Analysis of PDE6 function using chimeric PDE5/6 catalytic domains

Hakim Muradov; Kimberly K Boyd; Nikolai O Artemyev

doi:10.1016/j.visres.2005.09.015

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Analysis of PDE6 function using chimeric PDE5/6 catalytic domains

Journal article

Open access

Peer reviewed

Analysis of PDE6 function using chimeric PDE5/6 catalytic domains

Hakim Muradov, Kimberly K Boyd and Nikolai O Artemyev

Vision research (Oxford), Vol.46(6-7), pp.860-868

03/2006

DOI: 10.1016/j.visres.2005.09.015

PMID: 16256165

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Abstract

cGMP-phosphodiesterases of the PDE6 family are expressed in retinal photoreceptor cells, where they mediate the phototransduction cascade. A system for expression of PDE6 in vitro is lacking, thus straining progress in understanding the structure-function relationships of the photoreceptor enzyme. Here, we report generation and characterization of bacterially expressed chimeric PDE5/6 catalytic domains which are highly soluble, catalytically active, and sensitive to inhibition by the PDE6 Pgamma subunit. Two flexible PDE6 loops, H and M, impart chimeric PDE5/6 catalytic domains with PDE6-like properties. The replacement of the PDE6 H-loop into the PDE5 catalytic domain increases the catalytic rate and the K(m) value for cGMP hydrolysis, whereas the substitution of the M-loop produces catalytic PDE domains responsive to Pgamma. Multiple PDE6 segments preventing functional expression of the catalytic domain are identified, supporting the requirement for specialized photoreceptor chaperones to assist PDE6 folding in vivo.

Catalytic Domain

Phosphoric Diester Hydrolases - metabolism

Cyclic Nucleotide Phosphodiesterases, Type 5

3',5'-Cyclic-GMP Phosphodiesterases - genetics

Molecular Sequence Data

Phosphoric Diester Hydrolases - genetics

Structure-Activity Relationship

Recombinant Fusion Proteins - metabolism

Phosphoric Diester Hydrolases - physiology

Sequence Alignment

3',5'-Cyclic-GMP Phosphodiesterases - metabolism

Animals

Cattle

Base Sequence

Cloning, Molecular - methods

Escherichia coli - metabolism

Cyclic Nucleotide Phosphodiesterases, Type 6

Catalysis

Light Signal Transduction - physiology

Details

Title: Subtitle: Analysis of PDE6 function using chimeric PDE5/6 catalytic domains
Creators: Hakim Muradov - Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City, IA 52242, USA
Kimberly K Boyd
Nikolai O Artemyev
Resource Type: Journal article
Publication Details: Vision research (Oxford), Vol.46(6-7), pp.860-868
DOI: 10.1016/j.visres.2005.09.015
PMID: 16256165
NLM abbreviation: Vision Res
ISSN: 0042-6989
eISSN: 1878-5646
Publisher: England
Grant note: R01 EY010843 / NEI NIH HHS EY-10843 / NEI NIH HHS
Language: English
Date published: 03/2006
Academic Unit: Molecular Physiology and Biophysics; Iowa Neuroscience Institute; Ophthalmology and Visual Sciences
Record Identifier: 9984070281702771

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