Removal of phosphorylation sites of subunit of phosphodiesterase 6 alters rod light response (original) (raw)
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Biochemical and Biophysical Research Communications, 2009
The γ subunit of rod-specific cGMP phosphodiesterase 6 (PDE6γ), an effector of the G-protein GNAT1, is a key regulator of phototransduction. The results of several in vitro biochemical reconstitution experiments conducted to examine the effects of phosphorylation of PDE6γ on its ability to regulate the PDE6 catalytic core have been inconsistent, showing that phosphorylation of PDE6γ may increase or decrease the ability of PDE6γ to deactivate phototransduction. To resolve role of phosphorylation of PDE6γ in living photoreceptors, we generated transgenic mice in which either one or both Threonine (T) sites in PDE6γ (T22 and T35), which are candidates for putative regulatory phosphorylation, were substituted with alanine (A). Phosphorylation of these sites was examined as a function of light exposure. We found that phosphorylation of T22 increases with light exposure in intact mouse rods while constitutive phosphorylation of T35 is unaffected by light in intact mouse rods and cones. Phosphorylation of the cone isoform of PDE6γ, PDE6H, is constitutively phosphorylated at the T20 residue. Light-induced T22 phosphorylation was lost in T35A transgenic rods, and T35 phosphorylation was extinguished in T22A transgenic rods. The interdependency of phosphorylation of T22 and T35 suggests that light-induced, post-translational modification of PDE6γ is essential for the regulation of G-protein signaling.
Modulation of Phosphodiesterase6 Turnoff during Background Illumination in Mouse Rod Photoreceptors
Journal of Neuroscience, 2008
In rod photoreceptors of wild-type mice, background light produces an acceleration of the decay of responses to brief flashes, accompanied by a decrease in the rate-limiting time constant for response decay. In rods in which phosphodiesterase gamma (PDEgamma) lacks one of its sites of phosphorylation (T35A rods), both the waveform of response decay and the rate-limiting time constant are nearly unaffected by backgrounds. These effects are not the result of the removal of the phosphorylation site per se, because rods lacking both of the phosphorylation sites of PDEgamma (T22A/T35A rods) adapt to light in a nearly normal manner. Because PDEgamma is one of the proteins of the GTPase activating protein (GAP) complex, our experiments argue for a novel mechanism of photoreceptor light adaptation produced by modulation of GAP-dependent hydrolysis of transducin alpha GTP. In PDEgamma T35A rods, a change in the conformation of the PDEgamma subunit may hinder or mask this mechanism, which in mammals appears to be primarily responsible for the quickening of the temporal resolution of the rod response in backgrounds. Modulation of PDE turnoff also helps to prevent premature saturation of the rod in bright backgrounds, thus making an important contribution to light adaptation. Our experiments provide evidence for modulation of GAP protein-dependent response turnoff, which may also play a role in controlling signal duration at hormone receptors and synapses in the CNS.
Cellular Signalling, 2012
The light-dependent decrease in cyclic guanosine monophosphate (cGMP) in the rod outer segment is produced by a phosphodiesterase (PDE6), consisting of catalytic α and β subunits and two inhibitory γ subunits. The molecular mechanism of PDE6γ regulation of the catalytic subunits is uncertain. To study this mechanism in vivo, we introduced a modified Pde6g gene for PDE6γ into a line of Pde6g tm1 /Pde6g tm1 mice that does not express PDE6γ. The resulting ILE86TER mice have a PDE6γ that lacks the two final carboxyl-terminal Ile 86 and Ile 87 residues, a mutation previously shown in vitro to reduce inhibition by PDE6γ. ILE86TER rods showed a decreased sensitivity and rate of activation, probably the result of a decreased level of expression of PDE6 in ILE86TER rods. More importantly, they showed a decreased rate of decay of the photoresponse, consistent with decreased inhibition of PDE6 α and β by PDE6γ. Furthermore, ILE86TER rods had a higher rate of spontaneous activation of PDE6 than WT rods. Circulating current in ILE86TER rods that also lacked both guanylyl cyclase activating proteins (GCAPs) could be increased several fold by perfusion with 100 µM of the PDE6 inhibitor 3-isobutyl-1-methylxanthine (IBMX), consistent with a higher rate of dark PDE6 activity in the mutant photoreceptors. In contrast, IBMX had little effect on the circulating current of WT rods, unlike previous results from amphibians. Our results show for the first time that the Ile 86 and Ile 87 residues are necessary for normal inhibition of PDE6 catalytic activity in vivo, and that increased basal activity of PDE can be partially compensated by GCAP-dependent regulation of guanylyl cyclase.
Open Biology
We develop an improved quantitative model of mammalian rod phototransduction, and we apply it to the prediction of responses to bright flashes of light. We take account of the recently characterized dimeric nature of PDE6 activation, where the configuration of primary importance has two transducin molecules bound. We simulate the stochastic nature of the activation and shut-off reactions to generate the predicted kinetics of the active molecular species on the disc membrane surfaces, and then we integrate the differential equations for the downstream cytoplasmic reactions to obtain the predicted electrical responses. The simulated responses recover the qualitative form of bright-flash response families recorded from mammalian rod photoreceptors. Furthermore, they provide an accurate description of the relationship between the time spent in saturation and flash intensity, predicting the transition between first and second ‘dominant time constants’ to occur at an intensity around 5000...
Journal of Neuroscience, 2006
We have generated a mouse with rod photoreceptors overexpressing the ␥ inhibitory subunit (PDE6␥) of the photoreceptor G-protein effector cGMP phosphodiesterase (PDE6). PDE6␥ overexpression decreases the rate of rise of the rod response at dim intensities, indicating a reduction in the gain of transduction that may be the result of cytoplasmic PDE6␥ binding to activated transducin ␣ GTP (T ␣-GTP) before the T ␣-GTP binds to endogenous PDE6␥. Excess PDE6␥ also produces a marked acceleration in the falling phase of the light response and more rapid recovery of sensitivity and circulating current after prolonged light exposure. These effects are not mediated by accelerating GTP hydrolysis through the GAP (GTPase activating protein) complex, because the decay of the light response is also accelerated in rods that overexpress PDE6␥ but lack RGS9. Our results show that the PDE6␥ binding sites of PDE6 ␣ and  are accessible to excess (presumably cytoplasmic) PDE6␥ in the light, once endogenous PDE6␥ has been displaced from its binding site by T ␣-GTP. They also suggest that in the presence of T ␣-GTP, the PDE6␥ remains attached to the rest of the PDE6 molecule, but after conversion of T ␣-GTP to T ␣-GDP, the PDE6␥ may dissociate from the PDE6 and exchange with a cytoplasmic pool. This pool may exist even in wild-type rods and may explain the decay of rod photoresponses in the presence of nonhydrolyzable analogs of GTP.
Journal of Biological Chemistry, 2010
The central enzyme of the visual transduction cascade, cGMP phosphodiesterase (PDE6), is regulated by its ␥-subunit (P␥), whose inhibitory constraint is released upon binding of activated transducin. It is generally believed that the last four or five C-terminal amino acid residues of P␥ are responsible for blocking catalysis. In this paper, we showed that the last 10 C-terminal residues (P␥78 -87) are the minimum required to completely block catalysis. The kinetic mechanism of inhibition by the P␥ C terminus depends on which substrate is undergoing catalysis. We also discovered a second mechanism of P␥ inhibition that does not require this C-terminal region and that is capable of inhibiting up to 80% of the maximal cGMP hydrolytic rate. Furthermore, amino acids 63-70 and/or the intact ␣2 helix of P␥ stabilize binding of C-terminal P␥ peptides by 100-fold. When PDE6 catalytic subunits were reconstituted with portions of the P␥ molecule and tested for activation by transducin, we found that the C-terminal region (P␥63-87) by itself could not be displaced but that transducin could relieve inhibition of certain P␥ truncation mutants. Our results are consistent with two distinct mechanisms of P␥ inhibition of PDE6. One involves direct interaction of the C-terminal residues with the catalytic site. A second regulatory mechanism may involve binding of other regions of P␥ to the catalytic domain, thereby allosterically reducing the catalytic rate. Transducin activation of PDE6 appears to require interaction with both the C terminus and other regions of P␥ to effectively relieve its inhibitory constraint. . 2 The abbreviations used are: PDE6, photoreceptor cyclic nucleotide phosphodiesterase; P␣, catalytic dimer of PDE6 ␣and -subunits; P␥, inhibitory ␥ subunit of PDE6; T␣*, activated transducin ␣-subunit; GTP␥S, guanosine 5Ј-O-(thiotriphosphate).
Determination of basal phosphodiesterase activity in mouse rod photoreceptors with cGMP clamp
Scientific Reports
Light regulates cGMp concentration in the photoreceptor cytoplasm by activating phosphodiesterase (pDe) molecules through a G-protein signalling cascade. spontaneous pDe activity is present in rod outer segments even in darkness. this basal pDe activity (β dark) has not been determined in wild type mammalian photoreceptor cells although it plays a key role in setting the sensitivity and recovery kinetics of rod responses. We present a novel method for determination of β dark using local electroretinography (LeRG) from isolated mouse retinas. the method is based on the ability of pDe inhibitors to decrease β dark , which can be counterbalanced by increasing pDe activity with light. this procedure clamps cytoplasmic cGMp to its dark value. β dark can be calculated based on the amount of light needed for the "cGMp clamp" and information extracted from the registered rod photoresponses. Here we apply this method to determine β dark values for the first time in the mammalian rods and obtain the following estimates for different mouse models: 3.9 s −1 for wild type, 4.5 s −1 for guanylate cyclase activating proteins (GCAPs) knockout, and 4.4 s −1 for GCAps and recoverin double knockout mice. our results suggest that depletion of GCAPs or recoverin do not affect β dark. Photoreceptor cells convert light information to sensory signals in a process called phototransduction. When a photon is absorbed in a rhodopsin molecule in the rod outer segment disk membrane, the rhodopsin activates G-proteins, transducins, and the activated transducins bind to phosphodiesterase-6 molecules (PDE) forming enzyme complexes, which hydrolyse cyclic guanosine monophosphate (cGMP) at nearly a diffusion limited rate 1. A rapid drop in the cytoplasmic cGMP concentration leads to the closure of the cyclic nucleotide gated (CNG) channels in the outer segment plasma membrane, hyperpolarization of the cell membrane, change in the release rate of glutamate in the rod terminal and transmission of the light-generated signal to the inner retina (see e.g. 2,3). Thermal energy causes spontaneous activations of phototransduction molecules, which leads to fluctuations in the cytoplasmic level of cGMP. These fluctuations make up the main part the dark noise of photoreceptors 4. The dark noise consists mainly of three components: discrete spontaneous activations of rhodopsin, high frequency noise from fluctuations in the CNG channel conductance, and continuous noise from thermal activations of PDE 4. The amount of active PDE in darkness determines the rate constant for spontaneous cGMP hydrolysis, i.e. the basal PDE activity (β dark), which sets the steady state level and the turnover rate of cGMP. Hence, it is one of the main factors in setting the kinetics of photoresponse deactivation and spatial propagation of cGMP concentration drop during photoresponses 5. The basal PDE activity has been determined earlier for amphibian rod photoreceptors by abruptly blocking the activity of either PDE or guanylate cyclase 6-10. In the method, single photoreceptor outer segment is exposed to rapid solution changes while recording photoreceptor circulating dark current. However, this has turned out to be challenging with the fragile mammalian photoreceptors, and until now, no one has determined the β dark of wild type mammalian photoreceptors. Gross et al. (2012) demonstrated that when the calcium mediated feedback to guanylate cyclase is abolished by knocking out the guanylate cyclase activating proteins (GCAPs) and the lifetime of activated PDE is decreased by overexpressing RGS9, the basal PDE activity becomes the dominant factor determining the light response deactivation kinetics 5. In these circumstances, the late recovery of a single-photon response allows the determination of β dark. However, it is not known