INTERACTION SITES OF THE COOH-TERMINAL REGION OF THE GAMMA SUBUNIT OF CGMP PHOSPHODIESTERASE WITH THE GTP-BOUND ALPHA SUBUNIT OF TRANSDUCIN

Abstract In photoreceptor cells, visual transduction occurs through photoexcitation of rhodopsin, GTP activation of the α subunit of transducin, and interaction between GTP-bound transducin α subunit and the inhibitory γ subunit of phosphodiesterase. The γ subunit of phosphodiesterase, in turn, accelerates the hydrolysis of GTP on the α subunit of transducin. Within the COOH-terminal residues (46-87) of the phosphodiesterase γ subunit, Trp-70 has been implicated in phosphodiesterase activation, transducin α subunit-phosphodiesterase γ subunit interaction, and the GTP hydrolysis accelerating activity. We have derivatized the phosphodiesterase γ subunit with a reversible photoactivatable reagent, [125I]N-[(3-iodo-4-azidophenylpropionamido-S-(2-thiopyridyl)]cysteine ([125I]ACTP), at cysteine (Cys-68). A light-dependent, cross-linked complex of guanosine 5′-(γ-thio)triphosphate-bound transducin α subunit and ACTPderivatized phosphodiesterase γ subunit formed after photolysis of a 1:1 stoichiometic complex of the two proteins. The specificity of complex formation between the transducin α subunit and the phosphodiesterase γ subunit was demonstrated by specific protection by the C68A mutant of the phosphodiesterase γ subunit. The cross-linked complex was treated with β-mercaptoethanol to transfer the 125I photomoiety from the phosphodiesterase γ subunit to the transducin α subunit. Combined techniques involving electrophoresis, chemical and enzymatic cleavage, and chemical and radiosequencing were used to identify photoinsertion sites on the α3 and α4/β6 regions of the transducin α subunit. Three photo-labeled residues, His-244 (α3 helix), Met-308, and Arg-310 (α4/β6 interface), were specifically identified as photoinsertion sites. Utilizing the crystal structure coordinates of the GTP-bound transducin α subunit and molecular modeling, we conclude that Cys-68 of the phosphodiesterase γ subunit is located at a position between the exposed face of the α3 and α4 helices of the transducin α subunit. We propose that the phosphodiesterase γ subunit interacts with GTP-bound transducin α subunit at multiple sites in which the cysteine 68 to tryptophan 70 sequence of the phosphodiesterase γ subunit, which is critical for GTP hydrolysis accelerating activity, interacts in the α3/α4/β6 region of GTP-bound transducin α subunit.