title: Identification of a binding site on retinal transducin alpha for the phosphodiesterase inhibitory gamma subunit.
abstract: Transducin alpha (T alpha) activates retinal rod cyclic GMP phosphodiesterase (PDE) by interacting with and removing the inhibitory PDE gamma subunit. A T alpha-PDE gamma complex can be isolated in vitro, and our previous work [Morrison, Rider and Takemoto (1987) FEBS Lett. 222, 266-270; Morrison, Cunnick, Oppert and Takemoto (1989) J. Biol. Chem. 264, 11671-11681] has identified a region of PDE gamma, residues 24-45, that binds to T alpha. The C-terminal region of PDE gamma is the site that interacts with PDE alpha/beta and inhibits catalytic function. The site on T alpha that binds to the PDE gamma 24-45 region has not been identified. Synthetic peptides (15-mers) which span the bovine T alpha sequence were tested for binding to purified recombinant PDE gamma using a solid-phase assay. The peptides were also tested for ability to activate a PDE complex. We have identified a region, residues 250-275 of T alpha, which shows a high affinity of PDE gamma and for the PDE gamma (24-45) binding peptide. The peptide did not bind to the C-terminal residues 50-87 of PDE gamma. Likewise, a region of T alpha, 1-25 did not exhibit high-affinity binding to PDE gamma or to the 24-45 PDE gamma peptide. Specific binding of the 250-275 peptide to PDE gamma was confirmed by its ability to compete with T alpha binding to PDE gamma, although a higher concentration was required (10x). The T alpha-(250-275) peptide activated a fully inhibited PDE alpha beta gamma 2 complex in a dose-dependent manner. These results suggest that a region on T alpha that recognizes the PDE gamma-binding site is found within residues 250-275 of T alpha.
authors.affiliation: Department of Biochemistry, Kansas State University, Manhattan 66506.
authors.forename: J
authors.initials: J
authors.lastname: Cunnick
authors.forename: C
authors.initials: C
authors.lastname: Twamley
authors.forename: I
authors.initials: I
authors.lastname: Udovichenko
authors.forename: K
authors.initials: K
authors.lastname: Gonzalez
authors.forename: D J
authors.initials: DJ
authors.lastname: Takemoto
mesh_terms: D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001667:Binding, Competitive; D002417:Cattle; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D010446:Peptide Fragments / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012160:Retina / Q000737:chemistry*; D012374:Rod Cell Outer Segment / Q000737:chemistry; D015293:Transducin / Q000737:chemistry* / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015106:3',5'-Cyclic-GMP Phosphodiesterases; D015293:Transducin