Injection of GTP-binding protein or cyclic GMP phosphodiesterase hyperpolarizes retinal rods

Clack, James W.; Oakley, Bruce; Stein, Peter J.

doi:10.1038/305050a0

Cited by 33 publications

(12 citation statements)

References 20 publications

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“…However, it should be emphasized that these studies do not exclude the PDE deactivation by P␥ phosphorylation, because ATP was omitted from their experimental conditions (39), or under their conditions the P␥ phosphorylation might have been suppressed due to the use of a P␥ mutant that can not interact with GTP/T␣ (40). We also note that using hydrolysis-resistant GTP analogs, electrophysiological studies have already suggested a GTP hydrolysis-independent PDE turnoff (41,42). Under their conditions, P␥ phosphorylation by Cdk5 could be a mechanism for the GTP hydrolysis-independent PDE turnoff.…”

Section: Discussionmentioning

confidence: 99%

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Phosphorylation by Cyclin-dependent Protein Kinase 5 of the Regulatory Subunit of Retinal cGMP Phosphodiesterase

Hayashi¹,

Matsuura²,

Kachi³

et al. 2000

Journal of Biological Chemistry

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Retinal cGMP phosphodiesterase (PDE) is regulated by P␥, the regulatory subunit of PDE, and GTP/T␣, the GTP-bound ␣ subunit of transducin. In the accompanying paper (Matsuura, I., Bondarenko, V. A., Maeda, T., Kachi, S., Yamazaki, M., Usukura, J., Hayashi, F., and Yamazaki, A. (2000) J. Biol. Chem. 275, 32950 -32957), we have shown that all known P␥s contain a specific phosphorylation motif for cyclin-dependent protein kinase 5 (Cdk5) and that the unknown kinase is Cdk5 complexed with its activator. Here, using frog rod photoreceptor outer segments (ROS) isolated by a new method, we show that Cdk5 is involved in light-dependent P␥ phosphorylation in vivo. Under dark conditions only negligible amounts of P␥ were phosphorylated. However, under illumination that bleached less than 0.3% of the rhodopsin, ϳ4% of the total P␥ was phosphorylated in less than 10 s. P␥ dephosphorylation occurred in less than 1 s after the light was turned off. Analysis of the phosphorylated amino acid, inhibition of P␥ phosphorylation by Cdk inhibitors in vivo and in vitro, and twodimensional peptide map analysis of P␥ phosphorylated in vivo and in vitro indicate that Cdk5 phosphorylates a P␥ threonine in the same manner in vivo and in vitro. These observations, together with immunological data showing the presence of Cdk5 in ROS, suggest that Cdk5 is involved in light-dependent P␥ phosphorylation in ROS and that the phosphorylation is significant and reversible. In an homogenate of frog ROS, PDE activated by light/guanosine 5 -O-(3-thiotriphosphate) (GTP␥S) was inhibited by P␥ alone, but not by P␥ complexed with GDP/T␣ or GTP␥S/T␣. Under these conditions, P␥ phosphorylated by Cdk5 inhibited the light/ GTP␥S-activated PDE even in the presence of GTP␥S/ T␣. These observations suggest that phosphorylated P␥ interacts with and inhibits light/GTP␥S-activated PDE, but does not interact with GTP␥S/T␣ in the homogenate. Together, our results strongly suggest that after activation of PDE by light/GTP, P␥ is phosphorylated by Cdk5 and the phosphorylated P␥ inhibits GTP/T␣-activated PDE, even in the presence of GTP/T␣ in ROS.The hydrolysis of cGMP by PDE 1 in vertebrate ROS is directly involved in visual signal transduction (1, 2). The inactive PDE is composed of P␣␤, catalytic subunits, and two P␥s, regulatory subunits (3-6). In amphibian ROS, PDE is regulated similarly to that in mammalian ROS (7, 8): PDE catalytic activity is controlled by P␥ and GTP/T␣. In frog ROS membranes, bleached rhodopsin stimulates GTP/GDP exchange on T␣ (9), and the GTP/T␣ formed is released from membranebound T␤␥ (9, 10). The free GTP/T␣ interacts with P␣␤␥␥, and P␥ complexed with GTP/T␣ is released from P␣␤/membranes (10 -12). PDE is thereby activated. The release of the P␥ complex is detected even in an isotonic buffer containing Mg 2ϩ , and P␥ complexed with GTP␥S/T␣ can be isolated using sequential column chromatography (10). During PDE activation, P␥-less P␣␤ binds tightly to membranes. 2 In the recovery processes of frog ROS, after GTP hydrolysis by T␣, P␥ remai...

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Section: Discussionmentioning

confidence: 99%

“…We also need to explore the regulatory mechanism of the P␥ phosphorylation by Cdk5. Moreover, electrophysiological study is required to show that Cdk5 is involved in the GTP hydrolysisindependent PDE turnoff described previously (41,42). Cdk inhibitors we used in this study will be useful for the electrophysiological study.…”

Section: Discussionmentioning

confidence: 99%

Phosphorylation by Cyclin-dependent Protein Kinase 5 of the Regulatory Subunit of Retinal cGMP Phosphodiesterase

Hayashi¹,

Matsuura²,

Kachi³

et al. 2000

Journal of Biological Chemistry

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“…The number of Ga genes identified in most organisms is much greater than those identified for Go and G., which may be due to the relative ease of identification or perhaps to a lower number of G# and G. subunits due to the ability of different Ga subunits to interact with the same Gas complex. Some Ga subunits, such as the mammalian Gas or the Gat of transducin, appear to be the sole component required for activation of the effector and therefore the primary signal transducer of the heterotrimer (10,11). Many Ga genes have been identified from a diverse set of eukaryotic organisms (microbes, animals, and plants); however, in most cases relatively little is known about the signal transduction processes in which they function (refs.…”

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confidence: 99%

Identification of Dictyostelium G alpha genes expressed during multicellular development.

Hadwiger

Wilkie

Strathmann

et al. 1991

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

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Guanine nucleotide-binding protein (G protein)-mediated signal transduction constitutes a common mechanism by which cells receive and respond to a diverse set of environmental signals. Many of the signals involved in the developmental life cycle of the slime mold Dictyostelium have been postulated to be transduced by such pathways and, in some cases, these pathways have been demonstrated to be dependent on specific G proteins. Using the polymerase chain reaction, we have identified two additional Dictyostelium G alpha genes, G alpha 4 and G alpha 5, that are developmentally regulated. Transcripts from both of these genes are primarily expressed during the multicellular stages of development, suggesting possible roles in cell differentiation or morphogenesis. The entire G alpha 4 gene was sequenced and found to encode a protein consisting of 345 amino acids. The G alpha 4 subunit is homologous to other previously identified G alpha subunits, including the Dictyostelium G alpha 1 (43% identity) and G alpha 2 (41% identity) subunits. However, the G alpha 4 subunit contains some unusual sequence divergences in residues highly conserved among most eukaryotic G alpha subunits, suggesting that G alpha 4 may be a member of another class of G alpha subunits.

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“…Gt-p[NH]ppG is functionally closer to Gt-GTP than to Gt-GDP because i) p[NH]ppG potently extracts Gt from ROS (16), ii) a light triggered near-infrared light scattering "dissociation signal" (presumably of Gt from R*) is observed in the presence of either GTP, p[NH]ppG, or GTP-γ-S, but not in the presence of GDP (6,11), iii) the dissociation constants for the Gt/GTP and Gt/ p[NH]ppG interactions are similar (11), iv) Gt-p[NH]ppG activates PDE while Gt-GDP does not (1,17), and v) limited trypsin proteolysis patterns of Gt-p[NH]ppG and Gt-GTP-γ-S are identical, while the proteolysis pattern of Gt-GDP is distinct (15,18,19). It appears, therefore, that there is little structural or functional difference between Gt-GTP and Gt-p[NH]ppG.…”

Section: Resultsmentioning

confidence: 99%

“…Thus, it appears that Gt carrying a guanosine triphosphate moiety remains accessible to the activated receptor in both systems. It has been shown that Gt-p[NH]ppG, when injected into the toad rod outer segment, causes reversible membrane hyperpolarizations (17). Because p[NH]ppG is hydrolysis-resistant, one might expect that injection of Gt-p[NH]ppG would lead to a maintained activation of rod PDE and, thus, irreversibly hyperpolarize the membrane.…”

Section: Resultsmentioning

confidence: 99%

Affinity of transducin for photoactivated rhodopsin: dependence on nucleotide binding state

Clack¹

2008

BMB Reports

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The interaction of the rod GTP binding protein, Transducin (Gt), with bleached Rhodopsin (R*) was investigated by measuring radiolabeled guanine nucleotide binding to and release from soluble and/or membrane-bound Gt by reconstituting Gt containing bound GDP (Gt-GDP) or the hydrolysis-resistant GTP analog guanylyl imidodiphosphate (Gt-p[NH]ppG) with R* under physiological conditions. Release of GDP and p[NH]ppG from Gt occurred to the same extent and with the same light sensitivity both in the presence and absence of added GTP. Significant amounts of Gt without bound nucleotide (Gt-) were generated. When ROS containing bleached rhodopsin (R*) were centrifuged in low ionic strength buffer, Gt-remained associated with the membrane fraction, whereas Gt-GDP remained in the soluble fraction. These results suggest that Gt-GDP and Gt-p[NH]ppG have similar affinities for R*. The results also suggest that Gt-, rather than Gt-GDP, is the moiety which exhibits tight, "light-induced" binding to rhodopsin. [BMB reports 2008; 41(7): 555-560]

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Injection of GTP-binding protein or cyclic GMP phosphodiesterase hyperpolarizes retinal rods

Cited by 33 publications

References 20 publications

Phosphorylation by Cyclin-dependent Protein Kinase 5 of the Regulatory Subunit of Retinal cGMP Phosphodiesterase

Phosphorylation by Cyclin-dependent Protein Kinase 5 of the Regulatory Subunit of Retinal cGMP Phosphodiesterase

Identification of Dictyostelium G alpha genes expressed during multicellular development.

Affinity of transducin for photoactivated rhodopsin: dependence on nucleotide binding state

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