Rod phosphodiesterase-6 PDE6A and PDE6B subunits are enzymatically equivalent

Hakim Muradov; Kimberly K Boyd; Nikolai O Artemyev

doi:10.1074/jbc.M110.170068

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Rod phosphodiesterase-6 PDE6A and PDE6B subunits are enzymatically equivalent

Journal article

Open access

Peer reviewed

Rod phosphodiesterase-6 PDE6A and PDE6B subunits are enzymatically equivalent

Hakim Muradov, Kimberly K Boyd and Nikolai O Artemyev

The Journal of biological chemistry, Vol.285(51), pp.39828-39834

12/17/2010

DOI: 10.1074/jbc.M110.170068

PMCID: PMC3000964

PMID: 20940301

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Abstract

Phosphodiesterase-6 (PDE6) is the key effector enzyme of the phototransduction cascade in rods and cones. The catalytic core of rod PDE6 is a unique heterodimer of PDE6A and PDE6B catalytic subunits. The functional significance of rod PDE6 heterodimerization and conserved differences between PDE6AB and cone PDE6C and the individual properties of PDE6A and PDE6B are unknown. To address these outstanding questions, we expressed chimeric homodimeric enzymes, enhanced GFP (EGFP)-PDE6C-A and EGFP-PDE6C-B, containing the PDE6A and PDE6B catalytic domains, respectively, in transgenic Xenopus laevis. Similar to EGFP-PDE6C, EGFP-PDE6C-A and EGFP-PDE6C-B were targeted to the rod outer segments and concentrated at the disc rims. PDE6C, PDE6C-A, and PDE6C-B were isolated following selective immunoprecipitation of the EGFP fusion proteins. All three enzymes, PDE6C, PDE6C-A, and PDE6C-B, hydrolyzed cGMP with similar K(m) (20-23 μM) and k(cat) (4200-5100 s(-1)) values. Likewise, the K(i) values for PDE6C, PDE6C-A, and PDE6C-B inhibition by the cone- and rod-specific PDE6 γ-subunits (Pγ) were comparable. Recombinant cone transducin-α (Gα(t2)) and native rod Gα(t1) fully and potently activated PDE6C, PDE6C-A, and PDE6C-B. In contrast, the half-maximal activation of bovine rod PDE6 required markedly higher concentrations of Gα(t2) or Gα(t1). Our results suggest that PDE6A and PDE6B are enzymatically equivalent. Furthermore, PDE6A and PDE6B are similar to PDE6C with respect to catalytic properties and the interaction with Pγ but differ in the interaction with transducin. This study significantly limits the range of mechanisms by which conserved differences between PDE6A, PDE6B, and PDE6C may contribute to remarkable differences in rod and cone physiology.

Cell Line

Catalytic Domain

Cyclic Nucleotide Phosphodiesterases, Type 6 - metabolism

Animals, Genetically Modified

Humans

Xenopus laevis

Heterotrimeric GTP-Binding Proteins - metabolism

Cyclic Nucleotide Phosphodiesterases, Type 6 - genetics

Heterotrimeric GTP-Binding Proteins - genetics

Recombinant Fusion Proteins - metabolism

Animals

Eye Proteins - metabolism

Retinal Cone Photoreceptor Cells - enzymology

Cattle

Retinal Rod Photoreceptor Cells - cytology

Retinal Rod Photoreceptor Cells - enzymology

Recombinant Fusion Proteins - genetics

Eye Proteins - genetics

Enzyme Activation - physiology

Details

Title: Subtitle: Rod phosphodiesterase-6 PDE6A and PDE6B subunits are enzymatically equivalent
Creators: Hakim Muradov - Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242, USA
Kimberly K Boyd
Nikolai O Artemyev
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.285(51), pp.39828-39834
DOI: 10.1074/jbc.M110.170068
PMID: 20940301
PMCID: PMC3000964
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: United States
Grant note: EY-12682 / NEI NIH HHS R01 EY010843 / NEI NIH HHS R01 EY012682-12 / NEI NIH HHS R01 EY012682 / NEI NIH HHS EY-10843 / NEI NIH HHS R01 EY010843-18 / NEI NIH HHS
Language: English
Date published: 12/17/2010
Academic Unit: Molecular Physiology and Biophysics; Iowa Neuroscience Institute; Ophthalmology and Visual Sciences
Record Identifier: 9984025594802771

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