Amplification of phosphodiesterase activation is greatly reduced by rhodopsin phosphorylation

Miller, James L.; Fox, Deborah A.; Litman, Burton J.

doi:10.1021/bi00366a002

Cited by 100 publications

(61 citation statements)

References 26 publications

(31 reference statements)

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“…These results indicate that the nonphosphorylated P␥ retains its complex with GTP␥S/T␣ in the homogenate; however, after the P␥ is phosphorylated by Cdk5, the phosphorylated P␥ cannot keep its complex with GTP␥S/T␣ and inhibits effectively GTP␥S/T␣-activated PDE. We note that the effect of P␥ phosphorylation on the time course of light/GTP␥S-dependent PDE activation could not be measured by adding ATP to the homogenate, because rhodopsin in the homogenate is also phosphorylated, and the phosphorylated rhodopsin may affect the light/GTP␥S-activated PDE activity (33,34). We also note that 300 nM amounts of these P␥s, free or complexed with GDP/T␣ or GTP␥S/T␣, were used in these experiments, because by using the high concentration of P␥, we tried to clearly show that even a small portion of nonphosphorylated P␥ was not released from its complexes with GDP/T␣ or GTP␥S/T␣.…”

Section: Fig 4 Effect Of Cdk Inhibitors On P␥ Phosphorylation In VImentioning

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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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: Fig 4 Effect Of Cdk Inhibitors On P␥ Phosphorylation In VImentioning

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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“…This is the first study to date to correlate the reduced ability of the phosphorylated receptor to both bind and activate the G protein. Although it has been proposed that phosphorylation of rhodopsin reduces its binding affinity for transducin (18), evidence to support this is lacking. Multiple studies have demonstrated that the rhodopsin domains most critical for transducin binding are the second and third cytoplasmic loops (33)(34)(35)(36).…”

Section: Fig 2 Arrestin Binding To Rhodopsin and Phosphorhodopsinmentioning

confidence: 99%

“…Although several studies have demonstrated a modest to potent functional effect of receptor phosphorylation on G protein activation, it is not known whether this effect is due to attenuated G protein binding to the receptor or subsequent G protein activation. Phosphorylated rhodopsin was demonstrated to have a significantly lower light-induced phosphodiesterase activating capacity than nonphosphorylated rhodopsin (4,18), and it was subsequently proposed that rhodopsin phosphorylation reduces the binding affinity for transducin (18). ␤ARK phosphorylation of the ␤ 2 AR resulted in a modest functional inhibition of ␤ 2 AR-stimulated G o GTPase activity (12,15), and phosphorylation of the m2 muscarinic acetylcholine receptor by ␤ARK modestly reduced its capacity to stimulate GTP␥S binding to G o (19).…”

mentioning

confidence: 99%

Mechanism of Quenching of Phototransduction

Krupnick¹,

Gurevich²,

Benovic³

1997

Journal of Biological Chemistry

179

109

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Quenching of phototransduction in retinal rod cells involves phosphorylation of photoactivated rhodopsin by the enzyme rhodopsin kinase followed by binding of the protein arrestin. Although it has been proposed that the mechanism of arrestin quenching of visual transduction is via steric exclusion of transducin binding to phosphorylated light-activated rhodopsin (P-Rh * ), direct evidence for this mechanism is lacking. In this study, we investigated both the role of rhodopsin phosphorylation in modulating its interaction with arrestin and transducin and the proposed binding competition between arrestin and transducin for P-Rh * . While the ␤-adrenergic receptor kinase promotes significant arrestin binding to rhodopsin at a phosphorylation stoichiometry of 2 mol/mol, rhodopsin kinase promotes arrestin binding at a stoichiometry of ϳ0.9 mol/mol. Moreover, while ␤-adrenergic receptor kinase phosphorylation of rhodopsin only modestly decreases transducin binding and activation, rhodopsin kinase phosphorylation of rhodopsin significantly decreases transducin binding and activation. Finally, arrestin competes effectively with transducin for binding to P-Rh * (50% inhibition at ϳ1:1 molar ratio of arrestin:transducin) but has no effect on transducin binding to nonphosphorylated light-activated rhodopsin (Rh * ), paralleling the functional inhibition by arrestin on P-Rh * -stimulated transducin activation (50% inhibition at ϳ1.7:1 molar ratio of arrestin:transducin). These results demonstrate that a major role of rhodopsin phosphorylation is to promote high-affinity arrestin binding and decrease transducin binding thus allowing arrestin to effectively compete with transducin for binding to photoactivated rhodopsin.Phototransduction mediated by the photoreceptor rhodopsin and hormonal transduction mediated by the ␤-adrenergic receptor (␤AR) 1 serve as excellent model systems for investigation of the molecular events underlying agonist-induced receptor desensitization (reviewed in Refs. 1 and 2). Phosphorylation of light-activated rhodopsin and hormone-activated ␤AR by the G protein-coupled receptor kinases rhodopsin kinase and ␤-adrenergic receptor kinase (␤ARK), respectively, initiates desensitization and leads to partial uncoupling of receptor-G protein interaction. Rapid and complete uncoupling, however, is accomplished by the subsequent binding of the proteins arrestin and ␤-arrestin, respectively, to the phosphorylated form of the agonist-activated receptor.Many studies have investigated the effect of receptor phosphorylation on modulating interaction with arrestins. Lightinduced binding of visual arrestin to rhodopsin was highly enhanced by rhodopsin phosphorylation (3), and this binding suppressed light-induced cGMP phosphodiesterase activation by rhodopsin (4). Although it has been shown that highly phosphorylated rhodopsin is significantly impaired in its ability to activate transducin, addition of arrestin accelerated the recovery process and further suppressed phosphodiesterase activation, most likely by quenc...

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“…At the first step, this is due to rhodopsin phosphorylation that causes a considerable decrease in the rate of T activation [4][5][6][7][8]. The phosphorylation effect can be apparently enhanced as a result of the binding of an ROS cytoplasmic 48 kDa protein to phosphorylated rhodopsin [9].…”

Section: Introductionmentioning

confidence: 99%

On the role of transducin GTPase in the quenching of a phosphodiesterase cascade of vision

1987

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The rate of GTP hydrolysis in the active site of transducin and that of the release of the phosphate thus formed have been measured. The former step has been found to be a rate-limiting one. The rate constant for GTP hydrolysis is equal to 0.027 s -1 at 23°C, and 0.07 s 1 at 37°C. Bedsides, it has been shown that the rate of GTPase reaction on the transducin ~-subunit does not depend on the concentration of a complex of transducin fl-and y-subunits or on the presence of cGMP phosphodiesterase and a 48 kDa protein from rod outer segments. According to the results, GTP hydrolysis on transducin proceeds too slowly to account for the rapid quenching of a phosphodiesterase cascade in rod outer segments.

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Amplification of phosphodiesterase activation is greatly reduced by rhodopsin phosphorylation

Cited by 100 publications

References 26 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

Mechanism of Quenching of Phototransduction

On the role of transducin GTPase in the quenching of a phosphodiesterase cascade of vision

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