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Volume 271, Number 43, Issue of October 25, 1996 pp. 26900-26907
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Interaction Sites of the COOH-terminal Region of the  Subunit of cGMP Phosphodiesterase with the GTP-bound  Subunit of Transducin

(Received for publication, April 10, 1996, and in revised form, August 20, 1996)

Yu Liu , Vadim Y. Arshavsky ^§ and Arnold E. Ruoho

From the  Department of Pharmacology, University Wisconsin Medical School, Madison Wisconsin 53706 and the ^§ Harvard Medical School, Massachusetts Eye and Ear Infirmary, Boston, Massachusetts 02114

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

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