Interaction sites of the COOH-terminal region of the gamma subunit of cGMP phosphodiesterase with the GTP-bound alpha subunit of transducin
Liu, Y.; Arshavsky, V.Y.; Ruoho, A.E.
Journal of Biological Chemistry 271(43): 26900-26907
1996
ISSN/ISBN: 0021-9258
PMID: 8900174
Accession: 008893516
In photoreceptor cells, visual transduction occurs through photoexcitation of rhodopsin, GTP activation of the alpha subunit of transducin, and interaction between GTP-bound transducin alpha subunit and the inhibitory gamma subunit of phosphodiesterase. The gamma subunit of phosphodiesterase, in turn, accelerates the hydrolysis of GTP on the a subunit of transducin. Within the COOH-terminal residues (46-87) of the phosphodiesterase gamma subunit, Trp-70 has been implicated in phosphodiesterase activation, transducin alpha subunit-phosphodiesterase gamma subunit interaction, and the GTP hydrolysis accelerating activity. We have derivatized the phosphodiesterase gamma 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'-(gamma-thio)triphosphate-bound transducin alpha subunit and ACTP-derivatized phosphodiesterase gamma subunit formed after photolysis of a 1:1 stoichiometric complex of the two proteins. The specificity of complex formation between the transducin alpha subunit and the phosphodiesterase gamma subunit was demonstrated by specific protection by the C68A mutant of the phosphodiesterase gamma subunit. The cross-linked complex was treated with beta-mercaptoethanol to transfer the 125I photomoiety from the phosphodiesterase gamma subunit to the transducin alpha subunit. Combined techniques involving electrophoresis, chemical and enzymatic cleavage, and chemical and radiosequencing were used to identify photoinsertion sites on the alpha-3 and alpha-4/beta-6 regions of the transducin alpha subunit. Three photo-labeled residues, His-244 (alpha-3 helix), Met-308, and Arg-310 (alpha-4/beta-6 interface), were specifically identified as photoinsertion sites. Utilizing the crystal structure coordinates of the GTP-bound transducin alpha subunit and molecular modeling, we conclude that Cys-68 of the phosphodiesterase gamma subunit is located at a position between the exposed face of the alpha-3 and alpha-4 helices of the transducin alpha subunit. We propose that the phosphodiesterase gamma subunit interacts with GTP-bound transducin alpha subunit at multiple sites in which the cysteine 68 to tryptophan 70 sequence of the phosphodiesterase gamma subunit, which is critical for GTP hydrolysis accelerating activity, interacts in the alpha-3/alpha-4/beta-6 region of GTP-bound transducin alpha subunit.