Identification of the gamma subunit-interacting residues on photoreceptor cGMP phosphodiesterase, PDEG alpha '*

Photoreceptor cGMP phosphodiesterase (PDE6) is the effector enzyme in the G protein-mediated visual transduction cascade. In the dark, the activity of PDE6 is shut off by the inhibitory gamma subunit (P gamma), Chimeric prote ins between cone PDE6 alpha' and cGMP-binding and cGMP-specific PDE (PDE5) have been constructed and expressed in Sf9 cells to study the mechanism of inhibition of PDE6 catalytic activity by P gamma. Substitution of the segme nt PDE5-(773-820) by the corresponding PDE6 alpha'-(737-784) sequence in th e wild-type PDE5 or in a PDE5/PDE6 alpha' chimera containing the catalytic domain of PDE5 results in chimeric enzymes capable of inhibitory interactio n with P gamma. The catalytic properties of the chimeric PDEs remained simi lar to those of PDE5. Ala-scanning mutational analysis of the P gamma -bind ing region, PDE6 alpha'-(750-760), revealed PDE6 alpha' residues essential for the interaction. The M758A mutation markedly impaired and the Q752A mut ation moderately impaired the inhibition of chimeric PDE by P gamma. The an alysis of the catalytic properties of mutant PDEs and a model of the PDE6 c atalytic domain suggest that residues Met(758) and Gln(752) directly bind P gamma. A model of the PDE6 catalytic site shows that PDE6 alpha'-(750-760) forms a loop at the entrance to the cGMP-binding pocket. Binding of P gamm a to Met(758) would effectively block access of cGMP to the catalytic cavit y, providing a structural basis for the mechanism of PDE6 inhibition.