Phototransduction in a Transgenic Mouse Model of Nougaret Night Blindness

Moussaif, Mustapha; Rubin, William W.; Kerov, Vasily; Reh, Rebecca; Chen, Desheng; Lem, Janis; Chen, Ching Kang; Hurley, James B.; Burns, Marie E.; Artemyev, Nikolai O.

doi:10.1523/jneurosci.1322-06.2006

Cited by 20 publications

(41 citation statements)

References 44 publications

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“…1 A). The transgene sequence was mutated previously to introduce the Glu-Glu (EE) monoclonal antibody epitope to assess expression levels of mutant transducins in mouse retinas (Kerov et al, 2005;Moussaif et al, 2006). The G2A substitution was created using the QuickChange mutagenesis kit (Stratagene, La Jolla, CA).…”

Section: Methodsmentioning

confidence: 99%

“…1 A) (Kerov et al, 2005;Moussaif et al, 2006). The EE tag facilitates determination of the expression levels and localization of the G␣ t G2A mutant in the presence of native G␣ t in transgenic rods.…”

Section: Expression and Localization Of G␣ T G2a Mutant In Transgenicmentioning

confidence: 99%

“…The EE tag facilitates determination of the expression levels and localization of the G␣ t G2A mutant in the presence of native G␣ t in transgenic rods. The EE tag does not alter biochemical properties of transducin, and the flash responses from transgenic mouse rods expressing G␣ t EE are similar to those from the wild-type rods (Kerov et al, 2005;Moussaif et al, 2006). Several transgenic mouse lines expressing the EE-tagged G␣ t G2A mutant were established.…”

Section: Expression and Localization Of G␣ T G2a Mutant In Transgenicmentioning

confidence: 99%

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N-Terminal Fatty Acylation of Transducin Profoundly Influences Its Localization and the Kinetics of Photoresponse in Rods

Kerov¹,

Rubin²,

Natochin³

et al. 2007

J. Neurosci.

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N-terminal acylation of the ␣-subunits of heterotrimeric G-proteins is believed to play a major role in regulating the cellular localization and signaling of G-proteins, but physiological evidence has been lacking. To examine the functional significance of N-acylation of a well understood G-protein ␣-subunit, transducin (G␣ t ), we generated transgenic mice that expressed a mutant G␣ t lacking N-terminal acylation sequence (G␣ t G2A). Rods expressing G␣ t G2A showed a severe defect in transducin cellular localization. In contrast to native G␣ t , which resides in the outer segments of dark-adapted rods, G␣ t G2A was found predominantly in the inner compartments of the photoreceptor cells. Remarkably, transgenic rods with the outer segments containing G␣ t G2A at 5-6% of the G␣ t levels in wild-type rods showed only a sixfold reduction in sensitivity and a threefold decrease in the amplification constant. The much smaller than predicted reduction may reflect an increase in the lateral diffusion of transducin and an increased activation rate by photoexcited rhodopsin or more efficient activation of cGMP phosphodiesterase 6 by G␣ t G2A; alternatively, nonlinear relationships between concentration and the activation rate of transducin also potentially contribute to the mismatch between the amplification constant and quantitative expression analysis of G␣ t G2A rods. Furthermore, the G2A mutation reduced the GTPase activity of transducin and resulted in two to three times slower than normal recovery of flash responses of transgenic rods, indicating the role of G␣ t membrane tethering for its efficient inactivation by the regulator of G-protein signaling 9 GTPase-activating protein complex. Thus, N-acylation is critical for correct compartmentalization of transducin and controls the rate of its deactivation.

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Section: Methodsmentioning

confidence: 99%

Section: Expression and Localization Of G␣ T G2a Mutant In Transgenicmentioning

confidence: 99%

Section: Expression and Localization Of G␣ T G2a Mutant In Transgenicmentioning

confidence: 99%

See 1 more Smart Citation

N-Terminal Fatty Acylation of Transducin Profoundly Influences Its Localization and the Kinetics of Photoresponse in Rods

Kerov¹,

Rubin²,

Natochin³

et al. 2007

J. Neurosci.

Self Cite

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“…Furthermore, a single amino acid substitution in the transducin ␣-subunit (i.e. G38D) disrupts the ability of transducin to bind P␥ (52,53) and is known to cause the Nougaret form of dominant stationary night blindness (54). The results in this study may provide a molecular basis for understanding how alterations in different regions of the P␥ molecule may adversely affect the transducin-mediated regulation of PDE6, as well as the ability of PDE6 to regulate cGMP homeostasis in the dark-adapted state and/or during visual excitation, recovery, and adaptation of photoreceptors.…”

Section: Transducin Interaction With the N-terminal Half Of P␥ Is Reqmentioning

confidence: 99%

Structural Requirements of the Photoreceptor Phosphodiesterase γ-Subunit for Inhibition of Rod PDE6 Holoenzyme and for Its Activation by Transducin

Zhang

Skiba²,

Cote³

2010

Journal of Biological Chemistry

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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. The photoreceptor cyclic nucleotide phosphodiesterase (PDE6)2 is the central enzyme in the vertebrate visual signaling pathway in rods and cones. Phototransduction is initiated when light induces the isomerization of the 11-cis-retinal chromophore of rhodopsin, which leads to activation of the photoreceptor-specific G-protein, transducin. Transducin binds GTP and releases its activated ␣-subunit (T␣*-GTP) to activate membrane-associated rod PDE6 by relieving the inhibition of the ␥-subunit at the active sites. The activation of PDE6 results in rapid lowering of cGMP levels, closure of cGMP gated ion channels, and hyperpolarization of the cell membrane (1-5).The PDE6 holoenzyme consists of a catalytic dimer of ␣-and ␤-subunits (P␣␤) and two inhibitory ␥-subunits (P␥) that are tightly bound to P␣␤. Considering its small size, the P␥ subunit of rod and cone PDE6 serves a remarkable number of functions (reviewed in Refs. (6 and 7): 1) a primary function of the P␥ subunit is to inhibit catalysis of cGMP by binding to the catalytic domain of PDE6; 2) the P␥ subunit also enhances the binding affinity of cGMP to the regulatory GAF (cGMP-regulated PDEs, certain adenylate cyclases, and the transcription factor Fh1A of bacteria) domain; 3) activated transducin binds to the P␥ subunit, leading to deinhibition of PDE6 at its active site; and 4) during deactivation, the ␥-subunit participates in a protein complex with the RGS9 and o...

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“…With the advance of genetic engineering, it was no longer necessary to wait; these animals could be produced with the desired alterations, since the genetic causes of the disease in question were known. So, animals were produced that serve as models for studies of different types of cancers (Santos et al, 2008), heart disease (Moon, 2008), hypertension (Zadelaar et al, 2007), diabetes (Leroith and Gavrilova, 2006), obesity (Blüher, 2005), osteoporosis (Klein, 2008), glaucoma (Zhou et al, 2008), blindness (Moussaif et al, 2006), deafness (Leibovici et al, 2008), Huntington's syndrome (Heng et al, 2008), Down's syndrome (Patterson, 2009), Parkinson's disease (Harvey et al, 2008), Alzheimer's disease (Gotz et al, 2009), anxiety (Kalueff et al, 2007, and depression (Kalueff et al, 2007).…”

Section: Use Of Genetically Modified Animals In Scientific Researchmentioning

confidence: 99%

Review article Genetically modified animals for use in research and biotechnology

Chaible¹,

Corat²,

Abdelhay³

et al. 2010

Genet. Mol. Res.

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ABSTRACT. Transgenic animals are used extensively in the study of in vivo gene function, as models for human diseases and in the production of biopharmaceuticals. The technology behind obtaining these animals involves molecular biology techniques, cell culture and embryo manipulation; the mouse is the species most widely used as an experimental model. In scientific research, diverse models are available as tools for the elucidation of gene function, such as transgenic animals, knockout and conditional knockout animals, knock-in animals, humanized animals, and knockdown animals. We examined the evolution of the science for the development of these animals, as well as the techniques currently used in obtaining these animal models. We review the phenotypic techniques used for elucidation of alterations caused by genetic modification. We also investigated the role of genetically modified animals in the biotechnology industry, where they promise a revolution in obtaining heterologous proteins through natural secretions, such as milk, increas- ing the scale of production and facilitating purification, thereby lowering the cost of production of hormones, growth factors and enzymes.

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Phototransduction in a Transgenic Mouse Model of Nougaret Night Blindness

Cited by 20 publications

References 44 publications

N-Terminal Fatty Acylation of Transducin Profoundly Influences Its Localization and the Kinetics of Photoresponse in Rods

N-Terminal Fatty Acylation of Transducin Profoundly Influences Its Localization and the Kinetics of Photoresponse in Rods

Structural Requirements of the Photoreceptor Phosphodiesterase γ-Subunit for Inhibition of Rod PDE6 Holoenzyme and for Its Activation by Transducin

Review article Genetically modified animals for use in research and biotechnology

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