for ORF74-EHV2.
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PMID:High constitutive activity of a virus-encoded seven transmembrane receptor in the absence of the conserved DRY motif (Asp-Arg-Tyr) in transmembrane helix 3. 1585 6

Recent studies have identified four receptors that are the physiological targets for relaxin family peptides. All are class I (rhodopsin like) G-protein-coupled receptors with LGR7 (RXFP1) and LGR8 (RXFP2) being type C leucine-rich repeat-containing receptors, whereas GPCR135 (RXFP3) and GPCR142 (RXFP4) resemble receptors that respond to small peptides such as somatostatin and angiotensin II. The cognate ligands for the receptors have been identified: relaxin for RXFP1; INSL3 for RXFP2; relaxin 3 for RXFP3 and INSL5 for RXFP4. RXFP1 and RXFP2 receptors produce increases in intracellular cAMP levels upon stimulation, although the response is complex and contains a component sensitive to PI-3-kinase inhibitors. There is also evidence that RXFP1 can activate Erk1/2 and nitric oxide synthase, and relaxin has been reported to enter cells and activate glucocorticoid receptors. In contrast, RXFP3 and RXFP4 couple to Gi by a pertussis toxin-sensitive mechanism to cause inhibition of cAMP production. Now that the receptors for relaxin family peptides and their cognate ligands have been identified, we suggest a nomenclature for both the peptides and the receptors that we hope will be helpful to researchers in this rapidly advancing field.
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PMID:Receptors for relaxin family peptides. 1595 88

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