Characterization and Analysis of Frog Photoreceptor Membranes

Bownds, Deric; Gordon-Walker, Ann; Gaide-Huguenin, Anne-Claude; Robinson, William E.

doi:10.1085/jgp.58.3.225

Cited by 180 publications

(69 citation statements)

References 28 publications

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“…Quantitative Western blotting to compare the R9AP and RGS9-1 contents of wild type and transgenic retinal samples after normalizing to rhodopsin concentration was performed as previously described (Krispel et al, 2006). Rhodopsin concentration was determined by difference spectroscopy at 500 nm before and after bleaching the sample, as described in (Bownds et al, 1971) using the molar extinction coefficient of 40,500.…”

Section: Methodsmentioning

confidence: 99%

Membrane Attachment Is Key to Protecting Transducin GTPase-Activating Complex from Intracellular Proteolysis in Photoreceptors

Gospe¹,

Baker²,

Kessler³

et al. 2011

J. Neurosci.

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The members of the R7 RGS protein sub-family are versatile regulators of G protein signaling throughout the nervous system. Recent studies indicate that they are often found in complexes with membrane anchor proteins which serve as versatile modulators of their activity, intracellular targeting and stability. One striking example is the interplay between the membrane anchor R9AP and the RGS9-1·Gβ5 GTPase activating complex responsible for the rapid inactivation of the G protein transducin in vertebrate photoreceptor cells during their recovery from light excitation. The amount of this complex in photoreceptors sets their temporal resolution and is precisely regulated by the expression level of R9AP, which serves to protect the RGS9-1 and Gβ5 subunits from intracellular proteolysis. In this study, we investigated the mechanism by which R9AP performs its protective function in mouse rods and found that it is entirely confined to recruiting RGS9-1·Gβ5 to cellular membranes. Furthermore, membrane attachment of RGS9-1·Gβ5 is sufficient for its stable expression in rods even in the absence of R9AP. Our second finding is that RGS9-1·Gβ5 possesses targeting information that specifies its exclusion from the outer segment and that this information is neutralized by association with R9AP to allow outer segment targeting. Finally, we demonstrate that the ability of R9AP·RGS9-1·Gβ5 to accelerate GTP hydrolysis on transducin is independent of its means of membrane attachment, since replacing the transmembrane domain of R9AP with a site for lipid modification did not impair the catalytic activity of this complex.

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Section: Methodsmentioning

confidence: 99%

Membrane Attachment Is Key to Protecting Transducin GTPase-Activating Complex from Intracellular Proteolysis in Photoreceptors

Gospe¹,

Baker²,

Kessler³

et al. 2011

J. Neurosci.

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“…A 100 l aliquot was mixed with 20 l of 200 mM hydroxylamine (titrated to pH 7.5 by NaOH) containing 10% n-octyl-␤-D-glucopyranoside. The sample was centrifuged in a tabletop microcentrifuge, and rhodopsin concentration in the supernatant was measured by difference spectrometry using the molar extinction coefficient of 40,500 (Bownds et al, 1971). The rest of the sonicated sample was mixed with 0.5 l of 4 mM 11-cis-retinal solubilized in ethanol, sonicated again, and incubated at 37°C for 40 min in complete darkness.…”

Section: Methodsmentioning

confidence: 99%

Arrestin Translocation Is Induced at a Critical Threshold of Visual Signaling and Is Superstoichiometric to Bleached Rhodopsin

Strissel¹,

Sokolov²,

Trieu³

et al. 2006

J. Neurosci.

133

190

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Light induces massive translocation of major signaling proteins between the subcellular compartments of photoreceptors. Among them is visual arrestin responsible for quenching photoactivated rhodopsin, which moves into photoreceptor outer segments during illumination. Here, for the first time, we determined the light dependency of arrestin translocation, which revealed two key features of this phenomenon. First, arrestin translocation is triggered when the light intensity approaches a critical threshold corresponding to the upper limits of the normal range of rod responsiveness. Second, the amount of arrestin entering rod outer segments under these conditions is superstoichiometric to the amount of photoactivated rhodopsin, exceeding it by at least 30-fold. We further showed that it is not the absolute amount of excited rhodopsin but rather the extent of downstream cascade activity that triggers translocation. Finally, we demonstrated that the total amount of arrestin in the rod cell is nearly 10-fold higher than previously thought and therefore sufficient to inactivate the entire pool of rhodopsin at any level of illumination. Thus, arrestin movement to the outer segment leads to an increase in the free arrestin concentration and thereby may serve as a powerful mechanism of light adaptation.

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“…Before each experiment, ROS were disrupted in a Potter-Elvehjem homogenizer, creating a membrane suspension with no structure detectable under light microscopy (Dumke et al, 1994). Rhodopsin concentration was determined spectrophotometrically according to Bownds et al (1971). All experiments were carried out at 22°C.…”

Section: Methodsmentioning

confidence: 99%

Onset of Feedback Reactions Underlying Vertebrate Rod Photoreceptor Light Adaptation

Calvert

Ho²,

LeFebvre³

et al. 1998

The Journal of General Physiology

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Light adaptation in vertebrate photoreceptors is thought to be mediated through a number of biochemical feedback reactions that reduce the sensitivity of the photoreceptor and accelerate the kinetics of the photoresponse. Ca 2� plays a major role in this process by regulating several components of the phototransduction cascade. Guanylate cyclase and rhodopsin kinase are suggested to be the major sites regulated by Ca 2� . Recently, it was proposed that cGMP may be another messenger of light adaptation since it is able to regulate the rate of transducin GTPase and thus the lifetime of activated cGMP phosphodiesterase. Here we report measurements of the rates at which the changes in Ca 2� and cGMP are followed by the changes in the rates of corresponding enzymatic reactions in frog rod outer segments. Our data indicate that there is a temporal hierarchy among reactions that underlie light adaptation. Guanylate cyclase activity and rhodopsin phosphorylation respond to changes in Ca 2� very rapidly, on a subsecond time scale. This enables them to accelerate the falling phase of the flash response and to modulate flash sensitivity during continuous illumination. To the contrary, the acceleration of transducin GTPase, even after significant reduction in cGMP, occurs over several tens of seconds. It is substantially delayed by the slow dissociation of cGMP from the noncatalytic sites for cGMP binding located on cGMP phosphodiesterase. Therefore, cGMP-dependent regulation of transducin GTPase is likely to occur only during prolonged bright illumination. tor outer segment (reviewed in Chabre and Deterre, regulation is guanylate cyclase, the enzyme responsible for cGMP synthesis. This regulation is conferred through 1989; Pugh and Lamb, 1990). Photoreceptors adapt to the Ca 2� binding proteins known as guanylate cyclase ambient light through feedback reactions that regulate activating proteins (GCAPs) (Palczewski et al., 1994; either the catalytic activity or the catalytic lifetime of individual components of the phototransduction cas- Dizhoon et al., 1995). Cyclase activity is low in darkness cade. It is well established that Ca 2� plays a significant and increases when Ca 2� levels drop in response to light. The third site is the regulation of the sensitivity of role in this process (reviewed by Lagnado and Baylor, the cationic channel to cGMP. Lowering Ca 2� causes 1992; Koutalos and Yau, 1993;Bownds and Arshavsky, 1995). Ca 2� declines in response to light because it can the channel to become more sensitive to cGMP due to no longer enter through the cationic channels while it the dissociation of calmodulin (CaM) or a closely related Ca 2� binding protein. This might result in the accontinues to be extruded through the Na/Ca,K excelerated recovery of the photoresponse by facilitating changer. There are at least three sites of Ca 2� regulathe reopening of channels. In addition, the gain of the tion in the cascade. The first is the regulation of the lifetime of light-activated rhodopsin. Rhodopsin is turned casc...

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Characterization and Analysis of Frog Photoreceptor Membranes

Abstract: Frog photoreceptor membranes contain 54,000 g of protein

Cited by 180 publications

References 28 publications

Membrane Attachment Is Key to Protecting Transducin GTPase-Activating Complex from Intracellular Proteolysis in Photoreceptors

Membrane Attachment Is Key to Protecting Transducin GTPase-Activating Complex from Intracellular Proteolysis in Photoreceptors

Arrestin Translocation Is Induced at a Critical Threshold of Visual Signaling and Is Superstoichiometric to Bleached Rhodopsin

Onset of Feedback Reactions Underlying Vertebrate Rod Photoreceptor Light Adaptation

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