Identification of a binding site on retinal transducin α for the phosphodiesterase inhibitory γ subunit

Abstract

Transducin alpha (Tα) activates retinal rod cyclic GMP phosphodiesterase (PDE) by interacting with and removing the inhibitory PDEγ subunit. A Tα-PDEγ complex can be isolated in vitro, and our previous work has identified a region of PDEγ, residues 24-45, that binds to Tα. The C-terminal region of PDEγ is the site that interacts with PDEα/β and inhibits catalytic function. The site on Tα that binds to the PDEγ 24-45 region has not been identified. Synthetic peptides (15-mers) which span the bovine Tα sequence were tested for binding to purified recombinant PDEγ 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 Tα, which shows a high affinity for PDEγ and for the PDEγ (24-45) binding peptide. The peptide did not bind to the C-terminal residues 50-87 of PDEγ. Likewise, a region of Tα, 1-25 did not exhibit high-affinity binding to PDEγ or to the 24-45 PDEγ peptide. Specific binding of the 250-275 peptide to PDEγ was confirmed by its ability to compete with Tα binding to PDEγ, although a higher concentration was required (10 x). The Tα-(250-275) peptide activated a fully inhibited PDE αβγ2 complex in a dose-dependent manner. These results suggest that a region on Tα that recognizes the PDEγ-binding site is found within residues 250-275 of Tα.