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At least 1,135 records · Page 63Linked to original sources
PubMed1999

Effect of protein hydration on receptor conformation: decreased levels of bound water promote metarhodopsin II formation.

Neutral solutes were used to investigate the effects of osmotic stress both on the ability of rhodopsin to undergo its activating conformation change and on acyl chain packing in the rod outer segment (ROS) disk membrane. The equilibrium concentration of metarhodopsin II (MII), the conformation of photoactivated rhodopsin, which binds and activates transducin, was increased by glycerol, sucrose, and stachyose in a manner which was linear with osmolality. Analysis of this shift in equilibrium in terms of the dependence of ln(Keq) on osmolality revealed that 20 +/- 1 water molecules are released during the MI-to-MII transition at 20 degrees C, and at 35 degrees C 13 +/- 1 waters are released. At 35 degrees C the average time constant for MII formation was increased from 1.20 +/- 0.09 ms to 1.63 +/- 0.09 ms by addition of 1 osmolal sucrose or glycerol. The effect of the neutral solutes on acyl chain packing in the ROS disk membrane was assessed via measurements of the fluorescence lifetime and anisotropy decay of 1,6-diphenyl-1,3,5-hexatriene (DPH). Analysis of the anisotropy decay of DPH in terms of the rotational diffusion model showed that the angular width of the equilibrium orientational distribution of DPH about the membrane normal was progressively narrowed by increased osmolality. The parameter fv, which is proportional to the overlap between the DPH orientational probability distribution and a random orientational distribution, was reduced by the osmolytes in a manner which was linear with osmolality. This study highlights the potentially opposing interplay between the effect of membrane surface hydration on both the lipid bilayer and integral membrane protein structure. Our results further demonstrate that the binding and release of water molecules play an important role in modulating functional conformational changes for integral membrane proteins, as well as for soluble globular proteins.

Animals↗
PubMed1997

Alkyl-substituted amino acid amides and analogous di- and triamines: new non-peptide G protein activators.

Synthesis and pharmacological properties of new potent direct activators of heterotrimeric G proteins are described. Compounds were synthesized from protected amino acids with alkylamines using coupling reagents (CDI, DCC, and EDC). Alkyl-substituted amino acid amides and their corresponding di- and triamines were subjected to structure-activity analysis. All compounds activated membrane-bound HL-60 GTPases in a pertussis toxin-sensitive fashion. This suggests a specific effect of compounds on the carboxy terminus of a defined subclass of heterotrimeric G proteins, i.e., members of the G alpha i subfamily. Elongation of the alkyl chain and increasing the number of amino groups enhanced the potency of compounds on HL-60 membrane-bound GTPase. N-(2,5-Diaminopentyl)dodecylamine (21) was selected to study its mode of action employing purified pertussis toxin-sensitive G proteins. It stimulated G alpha subunits by inducing the release of bound GDP. In contrast to receptors G beta gamma complexes were not required for 21-mediated activation of G alpha. Moderate isoform selectivity of its action was observed within a group of highly homologous members of the Gi subfamily with G alpha o1 being activated at lowest concentrations, whereas higher concentrations were necessary for the stimulation of G alpha i1 or transducin. We conclude that these compounds represent important tools for studying G protein-dependent cellular functions.

Amides↗
PubMedDate not supplied

[The effect of proteolytic removal of the C-terminal fragment of rhodopsin on its ability to activate visual cascade].

The role of the C-terminal domain of rhodopsin in the activation of transducin was studied. The treatment of photoreceptor membranes with trypsin, thermolysin, and Asp-N-endoprotease led to the respective rhodopsin species devoid of 9, 12-, or 19-aa C-terminal fragments. It was shown that the removal of 9-aa fragment by trypsin does not affect the catalytic activity of the receptor, whereas the thermolysin-induced truncation of the rhodopsin C-terminus by 12 aa about 1.5-fold enhances its activity. The Asp-N-endoprotease-assisted removal of 19 aa (i.e., the shortening by seven more C-terminal aa) virtually unchanges the rhodopsin catalytic activity compared to the preparation truncated with thermolysin. These results suggest that the part of the rhodopsin C-terminal fragment between the sites of its cleavage by trypsin and thermolysin (Val337-Ser338-Lys339) inhibits the signal transduction from rhodopsin to the next component of visual cascade. The English version of the paper.

Amino Acid Sequence↗
PubMed2002

The transcription co-repressor TLE1 interacted with the intracellular region of gpl30 through its Q domain.

As the common signal transducer for IL-6 family cytokines, gp 130 interacts with various signal molecules. Our previous work found the amino-terminal enhancer of split (AES) molecule interacts with gp130 intracellular region through its conserved glutamine-rich (Q) domain. The Q domain in AES shares high homology with those in the transcription co-repressor transducin-like enhancer of split (TLE) proteins. The yeast two-hybrid assay, gluthione S-transferase fusion protein pull-down assay and immuno-co-precipitation assay indicated that the Q domain of TLE1 is capable of binding gp130 intracellular domain, and the intracellular membrane proximal region of gp 130 containing conserved Box1 and Box2 motifs seemed essential for this interaction. The interaction between gp130 and TLE1 indicated that molecules of TLE family might play a role in gp 130 signaling.

Antigens, CD↗
PubMed2003

A rapid method to attain isotope labeled small soluble peptides for NMR studies.

A widely applicable strategy is presented for efficient and rapid production of small water soluble peptides expressed as fusion proteins with the immunoglobulin-binding domain of streptococcal protein G. A simple extraction and purification scheme that includes a protease cleavage step to release the target peptide is described. The yield of authentic target peptide exceeds 10 mg per liter of culture. Production of U-13C, 15N and highly deuterated U-13C, 15N isotope labeled peptide is demonstrated for the 11 residue S2 peptide, corresponding to the C-terminus of the alpha-subunit of transducin, and the coiled coil trimerization domain from cartilage matrix protein (CMPcc), respectively. Heteronuclear two-dimensional NMR spectra are used for initial peptide characterization.

Animals↗
PubMed1991

A cyclic GMP-activated channel in dissociated cells of the chick pineal gland.

Phototransduction in the vertebrate retina is dependent in part on a cyclic GMP-activated ionic channel in the plasma membrane of rods and cones. But other vertebrate cells are also photosensitive. Cells of the chick pineal gland have a photosensitive circadian rhythm in melatonin secretion that persists in dissociated cell culture. Exposure to light causes inhibition of melatonin secretion, and entrainment of the intrinsic circadian oscillator. Chick pinealocytes express several 'retinal' proteins, including arrestin, transducin and a protein similar to the visual pigment rhodopsin. Pinealocytes of lower vertebrates display hyperpolarizing responses to brief pulses of light. Thus it is possible that some of the mechanisms of phototransduction are similar in retinal and pineal photoreceptors. We report here the first recordings of cyclic GMP-activated channels in an extraretinal photoreceptor. Application of GMP, but not cyclic AMP, to excised inside-out patches caused activation of a 15-25 pS cationic channel. These channels may be essential for phototransduction in the chick pineal gland.

Animals↗
PubMed1992

Hormonal stimulation of adenylyl cyclase through Gi-protein beta gamma subunits.

Agonist-bound receptors activate heterotrimeric (alpha beta gamma) G proteins by catalysing replacement by GTP of GDP bound to the alpha subunit, resulting in dissociation of alpha-GTP from the beta gamma subunits. In most cases, alpha-GTP carries the signal to effectors, as in hormonal stimulation and inhibition of adenylyl cyclase by alpha s and alpha i respectively. By contrast, genetic evidence in yeast and studies in mammalian cells suggest that beta gamma subunits of G proteins may also regulate effector pathways. Indeed, of the four recombinant mammalian adenylyl cyclases available for study, two, adenylyl cyclases II and IV, are stimulated by beta gamma. This effect of beta gamma requires costimulation by alpha s-GTP. This conditional pattern of effector responsiveness led to the prediction that receptors coupled to many G proteins will mediate elevation of cellular cyclic AMP, provided that Gs is also active. We now confirm this prediction. Coexpression of mutationally active alpha s with adenylyl cyclase II converted agonists that act through 'inhibitory' receptors (coupled to Gi) into stimulators of cAMP synthesis. Experiments using pertussis toxin and a putative scavenger of beta gamma, the alpha subunit of transducin, suggest that beta gamma subunits of the Gi proteins mediated this stimulation. These findings assign a new signalling function to beta gamma subunits of Gi proteins, the conditional stimulation of cAMP synthesis by adenylyl cyclase II.

Adenylate Cyclase Toxin↗
PubMed1994

Rhodopsin mutation G90D and a molecular mechanism for congenital night blindness.

Mutations in the gene for the visual pigment rhodopsin cause retinitis pigmentosa (RP) and congenital night blindness. Inheritance of the diseases is generally autosomal dominant and about 40 different rhodopsin mutations have been documented. Although the cell death and retinal degeneration associated with RP have been suggested to result from improper folding and accumulation of the mutant proteins in rod photoreceptor cells, this may not account for the disease in all cases. For example, RP mutations at Lys 296, site of Schiff base linkage to the retinal chromophore, result in constitutive activation of the protein in vitro; that is, the mutants can catalytically activate the G protein transducin in the absence of chromophore and in the absence of light. Similarly, mutation of Ala 292-->Glu activates opsin in vitro and causes night blindness. We show here that the mutation Gly 90-->Asp (G90D) in the second transmembrane segment of rhodopsin, which causes congenital night blindness, also constitutively activates opsin. Furthermore, we show that Asp 90 can substitute for the Schiff base counterion, Glu 113, which is located in the third transmembrane segment of the protein. This demonstrates the proximity of Asp 90 and Lys 296 in the three-dimensional structure of rhodopsin and suggests that the constitutively activating mutations operate by a common molecular mechanism, disrupting a salt bridge between Lys 296 and the Schiff base counterion, Glu 113.

Cell Line↗
PubMed1994

Functional dissection of the yeast Cyc8-Tup1 transcriptional co-repressor complex.

DNA-binding repressor proteins mediate regulation of yeast genes by cell type (Mcm1/alpha 2 and a1/alpha 2), glucose (Mig1) and oxygen (Rox1) (refs 1-4 respectively). An unusual feature of all these regulatory pathways is that transcriptional repression requires two physically associated proteins that do not bind DNA Cyc8(Ssn6) and Tup1. The Cyc8-Tup1 complex has been proposed to be a co-repressor that is recruited to target promoters by pathway-specific DNA-binding proteins, but the specific functions of the individual proteins are unknown. Here we show that when it is bound upstream of a functional promoter through the LexA DNA-binding domain, Tup1 represses transcription in the absence of Cyc8. Deletion analysis indicates that Tup1 contains at least two non-overlapping transcriptional repression regions with minimal primary sequence similarity, and a separable Cyc8-interaction domain. These Tup1 domains, which do not include the beta-transducin motifs, are necessary and partially sufficient for Tup1 function. We suggest that Tup1 performs the repression function of the Cyc8-Tup1 co-repressor complex, and that Cyc8 serves as a link with the pathway-specific DNA-binding proteins.

Amino Acid Sequence↗
PubMed1997

Interaction of Wnt and a Frizzled homologue triggers G-protein-linked phosphatidylinositol signalling.

In Drosophila, members of the frizzled family of tissue-polarity genes encode proteins that are likely to function as cell-surface receptors of the type known as Wnt receptors, and to initiate signal transduction across the cell membrane, although how they do this is unclear. We show here that the rat protein Frizzled-2 causes an increase in the release of intracellular calcium which is enhanced by Xwnt-5a, a member of the Wnt family. This release of intracellular calcium is suppressed by an inhibitor of the enzyme inositol monophosphatase and hence of the phosphatidylinositol signalling pathway; this suppression can be rescued by injection of the compound myo-inositol, which overcomes the decrease in this intermediate caused by the inhibitor. Agents that inhibit specific G-protein subunits, pertussis toxin, GDP-beta-S and alpha-transducin also inhibit the calcium release triggered by Xwnt-5a and rat Frizzled-2. Our results indicate that some Wnt proteins work through specific Frizzled homologues to stimulate the phosphatidylinositol signalling pathway via heterotrimeric G-protein subunits.

Animals↗
PubMed1994

Pinopsin is a chicken pineal photoreceptive molecule.

In avian pinealocytes, an environmental light signal resets the phase of the endogenous circadian pacemaker that controls the rhythmic production of melatonin. Investigation of the pineal phototransduction pathway should therefore reveal the molecular mechanism of the biological clock. The presence of rhodopsin-like photoreceptive pigment, transducin-like immunoreaction, and cyclic GMP-dependent cation-channel activity in the avian pinealocytes suggests that there is a similarity between retinal rod cells and pinealocytes in the phototransduction pathway. We have now cloned chicken pineal cDNA encoding the photoreceptive molecule, which is 43-48% identical in amino-acid sequence to vertebrate retinal opsins. Pineal opsin, produced by transfection of complementary DNA into cultured cells, was reconstituted with 11-cis-retinal, resulting in formation of a blue-sensitive pigment (lambda max approximately 470 nm). In the light of this functional evidence and because the gene is specifically expressed only in the pineal gland, we conclude that it is a pineal photosensor and name it pinopsin.

Amino Acid Sequence↗
PubMed1994

A polyadenylation factor subunit is the human homologue of the Drosophila suppressor of forked protein.

Polyadenylation of messenger RNA precursors is a complex process that requires multiple protein factors (for reviews, see refs 1, 2). Cleavage stimulation factor (CstF) is one of these, functioning together with cleavage-polyadenylation specificity factor, two cleavage factors, and poly(A)+ polymerase. CstF is composed of three subunits of M(r) 77, 64 and 50K. The 64K and 50K subunits contain, respectively, an RNP-type RNA-binding domain that contacts the pre-mRNA and transducin repeats characteristic of G-protein beta-subunits. Here we report the cloning and characterization of the 77K subunit of human CstF (referred to as 77K). We show that the 77K subunit is required for formation of active CstF and bridges the 64K and 50K subunits. Sequence analyses indicate that the 77K subunit is the homologue of the protein encoded by the Drosophila melanogaster suppressor of forked (su(f)) gene. Mutations in su(f) can enhance or suppress the effects of transposable element insertions, and our data indicate that this is due to changes in polyadenylation. Both the 77K subunit and the su(f) protein share homology with Saccharomyces cerevisiae RNA14, previously shown to be involved in mRNA metabolism. Our results thus also indicate that components of the complex polyadenylation machinery are conserved from yeast to man.

Amino Acid Sequence↗
PubMed1996

Phosphorylation-dependent activation of the Ras-GRF/CDC25Mm exchange factor by muscarinic receptors and G-protein beta gamma subunits.

Muscarinic receptors activate Ras through a pathway distinct from that mediated through translocation of the exchange factor mSos1 by receptor tyrosine kinases. Here we report that muscarinic receptors can activate another Ras exchange factor, CDC25Mm, or p140Ras-GRF (refs 5,6). In NIH-3T3 cells expressing subtype 1 human muscarinic receptors (hm1), the agonist carbachol selectively increased the specific activity and phosphorylation state of epitope-tagged Ras-GRF. This stimulation was reversed by protein phosphatase 1 (PP1), and prevented by transducin alpha-subunits. Carbachol treatment of neonatal rat brain explants increasd Ras exchange factor activity and the phosphorylation state of endogenous Ras-GRF. In COS-7 cells, cotransfection of hm1 or hm2 receptors with Ras-GRF conferred carbachol-dependent increases in exchange-factor activity, whereas cotransfection with G-protein beta gamma subunits caused a constitutive activation that was sensitive to PP1. These results demonstrate a G-protein-coupled mechanism for Ras activation, mediated by p140 Ras-GRF.

3T3 Cells↗
PubMed1996

RGS family members: GTPase-activating proteins for heterotrimeric G-protein alpha-subunits.

Signaling pathways using heterotrimeric guanine-nucleotide-binding-proteins (G proteins) trigger physiological responses elicited by hormones, neurotransmitters and sensory stimuli. GTP binding activates G proteins by dissociating G alpha from G beta gamma subunits, and GTP hydrolysis by G alpha subunits deactivates G proteins by allowing heterotrimers to reform. However, deactivation of G-protein signalling pathways in vivo can occur 10- to 100-fold faster than the rate of GTP hydrolysis of G alpha subunits in vitro, suggesting that GTPase-activating proteins (GAPs) deactivate G alpha subunits. Here we report that RGS (for regulator of G-protein signalling) proteins are GAPs for G alpha subunits. RGS1, RGS4 and GAIP (for G alpha-interacting protein) bind specifically and tightly to G alphai and G alpha0 in cell membranes treated with GDP and AlF4(-), and are GAPs for G alphai, G alpha0 and transducin alpha-subunits, but not for G alphas. Thus, these RGS proteins are likely to regulate a subset of the G-protein signalling pathways in mammalian cells. Our results provide insight into the mechanisms that govern the duration and specificity of physiological responses elicited by G-protein-mediated signalling pathways.

Animals↗
PubMed1998

Structure of rhodopsin.

Two-dimensional crystals of rhodopsin were studied to determine the arrangement of the transmembrane alpha helices. A combination of electron cryo-microscopy, image processing and electron crystallography was used to extract amplitudes and phases from images, and a three-dimensional map to a resolution of 7.5 A was calculated. Density peaks for all seven transmembrane helices were observed and the helix axes for all seven helices could be estimated. Near the intracellular side, which interacts with the G protein transducin, we observed three layers of helices arranged differently from bacteriorhodopsin. The arrangement opens up towards the extracellular side forming a cavity that serves as the binding pocket for the retinal. This cavity is closed towards the intracellular side by the long and highly tilted helix 3, and must be closed towards the extracellular side by the loop linking helices 4 and 5 that is linked by a disulphide bridge to the extracellular end of helix 3.

Animals↗
PubMed2004

Defects in RGS9 or its anchor protein R9AP in patients with slow photoreceptor deactivation.

The RGS proteins are GTPase activating proteins that accelerate the deactivation of G proteins in a variety of signalling pathways in eukaryotes. RGS9 deactivates the G proteins (transducins) in the rod and cone phototransduction cascades. It is anchored to photoreceptor membranes by the transmembrane protein R9AP (RGS9 anchor protein), which enhances RGS9 activity up to 70-fold. If RGS9 is absent or unable to interact with R9AP, there is a substantial delay in the recovery from light responses in mice. We identified five unrelated patients with recessive mutations in the genes encoding either RGS9 or R9AP who reported difficulty adapting to sudden changes in luminance levels mediated by cones. Standard visual acuity was normal to moderately subnormal, but the ability to see moving objects, especially with low-contrast, was severely reduced despite full visual fields; we have termed this condition bradyopsia. To our knowledge, these patients represent the first identified humans with a phenotype associated with reduced RGS activity in any organ.

Adaptation, Physiological↗
PubMed2003

Spontaneous activity of opsin apoprotein is a cause of Leber congenital amaurosis.

Mutations in Rpe65 disrupt synthesis of the opsin chromophore ligand 11-cis-retinal and cause Leber congenital amaurosis (LCA), a severe, early-onset retinal dystrophy. To test whether light-independent signaling by unliganded opsin causes the degeneration, we used Rpe65-null mice, a model of LCA. Dark-adapted Rpe65-/- mice behaved as if light adapted, exhibiting reduced circulating current, accelerated response turn-off, and diminished intracellular calcium. A genetic block of transducin signaling completely rescued degeneration irrespective of an elevated level of retinyl ester. These studies clearly show that activation of sensory transduction by unliganded opsin, and not the accumulation of retinyl esters, causes light-independent retinal degeneration in LCA. A similar mechanism may also be responsible for degeneration induced by vitamin A deprivation.

Animals↗
PubMed2004

EGFR signaling attenuates Groucho-dependent repression to antagonize Notch transcriptional output.

Crosstalk between signaling pathways is crucial for the generation of complex and varied transcriptional networks. Antagonism between the EGF-receptor (EGFR) and Notch pathways in particular is well documented, although the underlying mechanism is poorly understood. The global corepressor Groucho (Gro) and its transducin-like Enhancer-of-split (TLE) mammalian homologs mediate repression by a myriad of repressors, including effectors of the Notch, Wnt (Wg) and TGF-beta (Dpp) signaling cascades. Given that there are genetic interactions between gro and components of the EGFR pathway (ref. 9 and P.H. et al., unpublished results), we tested whether Gro is at a crossroad between this and other pathways. Here we show that phosphorylation of Gro in response to MAPK activation weakens its repressor capacity, attenuating Gro-dependent transcriptional silencing by the Enhancer-of-split proteins, effectors of the Notch cascade. Thus, Gro is a new junction between signaling pathways, enabling EGFR signaling to antagonize transcriptional output by Notch and potentially other Gro-dependent pathways.

Animals↗