Analysis of dimerization determinants of PDE6 catalytic subunits.

An absolute majority of cyclic nucleotide phosphodiesterases (PDEs) form catalytic dimers. The structural determinants and functional significance of PDE dimerization are poorly understood. Furthermore, all known dimeric PDEs with the exception of retinal rod guanosine 3',5'-cyclic-monophosphate PDE (PDE6) are homodimeric enzymes. Rod PDE6 is a catalytic heterodimer composed of alpha- and beta-subunits. Gel filtration, sucrose gradient centrifugation, and immunoprecipitation are standard techniques used to study dimerization of proteins. We successfully applied these methods to investigate dimerization of chimeric proteins between PDE6alphabeta and PDE5, which allowed us to elucidate the structural basis for heterodimerization of rod PDE6. This chapter outlines approaches to the investigation of PDE6 dimerization that can be utilized in a broader analysis of PDE dimerization.

Mutation in rod PDE6 linked to congenital stationary night blindness impairs the enzyme inhibition by its gamma-subunit.

Photoreceptor cGMP phosphodiesterase (PDE6) is the effector enzyme in the vertebrate visual transduction cascade. The activity of rod PDE6 catalytic alpha- and beta-subunits is blocked in the dark by two inhibitory Pgamma-subunits. The inhibition is released upon light-stimulation of photoreceptor cells. Mutation H258N in PDE6beta has been linked to congenital stationary night blindness (CSNB) in a large Danish family (Rambusch pedigree) (Gal, A., Orth, U., Baehr, W., Schwinger, E., and Rosenberg, T. (1994) Nat. Genet. 7, 64-67.) We have analyzed the consequences of this mutation for PDE6 function using a Pgamma-sensitive PDE6alpha'/PDE5 chimera, Chi16. Biochemical analysis of the H257N mutant, an equivalent of PDE6betaH258N, demonstrates that this substitution does not alter the ability of chimeric PDE to dimerize or the enzyme's catalytic properties. The sensitivity of H257N to a competitive inhibitor zaprinast was also unaffected. However, the mutant displayed a significant impairment in the inhibitory interaction with Pgamma, which was apparent from a approximately 20-fold increase in the K(i) value (46 nM) and incomplete maximal inhibition. The inhibitory defect of H257N is not due to perturbation of noncatalytic cGMP binding to the PDE6alpha' GAF domains. The noncatalytic cGMP-binding characteristics of the H257N mutant were similar to those of the parent PDE6alpha'/PDE5 chimera. Since rod PDE6 in the Rambusch CSNB is a catalytic heterodimer of the wild-type PDE6alpha and mutant PDE6beta, Chi16 and H257N were coexpressed, and a heterodimeric PDE, Chi16/H257N, was isolated. It displayed two Pgamma inhibitory sites with the K(i) values of 5 and 57 nM. Our results support the hypothesis that mutation H258N in PDE6beta causes CSNB through incomplete inhibition of PDE6 activity by Pgamma, which leads to desensitization of rod photoreceptors.

The GAFa domains of rod cGMP-phosphodiesterase 6 determine the selectivity of the enzyme dimerization.

Retinal rod cGMP phosphodiesterase (PDE6 family) is the effector enzyme in the vertebrate visual transduction cascade. Unlike other known PDEs that form catalytic homodimers, the rod PDE6 catalytic core is a heterodimer composed of alpha and beta subunits. A system for efficient expression of rod PDE6 is not available. Therefore, to elucidate the structural basis for specific dimerization of rod PDE6, we constructed a series of chimeric proteins between PDE6alphabeta and PDE5, which contain the N-terminal GAFa/GAFb domains, or portions thereof, of the rod enzyme. These chimeras were co-expressed in Sf9 cells in various combinations as His-, myc-, or FLAG-tagged proteins. Dimerization of chimeric PDEs was assessed using gel filtration and sucrose gradient centrifugation. The composition of formed dimeric enzymes was analyzed with Western blotting and immunoprecipitation. Consistent with the selectivity of PDE6 dimerization in vivo, efficient heterodimerization was observed between the GAF regions of PDE6alpha and PDE6beta with no significant homodimerization. In addition, PDE6alpha was able to form dimers with the cone PDE6alpha' subunit. Furthermore, our analysis indicated that the PDE6 GAFa domains contain major structural determinants for the affinity and selectivity of dimerization of PDE6 catalytic subunits. The key dimerization selectivity module of PDE6 has been localized to a small segment within the GAFa domains, PDE6alpha-59-74/PDE6beta-57-72. This study provides tools for the generation of the homodimeric alphaalpha and betabeta enzymes that will allow us to address the question of functional significance of the unique heterodimerization of rod PDE6.

Direct interaction of the inhibitory gamma-subunit of Rod cGMP phosphodiesterase (PDE6) with the PDE6 GAFa domains.

Retinal rod and cone cGMP phosphodiesterases (PDE6 family) function as the effector enzyme in the vertebrate visual transduction cascade. The activity of PDE6 catalytic subunits is controlled by the Pgamma-subunits. In addition to the inhibition of cGMP hydrolysis at the catalytic sites, Pgamma is known to stimulate a noncatalytic binding of cGMP to the regulatory GAFa-GAFb domains of PDE6. The latter role of Pgamma has been attributed to its polycationic region. To elucidate the structural basis for the regulation of cGMP binding to the GAF domains of PDE6, a photoexcitable peptide probe corresponding to the polycationic region of Pgamma, Pgamma-21-45, was specifically cross-linked to rod PDE6alphabeta. The site of Pgamma-21-45 cross-linking was localized to Met138Gly139 within the PDE6alpha GAFa domain using mass spectrometric analysis. Chimeras between PDE5 and cone PDE6alpha', containing GAFa and/or GAFb domains of PDE6alpha' have been generated to probe a potential role of the GAFb domains in binding to Pgamma. Analysis of the inhibition of the PDE5/PDE6alpha' chimeras by Pgamma supported the role of PDE6 GAFa but not GAFb domains in the interaction with Pgamma. Our results suggest that a direct binding of the polycationic region of Pgamma to the GAFa domains of PDE6 may lead to a stabilization of the noncatalytic cGMP-binding sites.