Subunit Of Transducin Research Articles

Structural information and membrane binding of truncated RGS9-1 Anchor Protein and its C-terminal hydrophobic segment

Visual phototransduction takes place in photoreceptor cells. Light absorption by rhodopsin leads to the activation of transducin as a result of the exchange of its GDP for GTP. The GTP-bound ⍺-subunit of transducin then activates phosphodiesterase (PDE), which in turn hydrolyzes cGMP leading to photoreceptor hyperpolarization. Photoreceptors return to the dark state upon inactivation of these proteins. In particular, PDE is inactivated by the protein complex R9AP/RGS9-1/Gβ5. R9AP (RGS9-1 anchor protein) is responsible for the membrane anchoring of this protein complex to photoreceptor outer segment disk membranes most likely by the combined involvement of its C-terminal hydrophobic domain as well as other types of interactions. This study thus aimed to gather information on the structure and membrane binding of the C-terminal hydrophobic segment of R9AP as well as of truncated R9AP (without its C-terminal domain, R9AP∆TM). Circular dichroism and infrared spectroscopic measurements revealed that the secondary structure of R9AP∆TM mainly includes ⍺-helical structural elements. Moreover, intrinsic fluorescence measurements of native R9AP∆TM and individual mutants lacking one tryptophan demonstrated that W79 is more buried than W173 but that they are both located in a hydrophobic environment. This method also revealed that membrane binding of R9AP∆TM does not involve regions near its tryptophan residues, while infrared spectroscopy validated its binding to lipid vesicles. Additional fluorescence measurements showed that the C-terminal segment of R9AP is membrane embedded. Maximum insertion pressure and synergy data using Langmuir monolayers suggest that interactions with specific phospholipids could be involved in the membrane binding of R9AP∆TM.

Diurnal variation in opsin expression and common housekeeping genes necessitates comprehensive normalization methods for quantitative real-time PCR analyses.

To determine the visual sensitivities of an organism of interest, quantitative reverse transcription-polymerase chain reaction (qRT-PCR) is often used to quantify expression of the light-sensitive opsins in the retina. While qRT-PCR is an affordable, high-throughput method for measuring expression, it comes with inherent normalization issues that affect the interpretation of results, especially as opsin expression can vary greatly based on developmental stage, light environment or diurnal cycles. We tested for diurnal cycles of opsin expression over a period of 24hr at 1-hr increments and examined how normalization affects a data set with fluctuating expression levels using qRT-PCR and transcriptome data from the retinae of the cichlid Pelmatolapia mariae. We compared five methods of normalizing opsin expression relative to (a) the average of three stably expressed housekeeping genes (Ube2z, EF1-α and β-actin), (b) total RNA concentration, (c) GNAT2, (the cone-specific subunit of transducin), (d) total opsin expression and (e) only opsins expressed in the same cone type. Normalizing by proportion of cone type produced the least variation and would be best for removing time-of-day variation. In contrast, normalizing by housekeeping genes produced the highest daily variation in expression and demonstrated that the peak of cone opsin expression was in the late afternoon. A weighted correlation network analysis showed that the expression of different cone opsins follows a very similar daily cycle. With the knowledge of how these normalization methods affect opsin expression data, we make recommendations for designing sampling approaches and quantification methods based upon the scientific question being examined.

Gain-of-function screen of α-transducin identifies an essential phenylalanine residue necessary for full effector activation

Two regions on the α subunits of heterotrimeric GTP-binding proteins (G-proteins), the Switch II/α2 helix (which changes conformation upon GDP-GTP exchange) and the α3 helix, have been shown to contain the binding sites for their effector proteins. However, how the binding of Gα subunits to their effector proteins is translated into the stimulation of effector activity is still poorly understood. Here, we took advantage of a reconstituted rhodopsin-coupled phototransduction system to address this question and identified a distinct surface and an essential residue on the α subunit of the G-protein transducin (αT) that is necessary to fully activate its effector enzyme, the cGMP phosphodiesterase (PDE). We started with a chimeric G-protein α subunit (αT*) comprising residues mainly from αT and a short stretch of residues from the Gi1 α subunit (αi1), which only weakly stimulates PDE activity. We then reinstated the αT residues by systematically replacing the corresponding αi1 residues within αT* with the aim of fully restoring PDE stimulatory activity. These experiments revealed that the αG/α4 loop and a phenylalanine residue at position 283 are essential for conferring the αT* subunit with full PDE stimulatory capability. We further demonstrated that this same region and amino acid within the α subunit of the Gs protein (αs) are necessary for full adenylyl cyclase activation. These findings highlight the importance of the αG/α4 loop and of an essential phenylalanine residue within this region on Gα subunits αT and αs as being pivotal for their selective and optimal stimulation of effector activity.

Knockout ofRP2decreases GRK1 and rod transducin subunits and leads to photoreceptor degeneration in zebrafish

Retinitis pigmentosa (RP) affects about 1.8 million individuals worldwide. X-linked retinitis pigmentosa (XLRP) is one of the most severe forms of RP. Nearly 85% of XLRP cases are caused by mutations in the X-linked retinitis pigmentosa 2 (RP2) and RPGR. RP2 has been considered to be a GTPase activator protein for ARL3 and to play a role in the traffic of ciliary proteins. The mechanism of how RP2 mutations cause RP is still unclear. In this study, we generated an RP2 knockout zebrafish line using transcription activator-like effector nuclease technology. Progressive retinal degeneration could be observed in the mutant zebrafish. The degeneration of rods' outer segments (OSs) is predominant, followed by the degeneration of cones' OS. These phenotypes are similar to the characteristics of RP2 patients, and also partly consistent with the phenotypes of RP2 knockout mice and morpholino-mediated RP2 knockdown zebrafish. For the first time, we found RP2 deletion leads to decreased protein levels and abnormal retinal localizations of GRK1 and rod transducin subunits (GNAT1 and GNB1) in zebrafish. Furthermore, the distribution of the total farnesylated proteins in zebrafish retina is also affected by RP2 ablation. These molecular alterations observed in the RP2 knockout zebrafish might probably be responsible for the gradual loss of the photoreceptors' OSs. Our work identified the progression of retinal degeneration in RP2 knockout zebrafish, provided a foundation for revealing the pathogenesis of RP caused by RP2 mutations, and would help to develop potential therapeutics against RP in further studies.

The X-linked Retinitis Pigmentosa protein RP2 facilitates traffic of cilia target proteins

Photoreceptors are specialized ciliated sensory neurons and aberrant traffic of proteins to the outer segment causes photoreceptor cell death. RP2 is a GTPase activating protein (GAP) for the small GTPase Arl3 and both proteins facilitate protein trafficking to primary cilia. We used GST-RP2 pull down from retinal lysates and identified the Gβ subunit of transducin (Gβ1) as a novel RP2 interacting protein. RP2 competes with Gγ1 for Gβ1 binding and does not interact with the Gβ:Gγ heterodimer. In SK-N-SH cells, overexpression of Gβ1 resulted in the cytoplasmic accumulation of the protein, whereas co-expression of Gβ1 with either RP2 or Gγ1 restored membrane association of Gβ1. Depletion of RP2 in ARPE19 cells by siRNA resulted in a shift of Gβ1 from the membrane to the cytosol, confirming that RP2 facilitates the membrane association of Gβ1. This shift in Gβ1 localization was rescued by Gγ1 overexpression. Membrane targeting of Gβ1 required RP2 N-terminal myristoylation and occurs via the co-factor C (TBCC) homology domain. The interaction was disrupted by the pathogenic RP2 mutation R118H, which blocks Arl3 GAP activity. Arl3-Q71L competed with Gβ1 for RP2 binding suggesting that RP2 GAP activity on Arl3 would release Gβ1. RP2 stimulated the association of Gβ1 with Rab11, an important GTPase for post-Golgi vesicle trafficking of photoreceptor proteins. Collectively our data support a role for RP2 in facilitating membrane association and traffic of Gβ1. Combined with other recent evidence, this suggests that RP2 may co-operate with Arl3 and its effectors in cilia associated trafficking of G proteins.

Arl3 and RP2 mediated assembly and traffic of membrane associated cilia proteins

The traffic of proteins to the outer segment of photoreceptors is a fundamentally important process, which when perturbed results in photoreceptor cell death. Recent reports have revealed a novel pathway for the traffic of lipid-modified proteins involving the small GTPase Arl3 and its effectors PDEδ and Unc119. The retinitis pigmentosa protein RP2 is a GTPase activating protein (GAP) for Arl3 and also appears to regulate the assembly and traffic of membrane associated protein complexes. We recently identified the Gβ subunit of transducin (Gβ1) as a novel RP2 interacting protein. Our data support a role for RP2 in facilitating membrane association and traffic of Gβ1, potentially prior to the formation of the obligate Gβ:Gγ heterodimer. Here, we review the recent evidence that suggests that RP2 co-operates with Arl3 and its effectors in protein complex assembly and membrane specification for lipid-modified proteins. This is exemplified by the co-ordination of cilia associated traffic for heterotrimeric G proteins and we propose a model for the role of Arl3 and RP2 in this process.

Expansion of transducin subunit gene families in early vertebrate tetraploidizations

Hundreds of gene families expanded in the early vertebrate tetraploidizations including many gene families in the phototransduction cascade. We have investigated the evolution of the heterotrimeric G-proteins of photoreceptors, the transducins, in relation to these events using both phylogenetic analyses and synteny comparisons. Three alpha subunit genes were identified in amniotes and the coelacanth, GNAT1–3; two of these were identified in amphibians and teleost fish, GNAT1 and GNAT2. Most tetrapods have four beta genes, GNB1–4, and teleosts have additional duplicates. Finally, three gamma genes were identified in mammals, GNGT1, GNG11 and GNGT2. Of these, GNGT1 and GNGT2 were found in the other vertebrates. In frog and zebrafish additional duplicates of GNGT2 were identified. Our analyses show all three transducin families expanded during the early vertebrate tetraploidizations and the beta and gamma families gained additional copies in the teleost-specific genome duplication. This suggests that the tetraploidizations contributed to visual specialisations.

Characterization of Conformational Changes and Protein-Protein Interactions of Rod Photoreceptor Phosphodiesterase (PDE6)

As the central effector of visual transduction, the regulation of photoreceptor phosphodiesterase (PDE6) is controlled by both allosteric mechanisms and extrinsic binding partners. However, the conformational changes and interactions of PDE6 with known interacting proteins are poorly understood. Using a fluorescence detection system for the analytical ultracentrifuge, we examined allosteric changes in PDE6 structure and protein-protein interactions with its inhibitory γ-subunit, the prenyl-binding protein (PrBP/δ), and activated transducin. In solution, the PDE6 catalytic dimer (Pαβ) exhibits a more asymmetric shape (axial ratio of 6.6) than reported previously. The inhibitory Pγ subunit behaves as an intrinsically disordered protein in solution but binds with high affinity to the catalytic dimer to reconstitute the holoenzyme without a detectable change in shape. Whereas the closely related PDE5 homodimer undergoes a significant change in its sedimentation properties upon cGMP binding to its regulatory cGMP binding site, no such change was detected upon ligand binding to the PDE6 catalytic dimer. However, when Pαβ was reconstituted with Pγ truncation mutants lacking the C-terminal inhibitory region, cGMP-dependent allosteric changes were observed. PrBP/δ bound to the PDE6 holoenzyme with high affinity (K(D) = 6.2 nm) and induced elongation of the protein complex. Binding of activated transducin to PDE6 holoenzyme resulted in a concentration-dependent increase in the sedimentation coefficient, reflecting a dynamic equilibrium between transducin and PDE6. We conclude that allosteric regulation of PDE6 is more complex than for PDE5 and is dependent on interactions of regions of Pγ with the catalytic dimer.

Phosphorylation of Phosducin Accelerates Rod Recovery from Transducin Translocation

In rods saturated by light, the G protein transducin undergoes translocation from the outer segment compartment, which results in the uncoupling of transducin from its innate receptor, rhodopsin. We measured the kinetics of recovery from this adaptive cellular response, while also investigating the role of phosducin, a phosphoprotein binding transducin βγ subunits in its de-phosphorylated state, in regulating this process. Mice were exposed to a moderate rod-saturating light triggering transducin translocation, and then allowed to recover in the dark while free running. The kinetics of the return of the transducin subunits to the outer segments were compared in transgenic mouse models expressing full-length phosducin, and phosducin lacking phosphorylation sites serine 54 and 71, using Western blot analysis of serial tangential sections of the retina. In mice expressing normal phosducin, transducin α and βγ subunits returned to the outer segments with a half-time (t(1/2)) of ∼24 and 29 minutes, respectively. In the phosducin phosphorylation mutants, the transducin α subunit moved four times slower, with t(1/2) ∼95 minutes, while the movement of transducin βγ was less affected. We demonstrate that the recovery of rod photoreceptors from the ambient saturating levels of illumination, in terms of the return of the light-dispersed transducin subunits to the rod outer segments, occurs six times faster than reported previously. Our data also support the notion that the accumulation of transducin α subunit in the outer segment is driven by its re-binding to the transducin βγ dimer, because this process is accelerated significantly by phosducin phosphorylation.

The X-linked retinitis pigmentosa protein RP2 facilitates G protein traffic

The X-linked retinitis pigmentosa protein RP2 is a GTPase activating protein (GAP) for the small GTPase Arl3 and both proteins are implicated in the traffic of proteins to the primary cilia. Here, we show that RP2 can facilitate the traffic of the Gβ subunit of transducin (Gβ1). Glutathione S-transferase (GST)-RP2 pulled down Gβ from retinal lysates and the interaction was specific to Gβ1, as Gβ3 or Gβ5L did not bind RP2. RP2 did not appear to interact with the Gβ:Gγ heterodimer, in contrast Gγ1 competed with RP2 for Gβ binding. Overexpression of Gβ1 in SK-N-SH cells led to a cytoplasmic accumulation of Gβ1, while co-expression of RP2 or Gγ1 with Gβ1 restored membrane association of Gβ1. Furthermore, RP2 small interfering RNA in ARPE19 cells resulted in a reduction in Gβ1 membrane association that was rescued by Gγ1 overexpression. The interaction of RP2 with Gβ1 required RP2 N-terminal myristolyation and the co-factor C (TBCC) homology domain. The interaction was also disrupted by the pathogenic mutation R118H, which blocks Arl3 GAP activity. Interestingly, Arl3-Q71L competed with Gβ1 for RP2 binding, suggesting that Arl3-GTP binding by RP2 would release Gβ1. RP2 also stimulated the association of Gβ1 with Rab11 vesicles. Collectively, the data support a role for RP2 in facilitating the membrane association and traffic of Gβ1, potentially prior to the formation of the obligate Gβ:Gγ heterodimer. Combined with other recent evidence, this suggests that RP2 may co-operate with Arl3 and its effectors in the cilia-associated traffic of G proteins.

G PROTEIN‐COUPLED RECEPTORS

The invention provides human G-protein coupled receptors (GCREC) and polynucleotides which identify and encode GCREC. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating, or preventing disorders associated with aberrant expression of GCREC.

Distinct Kinetic and Spatial Patterns of Protein Kinase C (PKC)- and Epidermal Growth Factor Receptor (EGFR)-dependent Activation of Extracellular Signal-regulated Kinases 1 and 2 by Human Nicotinic Acid Receptor GPR109A

Nicotinic acid (niacin) has been widely used as a lipid-lowering drug for several decades, and recently, orphan G protein-coupled receptor GPR109A has been identified as a receptor for niacin. Mechanistic investigations have shown that, upon niacin activation, GPR109A couples to a G(i) protein and inhibits adenylate cyclase activity, leading to inhibition of liberation of free fatty acid. However, the underlying molecular mechanisms for GPR109A signaling remain largely unknown. Using CHO-K1 cells stably expressing GPR109A and A431 cells, which are a human epidermoid cell line with high levels of endogenous expression of functional GPR109A receptors, we found that activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) by niacin was rapid, peaking at 5 min, and was significantly blocked by pertussis toxin. Furthermore, time course experiments with different kinase inhibitors demonstrated that GPR109A induced ERK1/2 activation via the matrix metalloproteinase/epidermal growth factor receptor transactivation pathway at both early and later time points (2-5 min); this pathway was distinct from the PKC pathway-mediated ERK1/2 phosphorylation that occurs at early time points (≤2 min) in response to niacin. Overexpression of Gβγ subunit scavengers βARK1-CT and the Gα subunit of transducin led to a significant reduction of ERK1/2 phosphorylation, suggesting a critical role for βγ subunits in GPR109A-activated ERK1/2 phosphorylation. Using arrestin-2/3-specific siRNA and an internalization-deficient GPR109A mutant, we found that arrestin-2 and arrestin-3 were not involved in GPR109A-mediated ERK1/2 activation. In conclusion, our findings demonstrate that upon binding to niacin GPR109A receptors initially activate G(i), leading to dissociation of the Gβγ subunit from activated G(i), and subsequently induce ERK1/2 activation via two distinct pathways, one PKC-dependent pathway occurring at a peak time of ≤2 min and the other matrix metalloproteinase-dependent growth factor receptor transactivation occurring at both early and later time points (2-5 min).

The D153del mutation in GNB3 gene causes tissue specific signalling patterns and an abnormal renal morphology in Rge chickens.

BackgroundThe GNB3 gene is expressed in cone but not rod photoreceptors of vertebrates, where it acts as the β transducin subunit in the colour visual transduction process. A naturally occurring mutation ‘D153del’ in the GNB3 gene causes the recessively inherited blinding phenotype retinopathy globe enlarged (rge) disease in chickens. GNB3 is however also expressed in most other vertebrate tissues suggesting that the D153del mutation may exert pathological effects that outlie from eye.Principal Findings Recombinant studies in COS-7 cells that were transfected with normal and mutant recombinant GNB3 constructs and subjected to cycloheximide chase showed that the mutant GNB3d protein had a much shorter half life compared to normal GNB3. GNB3 codes for the Gβ3 protein subunit that, together with different Gγ and Gα subunits, activates and regulates phosphorylation cascades in different tissues. As expected, the relative levels of cGMP and cAMP secondary messengers and their activated kinases such as MAPK, AKT and GRK2 were also found to be altered significantly in a tissue specific manner in rge chickens. Histochemical analysis on kidney tissue sections, from rge homozygous affected chickens, showed the chickens had enlargement of the glomerular capsule, causing glomerulomegaly and tubulointerstitial inflammation whereas other tissues (brain, heart, liver, pancreas) were unaffected.SignificanceThese findings confirm that the D153del mutation in GNB3 gene targets GNB3 protein to early degradation. Lack of GNB3 signalling causes reduced phosphorylation activity of ERK2 and AKT leading to severe pathological phenotypes such as blindness and renal abnormalities in rge chickens.

STEREOLOGY AND SOME STRUCTURAL CORRELATES OF RETINAL AND PHOTORECEPTOR CELL FUNCTION

The retina is the part of the eye which detects light, transduces it into nerve impulses and plays a significant role in visual perception. Sensitivity to light is multi-factorial and depends on the properties of photopigment molecules, their synthesis and incorporation into photoreceptor membranes and the neural circuitry between photoreceptor cells, bipolar neurons and ganglion neurons. In addition, it depends on structural factors such as the absolute and relative numbers of different types of photoreceptor neurons, their subcellular morphology, their distribution across the retina and the physical dimensions (especially surface areas) and spatial arrangements of their photoreceptor membranes. At the molecular level, these membranes harbour photosensitive pigment molecules comprising transmembrane glycoproteins (opsins, which vary between photoreceptor cells) and a non-protein chromophore. Phototransduction involves a conformational change in the chromophore and activation of an opsin. A transducer G protein, transducin, lowers levels of cGMP and triggers changes in membrane ion permeability including the closure of Na+ channels. This causes the plasmalemma to become less depolarized and the relative hyperpolarization stimulates ganglion cells whose axons form the optic nerve. Phosducin is a light-regulated phosphoprotein located in inner and outer segments of rod photoreceptor cells. It modulates phototransduction by binding to beta and gamma subunits of transducin. This review briefly illustrates ways in which stereology can contribute to our understanding of these processes by providing quantitative data on photoreceptor number, disk membrane surface area and the subcellular immunolocalisation of key molecules.

Lipid second messengers and related enzymes in vertebrate rod outer segments

Rod outer segments (ROSs) are specialized light-sensitive organelles in vertebrate photoreceptor cells. Lipids in ROS are of considerable importance, not only in providing an adequate environment for efficient phototransduction, but also in originating the second messengers involved in signal transduction. ROSs have the ability to adapt the sensitivity and speed of their responses to ever-changing conditions of ambient illumination. A major contributor to this adaptation is the light-driven translocation of key signaling proteins into and out of ROS. The present review shows how generation of the second lipid messengers from phosphatidylcholine, phosphatidic acid, and diacylglycerol is modulated by the different illumination states in the vertebrate retina. Findings suggest that the light-induced translocation of phototransduction proteins influences the enzymatic activities of phospholipase D, lipid phosphate phosphatase, diacylglyceride lipase, and diacylglyceride kinase, all of which are responsible for the generation of the second messenger molecules.

Mechanism for the regulation of mammalian cGMP phosphodiesterase6. 1: Identification of its inhibitory subunit complexes and their roles

Cyclic GMP phosphodiesterase (PDE) in bovine rod photoreceptor outer segments (OS) comprises a catalytic subunit complex (Palphabeta) and two inhibitory subunits (Pgamma) and is regulated by the alpha subunit of transducin (Talpha). Here, we show an overall mechanism for PDE regulation by identifying Pgamma complexes in OS homogenates prepared with an isotonic buffer. Before Talpha activation, three Pgamma complexes exist in the soluble fraction. Complex a, a minor complex, contains Palphabeta, Talpha, and a protein named Pdelta. Complex b, Palphabetagammagamma( b ), has a PDE activity similar to that of membranous Palphabetagammagamma, Palphabetagammagamma( M ), and its level, although its large portion is Pdelta-free, is estimated to be 20-30% of the total Palphabetagammagamma. Complex c, (Pgamma.GDP-Talpha) (2) ( c ) , appears to be a dimer of Pgamma.GDP-Talpha. Upon Talpha activation, (1) complex a stays unchanged, (2) Palphabetagammagamma( b ) binds to membranes, (3) the level of (Pgamma.GDP-Talpha) (2) ( c ) is reduced as its GTP-form is produced, (4) complex d, Pgamma.GTP-Talpha( d ), is formed on membranes and its substantial amount is released to the soluble fraction, and (5) membranous Palphabetagammagamma, Palphabetagammagamma( M ) and/or Palphabetagammagamma( b ), becomes Pgamma-depleted. These observations indicate that Pgamma as a complex with GTP-Talpha dissociates from Palphabetagammagamma on membranes and is released to the soluble fraction and that Pgamma-depleted PDE is the GTP-Talpha-activated PDE. After GTP hydrolysis, both (Pgamma.GDP-Talpha) (2) ( c ) and Pgamma.GDP-Talpha( d ), without liberating Pgamma, deactivate Pgamma-depleted PDE. The preferential order to be used for the deactivation is membranous Pgamma.GDP-Talpha( d ), solubilized Pgamma.GDP-Talpha( d ) and (Pgamma.GDP-Talpha) (2) ( c ) . Release of Pgamma.GTP-Talpha complexes to the soluble fraction is relevant to light adaptation.

A nonsense mutation in Gnat1, encoding the α subunit of rod transducin, in spontaneous mouse models of retinal dysfunction

ICR-derived retinal dysfunction (IRD) 1 and IRD2 mice are new spontaneous mouse models of rod-cone and rod dysfunctions, respectively. In this study, we investigated the cause of rod dysfunction in IRD1 and IRD2 mice. Gene expression of rod phototransduction proteins was analyzed by quantitative real-time RT-PCR. mRNA levels of Gnat1, which encodes the α subunit of rod transducin (Trα), were severely reduced. Trα protein was immunohistochemically undetectable in both IRD1 and IRD2 mice. Sequencing of Trα cDNA revealed a 48-base pair (bp) insertion between exons 4 and 5 in both mutant strains. The insertion changed codon 150 (TAC) to a stop codon (TAG) (Tyr150Ter). The truncated Trα protein was undetectable in the retinas of both mutants by western blot analysis using a primary antibody against the N-terminal region. A 57-bp deletion was identified in intron 4 of the Gnat1 gene, which encodes the Trα protein, and included the last two bases of the splice donor site of intron 4. Thus our results showed that IRD1 and IRD2 mice harbor a nonsense mutation in the Gnat1 gene, resulting in the absence or suppressed expression of the Trα protein, which is the likely cause of rod dysfunction in both mutants.

Nr2e3-directed transcriptional regulation of genes involved in photoreceptor development and cell-type specific phototransduction

The retinal transcription factor Nr2e3 plays a key role in photoreceptor development and function. In this study we examine gene expression in the retina of Nr2e3 rd7/rd7 mutants with respect to wild-type control mice, to identify genes that are misregulated and hence potentially function in the Nr2e3 transcriptional network. Quantitative candidate gene real time PCR and subtractive hybridization approaches were used to identify transcripts that were misregulated in Nr2e3 rd7/rd7 mice. Chromatin immunoprecipitation assays were then used to determine which of the misregulated transcripts were direct targets of NR2E3. We identified 24 potential targets of NR2E3. In the developing retina, NR2E3 targets transcription factors such as Ror1, Rorg, and the nuclear hormone receptors Nr1d1 and Nr2c1. In the mature retina NR2E3 targets several genes including the rod specific gene Gnb1 and cone specific genes blue opsin, and two of the cone transducin subunits, Gnat2 and Gnb3. In addition, we identified 5 novel transcripts that are targeted by NR2E3. While mislocalization of proteins between rods and cones was not observed, we did observe diminished concentration of GNB1 protein in adult Nr2e3 rd7/rd7 retinas. These studies identified novel transcriptional pathways that are potentially targeted by Nr2e3 in the retina and specifically demonstrate a novel role for NR2E3 in regulating genes involved in phototransduction.

Preferential Binding Of cGMP Phosphodiesterase To Phospholipid Monolayers

The light-activated rhodopsin, metarhodopsin II, forms a complex with the alpha subunit of transducin (Tα). The exchange of GDP for GTP leads to a conformational change of Tα which is released from its beta-gamma subunits and rhodopsin. Tα-GTP then activates cGMP phosphodiesterase (PDE) which hydrolyzes cGMP and leads to the closure of the cGMP-gated channels and to the hyperpolarization of the rod photoreceptors. PDE is made of two catalytic (αβ) and two inhibitory (γ) subunits (Pαβγγ). PDE is acylated with a farnesyl and a geranylgeranyl and is thus membrane bound.

Interaction of retinal guanylate cyclase with the α subunit of transducin: potential role in transducin localization

Vertebrate phototransduction is mediated by cGMP, which is generated by retGC (retinal guanylate cyclase) and degraded by cGMP phosphodiesterase. Light stimulates cGMP hydrolysis via the G-protein transducin, which directly binds to and activates phosphodiesterase. Bright light also causes relocalization of transducin from the OS (outer segments) of the rod cells to the inner compartments. In the present study, we show experimental evidence for a previously unknown interaction between G(alphat) (the transducin alpha subunit) and retGC. G(alphat) co-immunoprecipitates with retGC from the retina or from co-transfected COS-7 cells. The retGC-G(alphat) complex is also present in cones. The interaction also occurs in mice lacking RGS9 (regulator of G-protein signalling 9), a protein previously shown to associate with both G(alphat) and retGC. The G(alphat)-retGC interaction is mediated primarily by the kinase homology domain of retGC, which binds GDP-bound G(alphat) stronger than the GTP[S] (GTPgammaS; guanosine 5'-[gamma-thio]triphosphate) form. Neither G(alphat) nor G(betagamma) affect retGC-mediated cGMP synthesis, regardless of the presence of GCAP (guanylate cyclase activating protein) and Ca2+. The rate of light-dependent transducin redistribution from the OS to the inner segments is markedly accelerated in the retGC-1-knockout mice, while the migration of transducin to the OS after the onset of darkness is delayed. Supplementation of permeabilized photoreceptors with cGMP does not affect transducin translocation. Taken together, these results suggest that the protein-protein interaction between G(alphat) and retGC represents a novel mechanism regulating light-dependent translocation of transducin in rod photoreceptors.