Identification of a binding site on retinal transducin alpha for the phosphodiesterase inhibitory gamma subunit.
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.
Cunnick
J
J
Department of Biochemistry, Kansas State University, Manhattan 66506.
Twamley
C
C
Udovichenko
I
I
Gonzalez
K
K
Takemoto
D J
DJ
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
D010446:Peptide Fragments; D011994:Recombinant Proteins; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D015106:3',5'-Cyclic-GMP Phosphodiesterases; D015293:Transducin