The N termini of the inhibitory -subunits of phosphodiesterase-6 (PDE6) from rod and cone photoreceptors differentially regulate transducin-mediated PDE6 activation

Phosphodiesterase-6 (PDE6) plays a central role in both rod and cone phototransduction pathways. In the dark, PDE6 activity is suppressed by its inhibitory -subunit (P). Rhodopsin-catalyzed activation of the G protein transducin relieves this inhibition and enhances PDE6 catalysis. We hypothesized that amino acid sequence differences between rod- and cone-specific Ps underlie transducin's ability to more effectively activate cone-specific PDE6 than rod PDE6. To test this, we analyzed rod and cone P sequences from all major vertebrate and cyclostome lineages and found that rod P loci are far more conserved than cone P sequences and that most of the sequence differences are located in the N-terminal region. Next we reconstituted rod PDE6 catalytic dimer (P) with various rod or cone P variants and analyzed PDE6 activation upon addition of the activated transducin -subunit (Gt*-GTPS). This analysis revealed a rod-specific P motif (amino acids 9-18) that reduces the ability of Gt*-GTPS to activate the reconstituted PDE6. In cone P, Asn-13 and Gln-14 significantly enhanced Gt*-GTPS activation of cone P truncation variants. Moreover, we observed that the first four amino acids of either rod or cone P contribute to Gt*-GTPS-mediated activation of PDE6. We conclude that physiological differences between rod and cone photoreceptor light responsiveness can be partially ascribed to ancient, highly conserved amino acid differences in the N-terminal regions of P isoforms, demonstrating for the first time a functional role for this region of P in the differential activation of rod and cone PDE6 by transducin.