Domain mapping of the retinal cyclic GMP phosphodiesterase y - subunit Function of the domains encoded by the three exons of the y - subunit gene

Retinal rod-outer-segment phosphodiesterase (PDE) is a heterotetramer consisting of two similar, but not identical, catalytic subunits (a and fi) and two identical inhibitory subunits (72). Previously, we have reported that the site of PDEa/# interaction with PDEy is located within residues 54-87 [Cunnick, Hurt, Oppert, Sakamoto & Takemoto (1990) Biochem. J. 271, 721-727]. The site for PDEy interaction with transducin a (Ta) was found to encompass residues 24-45 of PDEy [Morrison, Cunnick, Oppert & Takemoto (1989) J. Biol. Chem. 264, 11671-11681]. In order to identify binding sites and other functional domains of PDEy, the three peptides which are encoded by the three exons of the PDEy gene were synthesized chemically. These exons encode for residues 1-49, 50-62 and 63-87 of bovine PDEy [Piriev, Purishko, Khramtsov & Lipkin (1990) Dokl. Akad. Nauk. SSSR 315, 229-230]. The peptide encompassing residues 63-87 was inhibitory in a PDE assay, whereas peptides 1-49 and 50-62 had no effect. However, both peptides 1-49 and 63-87 bound to PDEa// in a solid-phase binding assay. Only peptide 1-49 bound to TacGTP[S] (GTP[S] is guanosine 5'-[ythio]triphosphate). These data confirm that the inhibitory region ofPDEy is encoded by exon 3 (residues 63-87), whereas a separate binding site for PDEa//J and for TaGTP[S] is encoded by exon 1 (residues 1-49). To study further the structure-function relationship of PDEy, this entire protein and two mutants were chemically synthesized. One mutant (CT) lacked residues 78-87, whereas another replaced tyrosine-84 with glycine (TYR-84). Whereas the synthetic PDEy inhibited PDEa/f catalytic activity, the -CT and TVR-84 mutants did not. All three synthetic proteins bound to both PDEa/, and and TaGTP[S]. These data confirm the presence of an alternative binding site on PDEy and demonstrate the importance of tyrosine-84 in PDEy inhibitory activity.