Interactions of a G-protein with its effector: transducin and cGMP phosphodiesterase in retinal rods

Pfister, Claude; Bennett, Nelly; Brückert, Franz; Catty, Patrice; Clerc, Armel; Pagès, Frédérique; Déterre, Philippe

doi:10.1016/0898-6568(93)90015-e

Cited by 78 publications

(30 citation statements)

References 124 publications

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“…ROS intactness was estimated by measuring the production of NADPH as described by Schnetkamp and Daemen (36). 2 Activity-Considering the discrepancy between ROS PLA 2 activity levels reported in the literature and that its role is still unknown, we have attempted to reproduce some of the reported results in order to further characterize this enzyme. We first tried to measure PLA 2 activity in the conditions described by Jelsema (5) because she reported the highest level of activity.…”

Section: C]arachidonic or [mentioning

confidence: 99%

“…Jelsema reported in 1987 (5) that phospholipase A 2 (PLA 2 ) activity was present in "crude ROS" and "partially purified ROS," and that this activity was stimulated by light and a non-hydrolyzable analog of GTP, GTP␥S. Moreover, Jelsema and Axelrod (6) demonstrated that T␤␥ was responsible for the activation of this ROS PLA 2 . These results led them to conclude that ROS PLA 2 could be involved in the phototransduction process.…”

Section: 163-168)mentioning

confidence: 99%

“…Moreover, the biochemical characteristics of this retinal PLA 2 have not been fully studied yet and its role is still unknown. We present here results showing that two subretinal fractions, namely RPE (enriched with retinal pigment epithelial cells) and P200 (presumably containing neuronal cells, Mü ller cells, and rod inner segments) are rich in PLA 2 activity having an alkaline pH optimum.…”

Section: 163-168)mentioning

confidence: 99%

“…For some experiments, it was rehomogenized with a tight-fitting (0.5 mm clearance) Potter-Elvehjem homogenizer before use. It was either assayed directly for PLA 2 (8). The total volume was 250 l. Incubations were done at 37°C under either dim red light or white light (1330 lx; we used a 250 watt tungsten lamp which practically does not emit in the UV range (31)).…”

Section: Isolation Of Ros By Hand Shaking (Hs-ros)-dark-adaptedmentioning

confidence: 99%

See 3 more Smart Citations

Presence of a Light-independent Phospholipase A2 in Bovine Retina but Not in Rod Outer Segments

Jacob

Weech

Salesse

1996

Journal of Biological Chemistry

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262, 163-168). This led that author to conclude that ROS PLA 2 could be involved in the phototransduction process, and raised the possibility of receptor-mediated activation of PLA 2 via G-proteins in cell types other than rods. However, the biochemical characteristics and the role of this PLA 2 have not been fully elucidated. We have tried to reproduce some of the results previously reported in order to further characterize this enzyme. We have found that, in our hands, there is neither light-dependent nor GTP-dependent PLA 2 activity in intact purified ROS. We also failed to detect PLA 1 activity in those ROS preparations. Nevertheless, we detected significant amounts of PLA 2 activity in two subretinal fractions adjacent to ROS: RPE (enriched with retinal pigment epithelial cells) and P200 (presumably containing neuronal cells, Mü ller cells, and rod inner segments). The enzyme present both in RPE and P200 is light-and GTP-independent, Ca 2؉ -and Mg 2؉ -independent, and seems to be optimally active in the alkaline pH range. Our results suggest that there is, if any, vanishingly little PLA 2 or PLA 1 activity in intact purified ROS and that the activity levels previously reported in the literature could have been due to a contamination by either RPE or P200. This is supported by our observation that some contaminated ROS preparations were "PLA 2 active."In the mammalian eye, rod outer segments (ROS) 1 consist of a stack of 1000 -2000 disks which contain the visual pigment, rhodopsin. ROS are thus responsible for the phototransduction process. It has been clearly shown that following absorption of light, photoexcited rhodopsin binds to and activates transducin (T␣␤␥), which is a member of the heterotrimeric GTP-binding protein family. During the activation of transducin, the GDP molecule (normally associated with the inactive state of the protein) is exchanged for GTP. As a consequence, transducin dissociates into T␣ and T␤␥ subunits. T␣ is well known to activate a cGMP-phosphodiesterase whose activity eventually leads to hyperpolarization of the rod through closure of Na ϩ / Ca 2ϩ cGMP-dependent channels (see Refs. 1-4 for reviews). Jelsema reported in 1987 (5) that phospholipase A 2 (PLA 2 ) activity was present in "crude ROS" and "partially purified ROS," and that this activity was stimulated by light and a non-hydrolyzable analog of GTP, GTP␥S. Moreover, Jelsema and Axelrod (6) demonstrated that T␤␥ was responsible for the activation of this ROS PLA 2 . These results led them to conclude that ROS PLA 2 could be involved in the phototransduction process. However, conflicting results have been reported since that time. In fact, although Zimmerman and Keys (8) detected phospholipase A activity in their ROS preparations, the activity that they measured, either PLA 2 or PLA 1 , was neither light-dependent nor GTP-dependent. It was rather stimulated by ATP and coenzyme A (CoA). Moreover, the maximum activity that they observed was approximately 1 order of magnitude lower than that reported by Jelsema (5). In addi...

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Section: C]arachidonic or [mentioning

confidence: 99%

Section: 163-168)mentioning

confidence: 99%

Section: 163-168)mentioning

confidence: 99%

Section: Isolation Of Ros By Hand Shaking (Hs-ros)-dark-adaptedmentioning

confidence: 99%

See 2 more Smart Citations

Presence of a Light-independent Phospholipase A2 in Bovine Retina but Not in Rod Outer Segments

Jacob

Weech

Salesse

1996

Journal of Biological Chemistry

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“…This results in activation of a photoreceptor-specific cGMP phosphodiesterase (PDE). 1 Increased cGMP hydrolysis lowers the cytoplasmic cGMP concentration which is believed to cause closure of the cGMP-gated ion channel in the plasma membrane and the generation of an electrical response (for reviews, see Refs. [1][2][3][4][5].…”

mentioning

confidence: 99%

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

Hebert¹,

Schwede²,

Jastorff³

et al. 1998

Journal of Biological Chemistry

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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.Visual excitation of vertebrate retinal photoreceptors begins with the absorption of light by the visual pigment (opsin) which is followed by activation of a G-protein, transducin. This results in activation of a photoreceptor-specific cGMP phosphodiesterase (PDE).1 Increased cGMP hydrolysis lowers the cytoplasmic cGMP concentration which is believed to cause closure of the cGMP-gated ion channel in the plasma membrane and the generation of an electrical response (for reviews, see Refs. 1-5).The rod photoreceptor PDE holoenzyme is a heterotetramer consisting of two non-identical catalytic subunits (␣ and ␤) and two small subunits (␥) that serve to inhibit catalytic activity. Cone photoreceptors have a highly homologous PDE that consists of two identical catalytic subunits (␣Ј) and two conespecific ␥Ј subunits. In addition to containing the active site for cGMP hydrolysis, the catalytic subunits of rod and cone PDEs also possess noncatalytic cGMP binding sites (6). Two other classes of PDEs, namely the cGMP-stimulated PDE (PDE2) and the cGMP-specific PDE (PDE5), are also known to contain noncatalytic binding sites for cGMP. In the case of PDE2, binding of cGMP at the noncatalytic site allosterically activates cyclic nucleotide hydrolysis at the active site (7,8). For PDE5, the...

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Mechanisms of Transmembrane Signaling

Wickman

Hedin

Perez‐Terzic

et al. 1997

Comprehensive Physiology

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Interactions of a G-protein with its effector: transducin and cGMP phosphodiesterase in retinal rods

Cited by 78 publications

References 124 publications

Presence of a Light-independent Phospholipase A2 in Bovine Retina but Not in Rod Outer Segments

Presence of a Light-independent Phospholipase A2 in Bovine Retina but Not in Rod Outer Segments

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

Mechanisms of Transmembrane Signaling

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