Loss of the Effector Function in a Transducin-α Mutant Associated with Nougaret Night Blindness

Muradov, Khakim G.; Artemyev, Nikolai O.

doi:10.1074/jbc.275.10.6969

Cited by 31 publications

(47 citation statements)

References 44 publications

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“…Instead, it could be the basis for a recessive stationary night blindness in which a-waves are absent from the ERG (39). Consistent with this prediction, the Nougaret form of stationary night blindness was traced to a mutation in Tr␣ (40) that reportedly leaves transducin unable to activate PDE (41). The dominant transmission of Nougaret disease has not been explained, however.…”

Section: Discussionsupporting

confidence: 60%

Phototransduction in transgenic mice after targeted deletion of the rod transducin α-subunit

Calvert

Krasnoperova

Lyubarsky

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

332

351

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Section: Discussionsupporting

confidence: 60%

Phototransduction in transgenic mice after targeted deletion of the rod transducin α-subunit

Calvert

Krasnoperova

Lyubarsky

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

332

351

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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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“…This effector competent Gtα* was generated previously, based on the Gtα/Giα Chi8 (23), by introducing two Gtα-specific residues H244 and N247 (15). The yields of purified Gtα*S43C and Gtα*S43N mutants were similar (∼1 mg/ L of culture).…”

Section: Resultsmentioning

confidence: 96%

“…Expression and trypsin sensitivity of the Gtα*S43C and Gtα*S43N mutants Two mutations, S43C and S43N, were introduced into the Gtα-like chimeric protein Gtα*, which is readily expressed in E. coli (15). This effector competent Gtα* was generated previously, based on the Gtα/Giα Chi8 (23), by introducing two Gtα-specific residues H244 and N247 (15).…”

Section: Resultsmentioning

confidence: 99%

Dominant Negative Mutants of Transducin-α That Block Activated Receptor

2006

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Mutations counterpart to dominant negative RasSer17Asn in the α-subunits of heterotrimeric Gproteins are known to also produce dominant negative effects. The mechanism of these mutations remains poorly understood. Here, we examined the effects and mechanism of the Ser43Cys and Ser43Asn mutants of transducin-like chimeric Gtα* in the visual signaling system. Our analysis showed that both mutants have reduced affinity for GDP and are likely to exist in an empty-or partially occupied-pocket state. S43C and S43N retained the ability to interact with Gtβγ and, as heterotrimeric proteins, bind to photoexcited rhodopsin (R*). The interaction with R* is unproductive as the mutants failed to bind GTPγS and become activated. S43C and S43N inhibited R*-dependent activation of Gtα* and Gtα, apparently by blocking R*. Finally, both Gtα* mutants lacked interaction with the γ-subunit of PDE6, an effector protein in phototransduction. These results indicate that the S43C and S43N mutants of Gtα* are dominant negative inhibitors that bind and block the activated receptor in a mechanism that parallels that of RasSer17Asn. Dominant negative mutants of Gtα sequestering R*, such as S43C and S43N, may become useful instruments in probing the mechanisms of visual dysfunctions caused by abnormal phototransduction signaling.

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Loss of the Effector Function in a Transducin-α Mutant Associated with Nougaret Night Blindness

Cited by 31 publications

References 44 publications

Phototransduction in transgenic mice after targeted deletion of the rod transducin α-subunit

Phototransduction in transgenic mice after targeted deletion of the rod transducin α-subunit

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

Dominant Negative Mutants of Transducin-α That Block Activated Receptor

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