AN INTERFACE OF INTERACTION BETWEEN PHOTORECEPTOR CGMP PHOSPHODIESTERASE CATALYTIC SUBUNITS AND INHIBITORY GAMMA-SUBUNITS

Cyclic guanosine 5'-monophosphate (cGMP) phosphodiesterase (PDE) regul ates the level of cGMP on transduction of a visual signal in vertebrat e photoreceptor cells. Two identical inhibitory PDE gamma subunits (P gamma s) block catalytic activity of PDE-alpha and -beta subunits (P a lpha beta) in the dark. The primary regions of P gamma involved in the interaction with P alpha beta are a central polycationic region, P ga mma-24-45, and a C-terminal region of P gamma. Recently, we have shown that the C-terminal region of P gamma, which is the major P gamma inh ibitory domain, blocks PDE activity by binding to the catalytic site o f PDE (Artemyev, N. O Natochin, M., Busman, M., Schey, K. L., and Hamm , H. E. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 5407-5412). Here, w e localize the site on the rod cGMP PDE alpha subunit that binds to th e central polycationic domain of P gamma. This site is located within a region that links a second noncatalytic cGMP binding site with the c atalytic domain of PDE. A polypeptide coresponding to this region, P a lpha-461-553, expressed as a glutathione S-transferase fusion protein in Escherichia coli and isolated after cleavage of the fusion protein with thrombin, blocks inhibition of PDE activity by P gamma. In additi on, P alpha-461-553 binds to the P gamma-24-45 region (K-d, 7 mu M), a s measured by a fluorescent increase in a P gamma-24-45Cys peptide lab eled with 3-(bromoacetyl)-7-diethylaminocoumarin. The P alpha-461-553 region was further characterized by using a set of synthetic peptides. A peptide corresponding to residues 517-541 of P alpha (P alpha-517-5 41) effectively suppressed inhibition of PDE activity by P gamma and b ound to P gamma-24-45Cys labeled with 3-(bromoacetyl) -7-diethylaminoc oumarin (K-d, 22 mu M). P alpha-517-541 also competes with the activat ed rod G-protein alpha-subunit for binding to P gamma labeled with luc ifer yellow vinyl sulfone. This suggests that light activation of rod PDE by the G-protein transducin involves competition between transduci n alpha-guanosine 5'-triphosphate and P alpha-517-541 for binding: to the P gamma-24-45 region. Based on the results, we propose a linear mo del of interactions between catalytic and inhibitory PDE subunits.