An Interface of Interaction between Photoreceptor cGMP Phosphodiesterase Catalytic Subunits and Inhibitory γ Subunits

Cyclic guanosine 5′-monophosphate (cGMP) phosphodiesterase (PDE) regulates the level of cGMP on transduction of a visual signal in vertebrate photoreceptor cells. Two identical inhibitory PDE γ subunits (Pγs) block catalytic activity of PDE-α and -β subunits (Pαβ) in the dark. The primary regions of...

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Bibliographic Details
Published in:The Journal of biological chemistry 1996-08, Vol.271 (33), p.19964-19969
Main Authors: Natochin, Michael, Artemyev, Nikolai O.
Format: Article
Language:English
Subjects:
3',5'-Cyclic-GMP Phosphodiesterases - chemistry
3',5'-Cyclic-GMP Phosphodiesterases - metabolism
Amino Acid Sequence
Animals
Binding Sites
Cattle
Cloning, Molecular
Enzyme Activation
GTP-Binding Proteins - chemistry
Molecular Sequence Data
Photoreceptor Cells - chemistry
Protein Binding
Rod Cell Outer Segment - chemistry
Sequence Alignment
Sequence Homology, Amino Acid
Vision, Ocular
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Summary:Cyclic guanosine 5′-monophosphate (cGMP) phosphodiesterase (PDE) regulates the level of cGMP on transduction of a visual signal in vertebrate photoreceptor cells. Two identical inhibitory PDE γ subunits (Pγs) block catalytic activity of PDE-α and -β subunits (Pαβ) in the dark. The primary regions of Pγ involved in the interaction with Pαβ are a central polycationic region, Pγ-24-45, and a C-terminal region of Pγ. Recently, we have shown that the C-terminal region of Pγ, which is the major Pγ inhibitory domain, blocks PDE activity by binding to the catalytic site of PDE (Artemyev, N. O., Natochin, M., Busman, M., Schey, K. L., and Hamm, H. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5407-5412). Here, we localize the site on the rod cGMP PDE α subunit that binds to the central polycationic domain of Pγ. This site is located within a region that links a second noncatalytic cGMP binding site with the catalytic domain of PDE. A polypeptide coresponding to this region, Pα-461-553, expressed as a glutathione S-transferase fusion protein in Escherichia coli and isolated after cleavage of the fusion protein with thrombin, blocks inhibition of PDE activity by Pγ. In addition, Pα-461-553 binds to the Pγ-24-45 region (Kd, 7 µM), as measured by a fluorescent increase in a Pγ-24-45Cys peptide labeled with 3-(bromoacetyl)-7-diethylaminocoumarin. The Pα-461-553 region was further characterized by using a set of synthetic peptides. A peptide corresponding to residues 517-541 of Pα (Pα-517-541) effectively suppressed inhibition of PDE activity by Pγ and bound to Pγ-24-45Cys labeled with 3-(bromoacetyl)-7-diethylaminocoumarin (Kd, 22 µM). Pα-517-541 also competes with the activated rod G-protein α-subunit for binding to Pγ labeled with lucifer yellow vinyl sulfone. This suggests that light activation of rod PDE by the G-protein transducin involves competition between transducin α-guanosine 5′-triphosphate and Pα-517-541 for binding to the Pγ-24-45 region. Based on the results, we propose a linear model of interactions between catalytic and inhibitory PDE subunits.
ISSN:0021-9258
1083-351X
DOI:10.1074/jbc.271.33.19964