THE CARBOXYL-TERMINUS OF THE GAMMA-SUBUNIT OF ROD CGMP PHOSPHODIESTERASE CONTAINS DISTINCT SITES OF INTERACTION WITH THE ENZYME CATALYTIC SUBUNITS AND THE ALPHA-SUBUNIT OF TRANSDUCIN

The interaction between the GTP-bound form of the transducin alpha-sub unit (G alpha(t)) and the gamma-subunit (P gamma) of cGMP phosphodiest erase (PDE) is a key event in effector activation during photon signal transduction. The carboxyl-terminal half of P gamma is involved in in teraction with G alpha(t), as well as in inhibition of PDE activity. H ere we have utilized a combination of synthetic peptide and mutagenesi s approaches to localize specific regions of the carboxyl-terminal reg ion of P gamma interacting with G alpha(t) and P alpha beta and have d etermined residues involved in inhibition of PDE activity. We found th at synthetic peptide corresponding to residues 68-87 of P gamma comple tely inhibit trypsin-activated PDE. The peptide P gamma-63-87 bound to G alpha(t)GTP gamma S with a K-d Of 2.5 mu m, whereas the binding of P gamma-68-87 60 G alpha(t)GTP gamma S was approximately 15-fold less (K-d = 40 mu M) suggesting that carboxyl-terminal P gamma region 68-87 contains a site for interaction with P alpha beta and also a part of the alpha(t) binding site. To map G alpha(t) and P alpha beta sites mo re precisely within the carboxyl-terminal region, a set of carboxyl-te rminal mutants was generated by site-directed mutagenesis. Deletion of residues 63-69 and 70-76 diminished the binding of mutants to alpha(t ) while binding to carboxyl-terminally truncated mutants lacking up to 11 amino acid residues was unchanged. In contrast, carboxyl-terminal truncations of P gamma from Delta 1 to Delta 11 resulted in a gradual decrease of its inhibitory activity. Thus, the extreme carboxyl-termin al hydrophobic sequence -Ile(86)-Ile(87) together with 9 adjacent resi dues provides inhibitory interaction of P gamma with P alpha beta. The carboxyl-terminal G alpha(t)GTP gamma S binding site of P gamma is di fferent from but adjacent to its PDE inhibitory site. During the visua l transduction process, G alpha(t)GTP Likely binds to this region of P gamma inducing a displacement of the extreme carboxyl terminus from t he inhibitory site on P alpha beta, leading to PDE activation.