Phosphodiesterase-6 (PDE6) plays a central role in both rod and cone phototransduction pathways. In the dark, PDE6 activity is suppressed by its inhibitory γ-subunit (Pγ). 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 Pγs underlie transducin's ability to more effectively activate cone-specific PDE6 than rod PDE6. To test this, we analyzed rod and cone Pγ sequences from all major vertebrate and cyclostome lineages and found that rod Pγ loci are far more conserved than cone Pγ sequences and that most of the sequence differences are located in the N-terminal region. Next we reconstituted rod PDE6 catalytic dimer (Pαβ) with various rod or cone Pγ variants and analyzed PDE6 activation upon addition of the activated transducin α-subunit (Gtα*-GTPγS). This analysis revealed a rod-specific Pγ motif (amino acids 9-18) that reduces the ability of Gtα*-GTPγS to activate the reconstituted PDE6. In cone Pγ, Asn-13 and Gln-14 significantly enhanced Gtα*-GTPγS activation of cone Pγ truncation variants. Moreover, we observed that the first four amino acids of either rod or cone Pγ contribute to Gtα*-GTPγS-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 Pγ isoforms, demonstrating for the first time a functional role for this region of Pγ in the differential activation of rod and cone PDE6 by transducin.
