The GAFa domains of rod cGMP-phosphodiesterase 6 determine the selectivity of the enzyme dimerization

Khakim G Muradov; Kimberly K Boyd; Sergio E Martinez; Joseph A Beavo; Nikolai O Artemyev

doi:10.1074/jbc.M208456200

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The GAFa domains of rod cGMP-phosphodiesterase 6 determine the selectivity of the enzyme dimerization

Journal article

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The GAFa domains of rod cGMP-phosphodiesterase 6 determine the selectivity of the enzyme dimerization

Khakim G Muradov, Kimberly K Boyd, Sergio E Martinez, Joseph A Beavo and Nikolai O Artemyev

The Journal of biological chemistry, Vol.278(12), pp.10594-10601

03/21/2003

DOI: 10.1074/jbc.M208456200

PMID: 12531898

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Abstract

Retinal rod cGMP phosphodiesterase (PDE6 family) is the effector enzyme in the vertebrate visual transduction cascade. Unlike other known PDEs that form catalytic homodimers, the rod PDE6 catalytic core is a heterodimer composed of alpha and beta subunits. A system for efficient expression of rod PDE6 is not available. Therefore, to elucidate the structural basis for specific dimerization of rod PDE6, we constructed a series of chimeric proteins between PDE6alphabeta and PDE5, which contain the N-terminal GAFa/GAFb domains, or portions thereof, of the rod enzyme. These chimeras were co-expressed in Sf9 cells in various combinations as His-, myc-, or FLAG-tagged proteins. Dimerization of chimeric PDEs was assessed using gel filtration and sucrose gradient centrifugation. The composition of formed dimeric enzymes was analyzed with Western blotting and immunoprecipitation. Consistent with the selectivity of PDE6 dimerization in vivo, efficient heterodimerization was observed between the GAF regions of PDE6alpha and PDE6beta with no significant homodimerization. In addition, PDE6alpha was able to form dimers with the cone PDE6alpha' subunit. Furthermore, our analysis indicated that the PDE6 GAFa domains contain major structural determinants for the affinity and selectivity of dimerization of PDE6 catalytic subunits. The key dimerization selectivity module of PDE6 has been localized to a small segment within the GAFa domains, PDE6alpha-59-74/PDE6beta-57-72. This study provides tools for the generation of the homodimeric alphaalpha and betabeta enzymes that will allow us to address the question of functional significance of the unique heterodimerization of rod PDE6.

Amino Acid Sequence

Protein Subunits

Catalytic Domain

Animals

Phosphoric Diester Hydrolases - metabolism

Cattle

Retinal Rod Photoreceptor Cells - enzymology

Molecular Sequence Data

Cyclic Nucleotide Phosphodiesterases, Type 6

Recombinant Fusion Proteins - chemistry

Dimerization

Phosphoric Diester Hydrolases - chemistry

Details

Title: Subtitle: The GAFa domains of rod cGMP-phosphodiesterase 6 determine the selectivity of the enzyme dimerization
Creators: Khakim G Muradov - Department of Physiology and Biophysics, University of Iowa College of Medicine, Iowa City 52242, USA
Kimberly K Boyd
Sergio E Martinez
Joseph A Beavo
Nikolai O Artemyev
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.278(12), pp.10594-10601
DOI: 10.1074/jbc.M208456200
PMID: 12531898
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Publisher: United States
Grant note: DK-25295 / NIDDK NIH HHS EY-10843 / NEI NIH HHS
Language: English
Date published: 03/21/2003
Academic Unit: Molecular Physiology and Biophysics; Iowa Neuroscience Institute; Ophthalmology and Visual Sciences
Record Identifier: 9984070885402771

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