Subunit Of Phosphodiesterase Research Articles

Transgenic rd mice harboring axokine gene by Rpe65 gene promoter does not rescue photoreceptor degeneration.

Neurotrophic factors have reported to rescue photoreceptor degeneration in dystrophic rats (Faktorovich et al., 1990), and variable strains of mice showing hereditary photoreceptor degeneration (LaVail et al., 1998). Axokine is a modified form of human CNTF to enhance its specific activity (Panayotatos et al., 1993) and its signaling pathway is reported in retinal neurons and glia via CNTF receptor (CNTFRa) (Peterson et al., 2000). In this study, we introduced Axokine gene in rd/rd mice, a mice reported to have mutation in s subunit of phosphodiesterase (PDE) gene (Pittler and Baehr, 1991) and examined its effect for retinal degeneration. The Axokine gene was introduced into retinal pigment epithelium (RPE) by Rpr65 promoter, which has acitivity of RPE specific expression

Phosphorylation by Cyclin-dependent Protein Kinase 5 of the Regulatory Subunit of Retinal cGMP Phosphodiesterase: II. ITS ROLE IN THE TURNOFF OF PHOSPHODIESTERASE IN VIVO

Retinal cGMP phosphodiesterase (PDE) is regulated by Pgamma, the regulatory subunit of PDE, and GTP/Talpha, the GTP-bound alpha 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 Pgammas 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 Pgamma phosphorylation in vivo. Under dark conditions only negligible amounts of Pgamma were phosphorylated. However, under illumination that bleached less than 0.3% of the rhodopsin, approximately 4% of the total Pgamma was phosphorylated in less than 10 s. Pgamma dephosphorylation occurred in less than 1 s after the light was turned off. Analysis of the phosphorylated amino acid, inhibition of Pgamma phosphorylation by Cdk inhibitors in vivo and in vitro, and two-dimensional peptide map analysis of Pgamma phosphorylated in vivo and in vitro indicate that Cdk5 phosphorylates a Pgamma 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 Pgamma 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) (GTPgammaS) was inhibited by Pgamma alone, but not by Pgamma complexed with GDP/Talpha or GTPgammaS/Talpha. Under these conditions, Pgamma phosphorylated by Cdk5 inhibited the light/GTPgammaS-activated PDE even in the presence of GTPgammaS/Talpha. These observations suggest that phosphorylated Pgamma interacts with and inhibits light/GTPgammaS-activated PDE, but does not interact with GTPgammaS/Talpha in the homogenate. Together, our results strongly suggest that after activation of PDE by light/GTP, Pgamma is phosphorylated by Cdk5 and the phosphorylated Pgamma inhibits GTP/Talpha-activated PDE, even in the presence of GTP/Talpha in ROS.

Phosphorylation by Cyclin-dependent Protein Kinase 5 of the Regulatory Subunit of Retinal cGMP Phosphodiesterase: I. IDENTIFICATION OF THE KINASE AND ITS ROLE IN THE TURNOFF OF PHOSPHODIESTERASE IN VITRO

Cyclic GMP phosphodiesterase (PDE) is an essential component in retinal phototransduction. PDE is regulated by Pgamma, the regulatory subunit of PDE, and GTP/Talpha, the GTP-bound alpha subunit of transducin. In previous studies (Tsuboi, S., Matsumoto, H. , Jackson, K. W., Tsujimoto, K., Williamas, T., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15016-15023; Tsuboi, S., Matsumoto, H., and Yamazaki, A. (1994) J. Biol. Chem. 269, 15024-15029), we showed that Pgamma is phosphorylated by a previously unknown kinase (Pgamma kinase) in a GTP-dependent manner in photoreceptor outer segment membranes. We also showed that phosphorylated Pgamma loses its ability to interact with GTP/Talpha, but gains a 10-15 times higher ability to inhibit GTP/Talpha-activated PDE than that of nonphosphorylated Pgamma. Thus, we propose that the Pgamma phosphorylation is probably involved in the recovery phase of phototransduction through shut off of GTP/Talpha-activated PDE. Here we demonstrate that all known Pgammas preserve a consensus motif for cyclin-dependent protein kinase 5 (Cdk5), a protein kinase believed to be involved in neuronal cell development, and that Pgamma kinase is Cdk5 complexed with p35, a neuronal Cdk5 activator. Mutational analysis of Pgamma indicates that all known Pgammas contain a P-X-T-P-R sequence and that this sequence is required for the Pgamma phosphorylation by Pgamma kinase. In three different column chromatographies of a cytosolic fraction of frog photoreceptor outer segments, the Pgamma kinase activity exactly coelutes with Cdk5 and p35. The Pgamma kinase activity ( approximately 85%) is also immunoprecipitated by a Cdk5-specific antibody, and the immunoprecipitate phosphorylates Pgamma. Finally, recombinant Cdk5/p35, which were expressed using clones from a bovine retina cDNA library, phosphorylates Pgamma in frog outer segment membranes in a GTP-dependent manner. These observations suggest that Cdk5 is probably involved in the recovery phase of phototransduction through phosphorylation of Pgamma complexed with GTP/Talpha in mature vertebrate retinal photoreceptors.

Suppression of GTP/T alpha-dependent activation of cGMP phosphodiesterase by ADP-ribosylation by its gamma subunit in amphibian rod photoreceptor membranes.

Our previous study has shown that P gamma, the regulatory subunit of cGMP phosphodiesterase (PDE), is ADP-ribosylated by endogenous ADP-ribosyltransferase when P gamma is free or complexed with the catalytic subunits of PDE in amphibian rod photoreceptor membranes. The P gamma domain containing ADP-ribosylated arginines was shown to be involved in its interaction with T alpha, a key interaction for PDE activation. In this study, we describe a possible function of the P gamma ADP-ribosylation in the GTP/T alpha-dependent PDE activation. When rod membranes were preincubated with or without NAD and washed with a buffer containing GTP, the PDE activity of NAD-preincubated membranes was increased by the GTP-washing only to approximately 50% of that of membranes preincubated without NAD. The P gamma release by the GTP-washing from these NAD-preincubated membranes was also suppressed to approximately 50% of that preincubated without NAD. Taking into consideration that approximately 50% of P gamma is ADP-ribosylated under these conditions, these observations suggest that the ADP-ribosylated P gamma cannot interact with GTP/T alpha. We have also shown that a soluble fraction of ROS contains an enzyme(s) to release the radioactivity of [32P]ADP-ribosylated P gamma in concentration- and time-dependent manners, suggesting that the P gamma ADP-ribosylation is reversible. Rod ADP-ribosyltransferase solubilized from membranes by phosphatidylinositol-specific phospholipase C was separated into two fractions by ion-exchange columns. Biochemical characterization of these two fractions, including measurement of the Km for NAD and P gamma, estimation of their molecular masses, ADP-ribosylation of P gamma arginine mutants, effects of ADP-ribosyltransferase inhibitors on the P gamma ADP-ribosylation, and effects of salts and pH on the P gamma ADP-ribosylation, indicates that rod ADP-ribosyltransferase contains two isozymes, and that these two isozymes have similar properties for the P gamma ADP-ribosylation. Our observations strongly suggest that the negative regulation of PDE through the reversible P gamma ADP-ribosylation may function in the phototransduction mechanism.

The α-Helical Domain of Gαt Determines Specific Interaction with Regulator of G Protein Signaling 9

RGS proteins (regulators of G protein signaling) are potent accelerators of the intrinsic GTPase activity of G protein alpha subunits (GAPs), thus controlling the response kinetics of a variety of cell signaling processes. Most RGS domains that have been studied have relatively little GTPase activating specificity especially for G proteins within the Gi subfamily. Retinal RGS9 is unique in its ability to act synergistically with a downstream effector cGMP phosphodiesterase to stimulate the GTPase activity of the alpha subunit of transducin, Galphat. Here we report another unique property of RGS9: high specificity for Galphat. The core (RGS) domain of RGS9 (RGS9) stimulates Galphat GTPase activity by 10-fold and Galphai1 GTPase activity by only 2-fold at a concentration of 10 microM. Using chimeric Galphat/Galphai1 subunits we demonstrated that the alpha-helical domain of Galphat imparts this specificity. The functional effects of RGS9 were well correlated with its affinity for activated Galpha subunits as measured by a change in fluorescence of a mutant Galphat (Chi6b) selectively labeled at Cys-210. Kd values for RGS9 complexes with Galphat and Galphai1 calculated from the direct binding and competition experiments were 185 nM and 2 microM, respectively. The gamma subunit of phosphodiesterase increases the GAP activity of RGS9. We demonstrate that this is because of the ability of Pgamma to increase the affinity of RGS9 for Galphat. A distinct, nonoverlapping pattern of RGS and Pgamma interaction with Galphat suggests a unique mechanism of effector-mediated GAP function of the RGS9.

Encapsidated adenovirus mini-chromosome-mediated delivery of genes to the retina: application to the rescue of photoreceptor degeneration.

First (DeltaE1/E3) and second (DeltaE1+DeltaE2/E3/E4) generation adenovirus (Ad) vectors have been shown previously to be of limited use in the treatment of human genetic diseases due to the induction of a host cytotoxic T-cell mediated immune response against virally expressed genes. In addition, a limited cloning capacity of approximately 8 kb does not cater for the incorporation of large upstream sequences essential for regulated tissue-specific expression or inclusion of multiple gene-expression cassettes. In this study we have exploited our recently developed Ad-based vector, the encapsidated adenovirus mini-chromosome (EAM) from which all of the viral genes have been deleted. EAMs contain only the inverted terminal repeats required for replication and five cis -acting Ad encapsidation signals necessary for packaging. We have shown previously that EAMs can efficiently transduce a variety of cell types in vitro. In this study we demonstrate that EAMs can transduce and rescue cells from the neurosensory retina in vivo. EAM-mediated delivery of the beta subunit of cyclic GMP phosphodiesterase (PDE) cDNA to mice affected with retinal degeneration (rd) allows prolonged transgene expression and rescue of rod photoreceptor cells. RT-PCR analysis from the injected retina indicates that transgene products are present for at least 18 weeks post-injection. Both the alpha and beta subunits of PDE could be detected up to 90 days postnatal in EAM-injected rd retina by western analysis. A maximal PDE activity of 150 nm/min/mg was detected at 33 days postnatal. Examination of outer nuclear thickness showed significant differences up to 12 weeks post-injection. These results demonstrate an improved level of rescue over first-generation adenoviral vectors and suggest the possibility of successful EAM-mediated treatment of some retinal diseases in humans.

Structural Features of the Noncatalytic cGMP Binding Sites of Frog Photoreceptor Phosphodiesterase Using cGMP Analogs

The cGMP-specific phosphodiesterase (PDE) of retinal photoreceptors is a central regulatory enzyme in the visual transduction pathway of vertebrate vision. Although the mechanism of activation of PDE by transducin is well understood, the role of the noncatalytic cGMP binding sites located on the catalytic subunits of PDE remains obscure. We report here for the first time the molecular basis of the noncovalent interactions between cGMP and the high affinity, noncatalytic cGMP binding sites of frog photoreceptor PDE. None of the tested cGMP analogs were able to bind with greater affinity than cGMP itself, and the noncatalytic sites were unable to bind cAMP. The major determinant for discrimination of cGMP over cAMP is in the N-1/C-6 region of the purine ring of cGMP where hydrogen bonding probably stabilizes the selective binding of cGMP. Substitutions at the C-2 position demonstrate that this region of the molecule plays a secondary but significant role in stabilizing cGMP binding to PDE through hydrogen bond interactions. The unaltered hydrogen at the C-8 position is also important for high affinity binding. A significant interaction between the binding pocket and the ribose ring of cGMP occurs at the 2'-hydroxyl position. Steric constraints were greatest in the C-8 and possibly the C-6/N-1 regions, whereas the C-2/N-3 and C-2' regions tolerated bulky substituents better. Several lines of evidence indicate that the noncatalytic site binds cGMP in the anti-conformation. The numerous noncovalent interactions between cGMP and the noncatalytic binding pocket of the photoreceptor PDE described in this study account for both the high affinity for cGMP and the high level of discrimination of cGMP from other cyclic nucleotides at the noncatalytic site.

Molecular origin of continuous dark noise in rod photoreceptors

Noise in the rod photoreceptors limits the ability of the dark-adapted visual system to detect dim lights. We investigated the molecular mechanism of the continuous component of the electrical dark noise in toad rods. Membrane current was recorded from intact, isolated rods or truncated, internally dialyzed rod outer segments. The continuous noise was separated from noise due to thermal activation of rhodopsin and to transitions in the cGMP-activated channels. Selectively disabling different elements of the phototransduction cascade allowed examination of their contributions to the continuous noise. These experiments indicate that the noise is generated by spontaneous activation of cGMP phosphodiesterase (PDE) through a process that does not involve transducin. The addition of recombinant gamma, the inhibitory subunit of PDE, did not suppress the noise, indicating that endogenous gamma does not completely dissociate from the catalytic subunit of PDE during spontaneous activation. Quantitative analysis of the noise provided estimates of the rate constants for spontaneous PDE activation and deactivation and the catalytic activity of a single PDE molecule in situ.

Subunit Structure of Rod cGMP-Phosphodiesterase

The rod cGMP phosphodiesterase (PDE) is the G-protein-activated effector enzyme that regulates the level of cGMP in vertebrate photoreceptor cells. Rod cGMP PDE is generally viewed as a heterotrimeric protein composed of catalytic alpha and beta subunits ( approximately90 kDa each) and two copies of the inhibitory subunit gamma ( approximately 10 kDa). However, the possibility that rod PDE could exist as distinct isoforms, such as alphaalphagamma2 and betabetagamma2 has not been ruled out. We have studied this question using cross-linking of PDE subunits with maleimidobenzoyl-N-hydroxysuccinimide ester and para-phenyldimaleimide. The cross-linking resulted in major products with molecular mass of 100 and 150 kDa, a doublet at approximately 180-190 kDa, and a doublet at approximately 210-220 kDa. Cross-linked products were analyzed using polyclonal-specific anti-PDEalphabeta, anti-PDEalpha, anti-PDEbeta, or anti-PDEgamma antibodies. The anti-PDEalpha and anti-PDEalphabeta antibodies recognized all the cross-linked products, whereas anti-PDEbeta and anti-PDEgamma antibodies did not interact with the 150-kDa band, indicating that the composition of this band is most likely alphaalpha. Similar analysis of cross-linked products of trypsin-treated PDE preparations revealed bands that are likely formed by PDEbeta subunit. The molecular size of holo-PDE and trypsin-activated PDE were studied using analytical ultracentrifugation in order to determine if oligomerization of PDE could account for the cross-linking of identical PDE subunits. The sedimentation analysis of both holo-PDE and ta-PDE revealed homogeneous samples with molecular masses of approximately220 and approximately150 kDa, respectively. These results indicate that PDE is likely a mixture of the major species alphabetagamma2, minor species alphaalphagamma2, and possibly betabetagamma2. Our data are consistent with the detection of low PDE activity in the rd mouse, which lacks any functional PDEbeta subunit.

Interaction Sites of the COOH-terminal Region of the γ Subunit of cGMP Phosphodiesterase with the GTP-bound α Subunit of Transducin

In photoreceptor cells, visual transduction occurs through photoexcitation of rhodopsin, GTP activation of the alpha subunit of transducin, and interaction between GTP-bound transducin alpha subunit and the inhibitory gamma subunit of phosphodiesterase. The gamma subunit of phosphodiesterase, in turn, accelerates the hydrolysis of GTP on the alpha subunit of transducin. Within the COOH-terminal residues (46-87) of the phosphodiesterase gamma subunit, Trp-70 has been implicated in phosphodiesterase activation, transducin alpha subunit-phosphodiesterase gamma subunit interaction, and the GTP hydrolysis accelerating activity. We have derivatized the phosphodiesterase gamma subunit with a reversible photoactivatable reagent, [125I]N-[(3-iodo-4-azidophenylpropionamido-S-(2-thiopyridyl) ]cysteine ([125I]ACTP), at cysteine (Cys-68). A light-dependent, cross-linked complex of guanosine 5'-(gamma-thio)triphosphate-bound transducin alpha subunit and ACTPderivatized phosphodiesterase gamma subunit formed after photolysis of a 1:1 stoichiometic complex of the two proteins. The specificity of complex formation between the transducin alpha subunit and the phosphodiesterase gamma subunit was demonstrated by specific protection by the C68A mutant of the phosphodiesterase gamma subunit. The cross-linked complex was treated with beta-mercaptoethanol to transfer the 125I photomoiety from the phosphodiesterase gamma subunit to the transducin alpha subunit. Combined techniques involving electrophoresis, chemical and enzymatic cleavage, and chemical and radiosequencing were used to identify photoinsertion sites on the alpha3 and alpha4/beta6 regions of the transducin alpha subunit. Three photo-labeled residues, His-244 (alpha3 helix), Met-308, and Arg-310 (alpha4/beta6 interface), were specifically identified as photoinsertion sites. Utilizing the crystal structure coordinates of the GTP-bound transducin alpha subunit and molecular modeling, we conclude that Cys-68 of the phosphodiesterase gamma subunit is located at a position between the exposed face of the alpha3 and alpha4 helices of the transducin alpha subunit. We propose that the phosphodiesterase gamma subunit interacts with GTP-bound transducin alpha subunit at multiple sites in which the cysteine 68 to tryptophan 70 sequence of the phosphodiesterase gamma subunit, which is critical for GTP hydrolysis accelerating activity, interacts in the alpha3/alpha4/beta6 region of GTP-bound transducin alpha subunit.

Phototransduction in transgenic mice

Transgenic mice provide a powerful tool for elucidating the molecular mechanisms of phototransduction. Mice expressing a phosphorylation-deficient rhodopsin and mice deficient in arrestin are being used to study shutoff of photoactivated rhodopsin. These in vivo mouse studies indicate that shutoff is partially mediated by rhodopsin phosphorylation alone, but complete deactivation on a physiological time scale requires arrestin. Work on other transgenic mutant mice to unravel the function of recoverin and phosducin and to further define the role of the γ subunit of phosphodiesterase is in progress. Transgenic mice are also being used to investigate how mutant proteins give rise to retinal disease and to develop therapeutic interventions.

An Interface of Interaction between Photoreceptor cGMP Phosphodiesterase Catalytic Subunits and Inhibitory γ Subunits

Cyclic guanosine 5'-monophosphate (cGMP) phosphodiesterase (PDE) regulates the level of cGMP on transduction of a visual signal in vertebrate photoreceptor cells. Two identical inhibitory PDE gamma subunits (Pgammas) block catalytic activity of PDE-alpha and -beta subunits (Palphabeta) in the dark. The primary regions of Pgamma involved in the interaction with Palphabeta are a central polycationic region, Pgamma-24-45, and a C-terminal region of Pgamma. Recently, we have shown that the C-terminal region of Pgamma, which is the major Pgamma inhibitory domain, blocks PDE activity by binding to the catalytic site of PDE (Artemyev, N. O., Natochin, M., Busman, M., Schey, K. L., and Hamm, H. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5407-5412). Here, we localize the site on the rod cGMP PDE alpha subunit that binds to the central polycationic domain of Pgamma. This site is located within a region that links a second noncatalytic cGMP binding site with the catalytic domain of PDE. A polypeptide coresponding to this region, Palpha-461-553, expressed as a glutathione S-transferase fusion protein in Escherichia coli and isolated after cleavage of the fusion protein with thrombin, blocks inhibition of PDE activity by Pgamma. In addition, Palpha-461-553 binds to the Pgamma-24-45 region (Kd, 7 microM), as measured by a fluorescent increase in a Pgamma-24-45Cys peptide labeled with 3-(bromoacetyl)-7-diethylaminocoumarin. The Palpha-461-553 region was further characterized by using a set of synthetic peptides. A peptide corresponding to residues 517-541 of Palpha (Palpha-517-541) effectively suppressed inhibition of PDE activity by Pgamma and bound to Pgamma-24-45Cys labeled with 3-(bromoacetyl)-7-diethylaminocoumarin (Kd, 22 microM). Palpha-517-541 also competes with the activated rod G-protein alpha-subunit for binding to Pgamma labeled with lucifer yellow vinyl sulfone. This suggests that light activation of rod PDE by the G-protein transducin involves competition between transducin alpha-guanosine 5'-triphosphate and Palpha-517-541 for binding to the Pgamma-24-45 region. Based on the results, we propose a linear model of interactions between catalytic and inhibitory PDE subunits.

Recovery phase of the murine rod photoresponse reconstructed from electroretinographic recordings

The activation and recovery phases of the murine rod photo-response were determined from corneal electroretinograms (ERGs) obtained in response to pairs of full-field flashes producing 50-10(5) photoisomerized rhodopsins (R*) per rod. The a-wave component of the ERG in response to the initial flash provided a well established measure of the activation phase of the rod response. The amplitude of the a-wave response to an intense second flash (45,000 R*) delivered 0.2-5 seconds (s) after the first flash was used to reconstruct the recovery phase of the response. For 160-3000 R* rod-1, recovery curves were isomorphic, translating on the time axis such that each e-fold increase in R* produced an incremental recovery delay of tau c = 210 +/- 50 ms (mean +/- SD). For initial flashes producing > 3000 R*, recovery curves lost their initial isomorphism and half-times had intensity dependence exceeding 1 s per e-fold increase in R*. We conclude that for flashes producing < 3000 R*, the effective lifetime of these R* is not > 210 ms. Two extant and non-mutually exclusive hypotheses are discussed that can account for the sharp increase in recovery times from flashes producing > 3000 R*. They are as follows: (1) approximately 0.03% of R* have a lifetime exceeding 1 s; and (2) the gamma subunit of phosphodiesterase (PDE gamma) serves as a GTPase-activating factor, and 3000 R* produce sufficient activated G-protein (G*) to exceed the total quantity of PDE gamma subunits such that excess G* must wait for unoccupied PDE gamma to inactivate via GTP hydrolysis.

Rod photoreceptor-specific gene expression in human retinoblastoma cells.

Retinoblastoma cells in culture have previously been shown to express cone-specific genes but not their rod counterparts. We have detected the messages for the rod alpha, beta, and gamma subunits of cGMP phosphodiesterase (PDE), the rod alpha subunit of transducin, rod opsin, and the cone alpha' subunit of PDE in RNA of human Y-79 retinoblastoma cells by reverse transcription-PCR. Quantitative analysis of the mRNAs for the rod alpha and cone alpha' PDE subunits revealed that they were expressed at comparable levels; however, the transcript encoding the rod beta PDE subunit was 10 times more abundant in these cells. Northern hybridization analysis of Y-79 cell RNA confirmed the presence of the transcripts for rod and cone PDE catalytic subunits. To test whether the transcriptional machinery required for the expression of rod-specific genes was endogenous in Y-79 retinoblastoma cells, cultures were transfected with a construct containing the promoter region of the rod beta PDE subunit gene attached to the firefly luciferase reporter vector. Significant levels of reporter enzyme activity were observed in the cell lysates. Our results demonstrate that the Y-79 retinoblastoma cell line is a good model system for the study of transcriptional regulation of rod-specific genes.

Biochemical analysis of the transducin-phosphodiesterase interaction

In vertebrate rod cells, the activated alpha-subunit of rod transducin interacts with the gamma (regulatory) subunits of phosphodiesterase to disinhibit the catalytic subunits. A 22-amino acid long region of rod transducin involved in phosphodiesterase activation has recently been identified. We have used peptides from this region of rod transducin and from several other G protein alpha-subunits to study the nature and specificity of the G protein alpha-effector interaction. Although peptides derived from rod transducin, cone transducin and gustducin are similar, only the rod peptide is capable of activating rod phosphodiesterase. Using substituted peptides we have identified five residues on one exposed face of rod transducin as important to phosphodiesterase activation. These results disagree with previous models which propose that loop regions of rod transducin interact with phosphodiesterase gamma.

Enhancement of rod outer segment GTPase accelerating protein activity by the inhibitory subunit of cGMP phosphodiesterase.

The cGMP phosphodiesterase (PDE) of retinal rod outer segments (ROS) is activated by the GTP-bound form of the G protein, transducin (Gt alpha). This activation can be reversed by the inhibitory gamma subunit of PDE through two distinct mechanisms: acceleration of GTP hydrolysis and direct inactivation independent of GTP hydrolysis. We have found that acceleration of Gt alpha GTPase by PDE gamma does not occur upon formation of a Gt alpha PDE gamma complex but rather reflects enhanced activity toward this complex of a membrane-bound GTPase accelerating protein. GTPase rate constants for Gt alpha in the presence of 3.3 microM PDE gamma were as high as 0.7 s-1 with hypotonically washed ROS membranes at 40 microM rhodopsin but were more than 10-fold lower when protein-free vesicles containing ROS lipids were substituted for ROS membranes. Acceleration of Gt alpha GTPase by PDE gamma was also barely detectable at low ROS concentrations (e.g. 4 microM rhodopsin) or if ROS treated with trypsin or urea were used. GTPase-independent inactivation by PDE gamma occurred efficiently at much lower membrane concentrations. Inhibition of Gt alpha-activated PDE was much slower than inhibition of PDE alpha beta by PDE gamma. Effects of PDE gamma upon successive additions of GTP suggested formation of a complex of PDE gamma and Gt alpha-GDP that is refractory to reactivation.

CGMP binding sites on photoreceptor phosphodiesterase: role in feedback regulation of visual transduction.

A central step in vertebrate visual transduction is the rapid drop in cGMP levels that causes cGMP-gated ion channels in the photoreceptor cell membrane to close. It has long been a puzzle that the cGMP phosphodiesterase (PDE) whose activation causes this decrease contains not only catalytic sites for cGMP hydrolysis but also noncatalytic cGMP binding sites. Recent work has shown that occupancy of these noncatalytic sites slows the rate of PDE inactivation. We report here that PDE activation induced by activated transduction lowers the cGMP binding affinity for noncatalytic sites on PDE and accelerates the dissociation of cGMP from these sites. These sites can exist in three states: high affinity (Kd = 60 nM) for the nonactivated PDE, intermediate affinity (Kd approximately 180 nM) when the enzyme is activated in a complex with transducin, and low affinity (Kd > 1 microM) when transducin physically removes the inhibitory subunits of PDE from the PDE catalytic subunits. Activation of PDE by transducin causes a 10-fold increase in the rate of cGMP dissociation from one of the two noncatalytic sites; physical removal of the inhibitory subunits from the PDE catalytic subunits further accelerates the cGMP dissociation rate from both sites > 50-fold. Because PDE molecules lacking bound cGMP inactivate more rapidly, this suggests that a prolonged cGMP decrease may act as a negative feedback regulator to generate the faster, smaller photoresponses characteristic of light-adapted photoreceptors.

Enhancement by phosphodiesterase subunits of the rate of GTP hydrolysis by transducin in bovine retinal rods. Essential role of the phosphodiesterase catalytic core.

Phosphodiesterase (PDE) in bovine retinal rod outer segments is activated when it forms a membrane-bound complex with the alpha-subunit of transducin loaded with GTP (T alpha*). At maximal activation, this complex contains two T alpha* and all the subunits of native PDE (PDE alpha, PDE beta, and two inhibitory PDE gamma). We observed previously (Pagès, F., Deterre, P., and Pfister, C. (1992) J. Biol. Chem. 267, 22018-22021) that the rate of GTP hydrolysis by transducin in a rod outer segment suspension is enhanced when T alpha* is bound to native PDE (PDE alpha beta gamma 2). In this article, we compare the effects of PDE species with different PDE gamma contents. We show that T alpha* hydrolyzes its GTP faster not only when bound to PDE alpha beta gamma 2, but also when bound to PDE alpha beta gamma or PDE alpha beta. Moreover, trypsin-treated PDE (PDE gamma-deprived soluble PDE) also induces an acceleration of GTP hydrolysis. On the contrary, addition of isolated PDE gamma alone does not accelerate GTP hydrolysis. The interaction between T alpha* and PDE gamma, which is essential for the activation of PDE by T alpha*, is apparently not responsible of the feedback of PDE on T alpha*. The interaction of primary importance for the acceleration of GTP hydrolysis would be that existing between T alpha* and PDE alpha beta.

Affinities of bovine photoreceptor cGMP phosphodiesterases for rod and cone inhibitory subunits

Rods and cones have analogous phototransduction components and cycles, but differ from each other in their physiological response to light. Differences between the affinities of rod and cone phosphodiesterase (PDE) catalytic subunits for their respective inhibitory subunits could potentially contribute to these physiological differences. To test this idea, we expressed both the 13 kDa PDE subunit, unique to a subset of bovine retinal cones [(1990) J. Biol. Chem. 265, 11259-11264], and the rod PDE 11 kDa inhibitory subunit in E. coll, purified them, and compared their abilities to inhibit rod and cone PDE catalytic subunits. Rod PDE has similar K i values (~80 pM) for both the rod and cone recombinant inhibitory subunits. Activated cone PDE has K i values of 200 pM for the cone 13 kDa subunit and 600 pM for rod PDEγ.

Molecular regulation of rat rod photoreceptor differentiation: Barnstable, C.J., Yu. X., and Ahmad, I. Department of Ophthalmology and Visual Science, Yale University, New Haven, CT 06510, USA

This chapter reviews the electrical and molecular properties of ion channels and the specific function they subserve during the light response. The cyclic GMP-gated channels of vertebrate rod and cone cells play a central role in phototransduction by controlling the flow of Na+ and Ca 2+ ions into the photoreceptor outer segment. Photoexcitation of the rod cell is initiated when a photon is captured by rhodopsin resulting in the conversion of the 11-cis retinal chromophore to its all-trans isomer. The resulting formation of Meta II rhodopsin triggers the activation of the visual enzyme cascade system and an amplification of the signal. Meta II rhodopsin catalyzes the exchange of guanosine diphosphate (GDP) for Guanosine-5'-triphosphate (GTP) on the -subunit of transducin. Interaction of this subunit with inhibitory subunit of phosphodiesterase (PDE) activates PDE leading to the hydrolysis of cGMP. As the intracellular concentration of cGMP decreases, the cGMP-gated channels close causing a hyperpolarization of the rod cell. This in turn results in an inhibition of glutamate transmitter release at the synapse and the transmission of the signal to other retinal neurons. Since the Na/Ca-K exchanger continues to extrude Ca 2+ from the outer segment, a marked decrease in intracellular Ca 2+ concentration (below 100 nM) also occurs. This reduction in Ca 2+ plays a central role in photorecovery and light adaptation.