The light-activated GTP-dependent cyclic GMP phosphodiesterase complex of bovine retinal rod outer segments. Dark resolution of the catalytic and regulatory proteins.

Kohnken, Russell E.; Eadie, D M; Revzin, Arnold; McConnell, David G.

doi:10.1016/s0021-9258(18)43303-0

Cited by 39 publications

(22 citation statements)

References 21 publications

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“…The binding of rhodopsin to transducin triggers the dissociation of a tightly bound molecule of GDP, which then enables GTP to bind and to trigger an 'activating' conformational change within the XT subunit. It is the activated a,T subunit that binds to the retinal effector enzyme, the cyclic GMP phosphodiesterase (a heterotrimeric protein with two larger subunits, apDE=fPDE of M, -85000, and a third smaller subunit, yPDE of Mr -14000 [6,7]). The activated aT subunit stimulates the phosphodiesterase (PDE) to hydrolyse cyclic GMP; this stimulation continues until the GTPase activity of aT converts the bound GTP back to GDP. Rhodopsin is a member of the large class of G-protein-coupled membrane receptors that includes the a-and fl-adrenergic receptors and the muscarinic acetylcholine receptors (see [8]).…”

Section: Introductionmentioning

confidence: 99%

A C-terminal peptide of bovine rhodopsin binds to the transducin α-subunit and facilitates its activation

Phillips

Cerione

1994

Biochemical Journal

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In order to investigate the possible roles of the intracellular domains of rhodopsin in the functional coupling of the photoreceptor to transducin, different peptides that correspond to parts of the known sequence of rhodopsin were synthesized. Since we have found tht the binding of rhodopsin to the alpha subunit of transducin (alpha T) increases the susceptibility of alpha T to phosphorylation by protein kinase C-beta 1, we used this phosphorylation reaction as an initial screen for peptides that mimic the actions of rhodopsin. The results of this screen indicated that a peptide from the C-terminal tail of rhodopsin (amino acids 325-338; KNPLGDDEASTTVS-amide; designated as peptide 3) was capable of interacting with the alpha T subunit. Evidence that peptide 3 binds to alpha T at a site that overlaps the rhodopsin-binding domain was obtained from experiments showing that peptide 3 inhibited the rhodopsin-stimulated GTPase activity of alpha T and that this inhibition was overcome at high levels of rhodopsin. A potentially important outcome of the peptide 3/alpha T interaction is the facilitation of the activation of the alpha T subunit. This was first demonstrated in fluorescence experiments where the binding of peptide 3 was shown to strongly promote the enhancement of the tryptophane emission of alpha T that is elicited by the addition of NaF. Specifically, the EC50 for NaF was shifted from approximately 4 mM in the absence of peptide 3 to below 0.5 mM in the presence of peptide 3. Further verification that peptide 3 facilitated the ability of NaF to activate the alpha T subunit was obtained from experiments measuring the alpha T GDP/NaF-stimulated hydrolysis of cyclic GMP by the cyclic GMP phosphodiesterase.

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Section: Introductionmentioning

confidence: 99%

A C-terminal peptide of bovine rhodopsin binds to the transducin α-subunit and facilitates its activation

Phillips

Cerione

1994

Biochemical Journal

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“…0006-2960/86/0425-0651$01.50/0 three subunits of Mt 88 000, 84 000, and 13 000 (Baehr et al, 1979;Kohnken et al, 1981) designated respectively as a, (3, and 7. The functions of the individual subunits of these two proteins and whether some of them leave the membrane and become "solubilized" during the activation process are a subject of investigation in several laboratories.…”

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confidence: 99%

Kinetic studies suggest that light-activated cyclic GMP phosphodiesterase is a complex with G-protein subunits

Sitaramayya¹,

Harkness²,

Parkes³

et al. 1986

Biochemistry

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Cyclic GMP phosphodiesterase (PDE) in rod disk membranes has three subunits of molecular weight 88 000 (alpha), 84 000 (beta), and 13 000 (gamma). Physiological activation of the enzyme by light is mediated by a GTP binding protein (G protein). The enzyme can also be activated by controlled digestion with trypsin, which destroys the gamma subunit, leaving the activated enzyme as PDE alpha beta [Hurley, J. B., & Stryer, L. (1982) J. Biol. Chem. 257, 11094-11099]. Addition of purified gamma subunit to PDE alpha beta inhibited the enzyme fully. This suggested the possibility that G protein could also activate PDE by removing the gamma subunit and leaving the active enzyme in the form of PDE alpha beta. Should this be true, the properties of light- and trypsin-activated enzymes should be comparable. We found this not to be the case. The Km of light-activated enzyme for cyclic GMP was about 0.9-1.4 mM while that of trypsin-activated enzyme was about 140 microM. The cyclic AMP Km was also different for the two enzymes: 6.7 mM for light-activated enzyme and 2.0 mM for trypsin-activated enzyme. The inhibition of both enzymes by the addition of purified gamma subunit also differed significantly. Trypsin-activated enzyme was fully inhibited by the addition of about 200 nM gamma, but light-activated enzyme could not be fully inhibited even with 2600 nM inhibitor subunit. The Ki of the trypsin-activated enzyme for gamma was 15 nM and of the light-activated enzyme 440 nM.(ABSTRACT TRUNCATED AT 250 WORDS)

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“…Miki et al (1974) showed that activation could occur if light preceded GTP treatment, but not vice versa.Experiments on activation by bleached rhodopsin further suggested a relatively stable, soluble activating agent (Pober & Bitensky, 1979).Reconstitution studies using purified components indicated that a GTP-binding protein was required for PDE activation. This protein has been called GTP-binding protein (Godchaux & Zimmerman, 1979), transducin (Fung et al, 1981), and G protein (Kohnken et al, 1981b;Baehr et al, 1982). Here we call it G protein.…”

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confidence: 99%

“…Here we call it G protein. G protein is composed of three nonidentical subunits with molecular weights of 40000 (GJ, 35000 (G^), and 8000 (Gy) (Kohnken et al, 1981b). This protein exhibits, in addition to PDE activation, GTPase activity afld GTP binding (Godchaux & Zimmerman, 1979) as well as a light-induced binding to ROS membranes that is reversed by GTP (Kuhn, 1980).…”

mentioning

confidence: 99%

Use of [.gamma.-32P]-8-azidoguanosine 5'-triphosphate as a probe of the guanosine 5'-triphosphate binding protein subunits in bovine rod outer segments

Kohnken¹,

Connell²

1985

Biochemistry

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In an in vitro incubation, 8-azidoguanosine 5'-[gamma-32P]triphosphate ( [gamma-32P]-8-azido-GTP) labeled bleached rhodopsin independent of ultraviolet light. Characterization of this labeling indicated that rhodopsin was phosphorylated with [gamma-32P]-8-azido-GTP as a phosphate donor. At low concentrations, ATP increased this labeling activity 5-fold. In the same incubation, [gamma-32P]-8-azido-GTP also labeled G alpha (Mr 40 000). This labeling was ultraviolet light dependent. G beta (Mr 35 000) was also labeled dependent for the most part upon ultraviolet light, but a smaller component of labeling appeared to result from phosphorylation. Differential labeling of G alpha and G beta was found to vary intricately with experimental conditions, especially prebleaching of rhodopsin, tonicity of the medium, and the presence or absence of 2-mercaptoethanol. Affinity labeling of G alpha and G beta by [gamma-32P]-8-azido-GTP in competition with ATP or GTP was kinetically complex, consistent with possible multiple binding sites for GTP on both subunits. Independent evidence for two or more binding sites on G alpha has been offered by other laboratories, and recently, at least one binding site on G beta and its analogues among the N proteins of adenylate cyclases has been identified.

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The light-activated GTP-dependent cyclic GMP phosphodiesterase complex of bovine retinal rod outer segments. Dark resolution of the catalytic and regulatory proteins.

Cited by 39 publications

References 21 publications

A C-terminal peptide of bovine rhodopsin binds to the transducin α-subunit and facilitates its activation

A C-terminal peptide of bovine rhodopsin binds to the transducin α-subunit and facilitates its activation

Kinetic studies suggest that light-activated cyclic GMP phosphodiesterase is a complex with G-protein subunits

Use of [.gamma.-32P]-8-azidoguanosine 5'-triphosphate as a probe of the guanosine 5'-triphosphate binding protein subunits in bovine rod outer segments

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