A third form of the G protein beta subunit. 1. Immunochemical identification and localization to cone photoreceptors.

Vertebrate retinal cones play a major role in both photopic vision and color perception. Although the molecular mechanism of visual excitation in the cone is not as well understood as in the rod, it is generally thought to involve a cone-specific G protein (cone transducin) that couples the cone visual pigment to a cGMP phosphodiesterase. Like all other G proteins, cone transducin is most likely a heterotrimer consisting of G alpha, G beta, and G gamma subunits. A G alpha subunit of cone transducin has been localized to the outer segment of bovine cones, but its associated G beta and G gamma subunits are unknown. To identify the G beta subunit involved in the phototransduction process of cones, we have developed a panel of antipeptide antisera against the most diverse region of the amino acid sequences encoded by G beta 1, G beta 2, and G beta 3 cDNAs and used them to determine the distribution of the G beta isoforms in different retinal preparations. We found that the G beta 3 subunit is present in bovine retinal transducin and phosducin-T beta gamma complex preparations which were previously thought to contain only G beta 1. Analysis of its subcellular distribution indicated that G beta 3 is predominantly cytoplasmic. Immunocytochemical staining of bovine retinal sections with the anti-G beta 3 antiserum further revealed a specific localization of G beta 3 in cones but not in rods. In contrast, anti-G beta 1 antiserum stained only the rods. These results suggest that G beta 3 is the G beta subunit of cone transducin and confirms the proposition that rods and cones utilize distinct signaling proteins for phototransduction.     

Role of membrane integrity on G protein-coupled receptors: Rhodopsin stability and function

Rhodopsin is a prototypical G protein-coupled receptor (GPCR) - a member of the superfamily that shares a similar structural architecture consisting of seven-transmembrane helices and propagates various signals across biological membranes. Rhodopsin is embedded in the lipid bilayer of specialized disk membranes in the outer segments of retinal rod photoreceptor cells where it transmits a light-stimulated signal. Photoactivated rhodopsin then activates a visual signaling cascade through its cognate G protein, transducin or Gt, that results in a neuronal response in the brain. Interestingly, the lipid composition of ROS membranes not only differs from that of the photoreceptor plasma membrane but is critical for visual transduction. Specifically, lipids can modulate structural changes in rhodopsin that occur after photoactivation and influence binding of transducin. Thus, altering the lipid organization of ROS membranes can result in visual dysfunction and blindness.     

Modeling of phototransduction processes in the photoreceptor disk membranes by the Monte Carlo method

The Monte Carlo method was used to model the diffusion behaviors of functionally important proteins of the phototransduction system in retinal rod outer segment disk membranes. The results expand our knowledge of the mechanisms of inactivation of the main phototransduction heterotrimeric GTP-binding protein transducin.     

Functional mapping of interacting regions of the photoreceptor phosphodiesterase (PDE6) γ-subunit with PDE6 catalytic dimer, transducin, and regulator of G-protein signaling9-1 (RGS9-1)

The cGMP phosphodiesterase (PDE6) involved in visual transduction in photoreceptor cells contains two inhibitory γ-subunits (Pγ) which bind to the catalytic core (Pαβ) to inhibit catalysis and stimulate cGMP binding to the GAF domains of Pαβ. During visual excitation, interaction of activated transducin with Pγ relieves inhibition. Pγ also participates in a complex with RGS9-1 and other proteins to accelerate the GTPase activity of activated transducin. We studied the structural determinants for these important functions of Pγ. First, we identified two important sites in the middle region of Pγ (amino acids 27-38 and 52-54) that significantly stabilize the overall binding affinity of Pγ with Pαβ. The ability of Pγ to stimulate noncatalytic cGMP binding to the GAF domains of PDE6 has been localized to amino acids 27-30 of Pγ. Transducin activation of PDE6 catalysis critically depends on the presence of Ile54 in the glycine-rich region of Pγ in order to relieve inhibition of catalysis. The central glycine-rich region of Pγ is also required for transducin to increase cGMP exchange at the GAF domains. Finally, Thr-65 and/or Val-66 of Pγ are critical residues for Pγ to stimulate GTPase activity of transducin in a complex with RGS9-1. We propose that the glycine-rich region of Pγ is a primary docking site for PDE6-interacting proteins involved in the activation/inactivation pathways of visual transduction. This functional mapping of Pγ with its binding partners demonstrates the remarkable versatility of this multifunctional protein and its central role in regulating the activation and lifetime of visual transduction.     

Visual responses in mice lacking critical components of all known retinal phototransduction cascades

The mammalian visual system relies upon light detection by outer-retinal rod/cone photoreceptors and melanopsin-expressing retinal ganglion cells. Gnat1(-/-);Cnga3(-/-);Opn4(-/-) mice lack critical elements of each of these photoreceptive mechanisms via targeted disruption of genes encoding rod α transducin (Gnat1); the cone-specific α3 cyclic nucleotide gated channel subunit (Cnga3); and melanopsin (Opn4). Although assumed blind, we show here that these mice retain sufficiently widespread retinal photoreception to drive a reproducible flash electroretinogram (ERG). The threshold sensitivity of this ERG is similar to that of cone-based responses, however it is lost under light adapted conditions. Its spectral efficiency is consistent with that of rod opsin, but not cone opsins or melanopsin, indicating that it originates with light absorption by the rod pigment. The TKO light response survives intravitreal injection of U73122 (a phospholipase C antagonist), but is inhibited by a missense mutation of cone α transducin (Gnat2(cpfl3)), suggesting Gnat2-dependence. Visual responses in TKO mice extend beyond the retina to encompass the lateral margins of the lateral geniculate nucleus and components of the visual cortex. Our data thus suggest that a Gnat1-independent phototransduction mechanism downstream of rod opsin can support relatively widespread responses in the mammalian visual system. This anomalous rod opsin-based vision should be considered in experiments relying upon Gnat1 knockout to silence rod phototransduction.     

Autophagy-mediated catabolism of visual transduction proteins prevents retinal degeneration

Autophagy is a lysosomal degradation pathway critical to preventing the accumulation of cytotoxic proteins. Deletion of the essential autophagy gene Atg5 from the rod photoreceptors of the retina (atg5^Δrod mouse) results in the accumulation of the phototransduction protein transducin and the degeneration of these neurons. The purpose of this study is to test the hypothesis that autophagic degradation of visual transduction proteins prevents retinal degeneration. Targeted deletion of both Gnat1 (a gene encoding the α subunit of the heterotrimeric G-protein transducin) and Atg5 in the rod photoreceptors resulted in a significantly decreased rate of rod cell degeneration as compared to the atg5^Δrod mouse retina, and considerable preservation of photoreceptors. Supporting this we used a novel technique to immunoprecipitate green fluorescent protein (GFP)-tagged autophagosomes from the retinas of the GFP-LC3 mice and demonstrated that the visual transduction proteins transducin and ARR/arrestin are associated with autophagosome-specific proteins. Altogether, this study shows that degradation of phototransduction proteins by autophagy is necessary to prevent retinal degeneration. In addition, we demonstrate a simple and easily reproducible immunoisolation technique for enrichment of autophagosomes from the GFP-LC3 mouse retina, providing a novel application to the study of autophagosome contents across different organs and specific cell types in vivo.     

Inhibition of retinal guanylyl cyclase by the RGS9-1 N-terminus

Cyclic GMP plays a key role in retinal phototransduction and its photoreceptor concentration is precisely controlled by the cooperative action of cGMP phosphodiesterase (PDE) and retinal guanylyl cyclase (retGC). However, studies of the relationship between these two systems have focused only on a Ca(2+)-mediated, indirect connection. Using a retinal "regulator of G-protein signaling" (RGS9-1) and its fragments, we show that the N-terminus of RGS9-1 inhibits retGC activity. We also indicate that the GGL domain and/or the RGS domain function as an internal suppressor against the N-terminus, suggesting that proteins bound to these domains regulate the inhibitory activity of the N-terminus. Direct interaction of retGC with RGS9-1 and its N-terminus is also proved by immunoprecipitation and an overlay technique. Since RGS9-1 also controls the lifetime of transducin-activated PDE through regulating GTPase activity of transducin, this study strongly suggests that RGS9-1 mediates the direct interaction between PDE and retGC systems, and that this ingenious mechanism plays an important role in tuning of cGMP concentration in photoreceptors.     

Similarities between G-proteins in visual cells of Sepia and cattle

In contrast to antisera against native transducin a polyclonal antiserum raised against heat-denatured bovine transducin crossreacts with the G-protein from Sepia visual cells. This antiserum recognizes a 44 kDa (G alpha) and a 36 kDa (G beta) protein band from Sepia photosensory membrane preparation. Furthermore we purified the antibody-binding G-protein from Sepia by binding it to light-activated rhodopsin of Sepia and GTP-induced extraction, similar to the purification of bovine transducin. This G-protein is probably involved in the phototransduction process. The purified Sepia G-protein did bind to vertebrate photosensoric membrane upon illumination, but was not eluted by GTP-containing buffer solution. After extensive bleaching, the G-protein became soluble.     

Conformations of the active and inactive states of opsin

The signaling state metarhodopsin II of the visual pigment rhodopsin decays to the apoprotein opsin and all-trans retinal, which are then regenerated to rhodopsin by the visual cycle. Opsin is known to have at neutral pH only a small residual constitutive activity toward its G protein transducin, which is thought to play a considerable role in light adaptation (bleaching desensitization). In this study we show with Fourier-transform infrared spectroscopy that after metarhodopsin II decay, opsin exists in two conformational states that are in a pH-dependent equilibrium at 30 degrees C with a pK of 4.1 in the presence of hydroxylamine scavenging the endogenous all-trans retinal. Despite the lack of the native agonist in its binding pocket, the low pH opsin conformation is very similar to that of metarhodopsin II and is likewise stabilized by peptides derived from rhodopsin's cognate G protein, transducin. The high pH form, on the other hand, has some conformational similarity to the inactive metarhodopsin I state. We therefore conclude that the opsin apoprotein displays intrinsic conformational states that are merely modulated by bound all-trans retinal.     

Arrestin of bovine photoreceptors reveals strong ATP binding

The soluble protein arrestin (also named 48K-protein or retinal-S-antigen) is involved in controlling light-dependent transducin and cGMP phosphodiesterase activity in retinal rods. It is also known for its ability to induce autoimmune reactions in the eye causing the eye disease uveitis. We report here a rapid binding of ATP to arrestin with KA = 2 x 10(21) (l/mol)3 and a coordination number n = 3. This ATP binding to arrestin supports the notion of a nucleotide exchange which initiates the rapid inhibitory action of this enzyme during the primary step of vertebrate phototransduction.     

Absence of the RGS9.Gbeta5 GTPase-activating complex in photoreceptors of the R9AP knockout mouse

Timely termination of the light response in retinal photoreceptors requires rapid inactivation of the G protein transducin. This is achieved through the stimulation of transducin GTPase activity by the complex of the ninth member of the regulator of G protein signaling protein family (RGS9) with type 5 G protein beta subunit (Gbeta5). RGS9.Gbeta5 is anchored to photoreceptor disc membranes by the transmembrane protein, R9AP. In this study, we analyzed visual signaling in the rods of R9AP knockout mice. We found that light responses from R9AP knockout rods were very slow to recover and were indistinguishable from those of RGS9 or Gbeta5 knockout rods. This effect was a consequence of the complete absence of any detectable RGS9 from the retinas of R9AP knockout mice. On the other hand, the level of RGS9 mRNA was not affected by the knockout. These data indicate that in photoreceptors R9AP determines the stability of the RGS9.Gbeta5 complex, and therefore all three proteins, RGS9, Gbeta5 , and R9AP, are obligate members of the regulatory complex that speeds the rate at which transducin hydrolyzes GTP.     

Gene replacement therapy for retinal CNG channelopathies

Visual phototransduction relies on the function of cyclic nucleotide-gated channels in the rod and cone photoreceptor outer segment plasma membranes. The role of these ion channels is to translate light-triggered changes in the second messenger cyclic guanosine 3′–5′-monophosphate levels into an electrical signal that is further processed within the retinal network and then sent to higher visual centers. Rod and cone photoreceptors express distinct CNG channels. The rod photoreceptor CNG channel is composed of one CNGB1 and three CNGA1 subunits, whereas the cone channel is formed by one CNGB3 and three CNGA3 subunits. Mutations in any of these channel subunits result in severe and currently untreatable retinal degenerative diseases like retinitis pigmentosa or achromatopsia. In this review, we provide an overview of the human diseases and relevant animal models of CNG channelopathies. Furthermore, we summarize recent results from preclinical gene therapy studies using adeno-associated viral vectors and discuss the efficacy and translational potential of these gene therapeutic approaches.     

Unknown mechanisms of the GPCR-signaling cascade in vertebrate photoreceptors

Among the GPCR-signaling cascades, phototransduction in vertebrate retinal photoreceptors has been characterized in unprecedented details. It is believed that basic mechanisms of phototransduction and adaptation are reliably and completely established, and phototransduction may serve as a benchmark for understanding other G-protein-coupled systems. In this review, we compare present scheme of phototransduction with other GPCR-cascades in order to reveal their similarities and specific features. We show, based mainly on our physiological and biophysical data, that the existing scheme misses a few important regulations whose molecular basis is unknown. There exists a fast and efficient mechanism that accelerates the turn-off of the activated G-protein (transducin) during light adaptation. A few slowly acting processes result in a long-lasting modification of the cascade's components and regulate the speed of rhodopsin and transducin quenching. Similarly to other GPCR-cascades, one may suggest that there are multiple signalling pathways that start from photoactivated rhodopsin and rely on different secondary messengers (e.g. cAMP vs. cGMP). We also show that rhodopsin in retinal rods may form areas of paracristalline organization, and that the oligomerization might be a mechanism for controlling the amplification of the signalling cascade. The missing mechanisms are by no means minor, and could ensure sensitivity regulation within two orders of magnitude range.     

Low Activation and Fast Inactivation of Transducin in Carp Cones

Cone photoreceptors show lower light sensitivity and briefer light responses than rod photoreceptors. The light detection signal in these cells is amplified through a phototransduction cascade. The first step of amplification in the cascade is the activation of a GTP-binding protein, transducin (Tr), by light-activated visual pigment (R*). We quantified transducin activation by measuring the binding of GTPγS in purified carp rod and cone membrane preparations with the use of a rapid quench apparatus and found that transducin activation by an R* molecule is ∼5 times less efficient in cones than in rods. Transducin activation terminated in less than 1 s in cones, more quickly than in rods. The rate of GTP hydrolysis in Tr*, and thus the rate of Tr* inactivation, was ∼25 times higher in cones than in rods. This faster inactivation of Tr* ensures briefer light responses in cones. The expression level of RGS9 was found to be ∼20 times higher in cones than in rods, which explains higher GTP hydrolytic activity and, thus, faster Tr* inactivation in cones than in rods. Although carp rods and cones express rod- or cone-versions of visual pigment and transducin, these molecules themselves do not seem to induce the differences significantly in the transducin activation and Tr* inactivation in rods and cones. Instead, the differences seem to be brought about in a rod or cone cell-type specific manner.     

Dynamics-based community analysis and perturbation response scanning of allosteric interaction networks in the TRAP1 chaperone structures dissect molecular linkage between conformational asymmetry and sequential ATP hydrolysis

Allosteric interactions of the Hsp90 chaperones with cochaperones and diverse protein clients can often exhibit distinct asymmetric features that determine regulatory mechanisms and cellular functions in many signaling networks. The recent crystal structures of the mitochondrial Hsp90 isoform TRAP1 in complexes with ATP analogs have provided first evidence of significant asymmetry in the closed dimerized state that triggers independent activity of the chaperone protomers, whereby preferential hydrolysis of the buckled protomer is followed by conformational flipping between protomers and hydrolysis of the second protomer. Despite significant insights in structural characterizations of the TRAP1 chaperone, the atomistic details and mechanics of allosteric interactions that couple sequential ATP hydrolysis with asymmetric conformational switching in the TRAP1 protomers remain largely unknown. In this work, we explored atomistic and coarse-grained simulations of the TRAP1 dimer structures in combination with the ensemble-based network modeling and perturbation response scanning of residue interaction networks to probe salient features underlying allosteric signaling mechanism. This study has revealed that key effector sites that orchestrate allosteric interactions occupy the ATP binding region and N-terminal interface of the buckled protomer, whereas the main sensors of allosteric signals that drive functional conformational changes during ATPase cycle are consolidated near the client binding region of the straight protomer, channeling the energy of ATP hydrolysis for client remodeling. The community decomposition analysis of the interaction networks and reconstruction of allosteric communication pathways in the TRAP1 structures have quantified mechanism of allosteric regulation, revealing control points and interactions that coordinate asymmetric switching during ATP hydrolysis.     

Farnesylation of retinal transducin underlies its translocation during light adaptation

G proteins are posttranslationally modified by isoprenylation: either farnesylation or geranylgeranylation. The gamma subunit of retinal transducin (Talpha/Tbetagamma) is selectively farnesylated, and the farnesylation is required for light signaling mediated by transducin in rod cells. However, whether and how this selective isoprenylation regulates cellular functions remain poorly understood. Here we report that knockin mice expressing geranylgeranylated Tgamma showed normal rod responses to dim flashes under dark-adapted conditions but exhibited impaired properties in light adaptation. Of note, geranylgeranylation of Tgamma suppressed light-induced transition of Tbetagamma from membrane to cytosol, and also attenuated its light-dependent translocation from the outer segment to the inner region, an event contributing to retinal light adaptation. These results indicate that, while the farnesylation of transducin is interchangeable with the geranylgeranylation in terms of the light signaling, the selective farnesylation is important for visual sensitivity regulation by providing sufficient but not excessive membrane anchoring of Tbetagamma.     

Altered functionality in rhodopsin point mutants associated with retinitis pigmentosa

Point mutations found in rhodopsin associated with the retinal degenerative disease retinitis pigmentosa have been expressed in mammalian COS-1 cells, purified, and characterised. The mutations characterised-most of them for the first time-have been Met44Thr, Gly114Asp, Arg135Leu, Val137Met, and Pro171Leu in the transmembrane domain; Leu328Pro and Ala346Pro in the C-terminal tail of the cytoplasmic domain; and Gly106Trp in the intradiscal domain. Several of these mutations cause misfolding which results in impaired 11-cis-retinal binding. Two of them, Met44Thr and Val137Met, show spectral and structural features similar to those of wild type rhodopsin (Type I mutants) but significantly increased transducin initial activation rates. We propose that, in the case of these mutants, abnormal functioning resulting in faster activation kinetics could also play a role in retinitis pigmentosa by altering the stoichiometric balance of the different proteins involved in the phototransduction biochemical reactions.     

Light-driven translocation of signaling proteins in vertebrate photoreceptors

The dynamic localization of proteins within cells is often determined by environmental stimuli. In retinal photoreceptors, light exposure results in the massive translocation of three key signal transduction proteins, transducin, arrestin and recoverin, into and out of the outer segment compartment where phototransduction takes place. This phenomenon has rapidly taken the center stage of photoreceptor cell biology, thanks to the introduction of new quantitative and transgenic approaches. Here, we discuss evidence that intracellular protein translocation contributes to adaptation of photoreceptors to diurnal changes in ambient light intensity and summarize the current debate on whether it is driven by diffusion or molecular motors.