Flow of information in the light-triggered cyclic nucleotide cascade of vision.

Fung, Bernard K.-K.; Hurley, James B.; Stryer, Lubert

doi:10.1073/pnas.78.1.152

Cited by 680 publications

(295 citation statements)

References 21 publications

(10 reference statements)

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“…Light absorbed by rhodopsin in the rod outer segments causes conformational changes that activate the cascade through a catalyzed exchange of GDP for GTP on the R subunit of transducin (60,61). GDP-GTP exchange releases the γ complex of transducin, freeing GTP-R-transducin to interact with a phosphodiesterase enzyme that hydrolyzes cGMP, and releasing the transducin γ complex to interact with phosducin.…”

Section: Discussionmentioning

confidence: 99%

Light-Driven Translocation of the Protein Phosphatase 2A Complex Regulates Light/Dark Dephosphorylation of Phosducin and Rhodopsin

et al. 2002

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In steps of protein purification of bovine retinal protein phosphatase 2A (PP2A), phosducin dephosphorylation activity peaks coelute with a PP2A enzyme complex, shown by peptide sequence analysis to contain a B′ subunit, B56epsilon. Other PP2A complexes with a slightly larger (56.5 kDa) B′ subunit (sequenced to be B56alpha) or with the BR regulatory subunit have no phosducin dephosphorylation activity. Upon exposure to light, a significant increase in the immunoreactive protein level of the A, C, and B56epsilon PP2A subunits is observed in the cytosolic fraction of mouse retina, the phosducin dephosphorylation of which occurs rapidly. During dark exposure, these subunits translocate to the membrane fraction where rhodopsin is slowly dephosphorylated. This PP2A redistribution occurs in less than 1.5 min and is dependent upon light and not upon an intrinsic circadian rhythm. Forty times more of the A subunit (∼20 ng/mouse retina) and 9 times more of the C subunit (∼4 ng/mouse retina) than of the B56epsilon subunit (∼0.45 ng/mouse retina) redistribute, which suggests that the predominant form of the PP2A enzyme complex on the membrane in the dark is a dimer, consisting of only A and C subunits. We observe that the dimer favors phosphorylated opsin as a substrate, while the trimer, particularly the enzyme complex with the B56epsilon subunit, greatly prefers phosphorylated phosducin, with an activity several hundred times those of other substrates that were tested. This light-driven PP2A translocation provides a potential mechanism for efficient dephosphorylation of two critical photoreceptor transduction proteins, cytosolic phosducin and membrane-bound rhodopsin, by the same enzyme.

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Section: Discussionmentioning

confidence: 99%

Light-Driven Translocation of the Protein Phosphatase 2A Complex Regulates Light/Dark Dephosphorylation of Phosducin and Rhodopsin

et al. 2002

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“…In certain cases, e.g. stimulation of various types of adenylyi cyclase or activation of the retinal cGMP phosphodiesterase, the ~z subunit alone is capable of effector regulation [3][4][5]. In other cases, however, free/J?'…”

Section: Introductionmentioning

confidence: 99%

Expression, characterization and purification of soluble G‐protein βγ dimers composed of defined subunits in baculovirus‐infected insect cells

et al. 1992

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Recombinant β1γ2 dimers of signal‐transducing guanine nucleotide‐binding proteins (G‐proteins) carrying a mutation known to block isoprenylation of the γ2 subunit were expressed as a soluble protein in baculovirus‐infected insect cells. The soluble βγ dimer was analyzed by sucrose density gradient centrifugation and purified to near homogeneity in the absence of detergents. The sedimentation velocity studies gave an s 20,w value of 4.1 ± 0.4 S. The two subunits segregated as a dimer upon sucrose density gradient centrifugation and purification by sequential ion exchange and hydroxylapatite chromatography. The results show that baculovirus‐infected insect cells can be employed for high level production of pure G‐protein βγ dimers suitable for functional and structural characterization.

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“…Both G␣ and G␤␥ subunits can communicate receptor signals (1)(2)(3)(4)(5). Regions of G␤␥ complex involved in communicating the signal to effectors have not been well characterized.…”

mentioning

confidence: 99%

A surface on the G protein β-subunit involved in interactions with adenylyl cyclases

Chen¹,

Weng²,

Li³

et al. 1997

Proc. Natl. Acad. Sci. U.S.A.

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Receptor activation of heterotrimeric G proteins dissociates G␣ from the G␤␥ complex, allowing both to regulate effectors. Little is known about the effectorinteraction regions of G␤␥. We had used molecular modeling to dock a peptide encoding the region of residues 956-982 of adenylyl cyclase (AC) 2 onto G␤ to identify residues on G␤ that may interact with effectors. Based on predictions from the model, we synthesized peptides encoding sequences of residues 86-105 (G␤86-105) and 115-135 (G␤115-135) from G␤. The G␤86-105 peptide inhibited G␤␥ stimulation of AC2 and blocked G␤␥ inhibition of AC1 and by itself inhibited calmodulin-stimulated AC1, thus displaying partial agonist activity. Substitution of Met-101 with Asn in this peptide resulted in the loss of both the inhibitory and partial agonist activities. Most activities of the G␤115-135 peptide were similar to those of G␤86-105 but G␤115-135 was less efficacious in blocking G␤␥ inhibition of AC1. Substitution of Tyr-124 with Val in the G␤115-135 peptide diminished all of its activities. These results identify the region encoded by amino acids 84-143 of G␤ as a surface that is involved in transmitting signals to effectors.Heterotrimeric G proteins serve as signal transducers for a wide variety of receptors. Both G␣ and G␤␥ subunits can communicate receptor signals (1-5). Regions of G␤␥ complex involved in communicating the signal to effectors have not been well characterized. We had identified the region of residues 956-982 of adenylyl cyclase (AC) 2 as being involved in receiving signals from G␤␥ (6). By using the yeast twohybrid system, the AC2 region of residues 956-982 has been subsequently shown to interact with G␤ but not G␥ subunits (7). In recent studies we found that the peptide encoding residues 956-982 of AC2 can be crosslinked to G␤ when it is part of the free G␤␥ complex but not when it is part of the heterotrimer, indicating that the putative binding surface on G␤ for the AC2 peptide is occluded by interactions with G␣. On the basis of constraints deduced from the crosslinking studies and other biophysical criteria, we docked the AC2 peptide containing residues 956-982 onto the crystal structure of G␤ by using molecular modeling techniques (8). From this docking model, we have identified the regions of G␤ that are predicted to interact with the AC2 peptide. Herein we have tested whether peptides encoding the effector-interaction surface of G␤ predicted from the modeling (8) can modulate G␤␥ regulation of AC1 and AC2. MATERIALS AND METHODSMaterials. Reagents for peptide synthesis were from Bachem. [␣-32 P]ATP was from New England Nuclear. Tissue culture reagents and fetal calf serum was from GIBCO. All other chemicals used were the highest grade available.Peptide Synthesis. Peptides were synthesized on an Applied Biosystems peptide synthesizer (model 431A) and purified by HPLC on acetonitrile gradients. Purified peptides were lyophilized and stored at Ϫ20ЊC. When required peptides were dissolved in water to a final concentration of 1-3 mM....

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Flow of information in the light-triggered cyclic nucleotide cascade of vision.

Cited by 680 publications

References 21 publications

Light-Driven Translocation of the Protein Phosphatase 2A Complex Regulates Light/Dark Dephosphorylation of Phosducin and Rhodopsin

Light-Driven Translocation of the Protein Phosphatase 2A Complex Regulates Light/Dark Dephosphorylation of Phosducin and Rhodopsin

Expression, characterization and purification of soluble G‐protein βγ dimers composed of defined subunits in baculovirus‐infected insect cells

A surface on the G protein β-subunit involved in interactions with adenylyl cyclases

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