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Abstract
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 gamma subunits (Pgammas) block catalytic activity of PDE-alpha and -beta subunits (Palphabeta) in the dark. The primary regions of Pgamma involved in the interaction with Palphabeta are a central polycationic region, Pgamma-24-45, and a C-terminal region of Pgamma. Recently, we have shown that the C-terminal region of Pgamma, which is the major Pgamma 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 alpha subunit that binds to the central polycationic domain of Pgamma. 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, Palpha-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 Pgamma. In addition, Palpha-461-553 binds to the Pgamma-24-45 region (Kd, 7 microM), as measured by a fluorescent increase in a Pgamma-24-45Cys peptide labeled with 3-(bromoacetyl)-7-diethylaminocoumarin. The Palpha-461-553 region was further characterized by using a set of synthetic peptides. A peptide corresponding to residues 517-541 of Palpha (Palpha-517-541) effectively suppressed inhibition of PDE activity by Pgamma and bound to Pgamma-24-45Cys labeled with 3-(bromoacetyl)-7-diethylaminocoumarin (Kd, 22 microM). Palpha-517-541 also competes with the activated rod G-protein alpha-subunit for binding to Pgamma labeled with lucifer yellow vinyl sulfone. This suggests that light activation of rod PDE by the G-protein transducin involves competition between transducin alpha-guanosine 5'-triphosphate and Palpha-517-541 for binding to the Pgamma-24-45 region. Based on the results, we propose a linear model of interactions between catalytic and inhibitory PDE subunits.
Highlights
The photoreceptor PDEs belong to a broader group of cGMPbinding PDEs, which contain two noncatalytic cGMP binding binds to the central polycationic domain of P␥
Using a cross-linking approach we have demonstrated that the C terminus of P␥ interacts with region P␣-751–763 located within the PDE catalytic domain (Artemyev et al, 1996)
Solubilization of inclusion bodies in 6 M urea, followed by removal of urea and cleavage of GST fusion proteins with thrombin, resulted in the formation of recombinant P␣ polypeptides with their expected molecular masses
Summary
Two regions of P␥, polycationic region P␥-24 – 45 and the C terminus of P␥, have been shown to participate in the interaction with P␣ (Lipkin et al, 1988; Artemyev and Hamm, 1992; Brown, 1992; Takemoto et al, 1992) Both of these P␥ domains bind to P␣, allowing effective inhibition of PDE activity by the P␥ C terminus. Fusion proteins contained a region unique for photoreceptor PDEs that links a second noncatalytic cGMP binding site with the catalytic domain. This protein was used to obtain a polypeptide, P␣-461–553, which blocks inhibition of PDE activ-
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