Evaluation of the 17-kDa prenyl-binding protein as a regulatory protein for phototransduction in retinal photoreceptors

Abstract

The mammalian rod photoreceptor phosphodiesterase (PDE6) holoenzyme is isolated in both a membrane-associated and a soluble form. Membrane binding is a consequence of prenylation of PDE6 catalytic subunits, whereas soluble PDE6 is purified with a 17-kDa prenyl-binding protein (PDES) tightly bound. This protein, here termed PrBP/δ, has been hypothesized to reduce activation of PDE6 by transducin, thereby desensitizing the photoresponse. To test the potential role of PrBP/δ in regulating phototransduction, we examined the abundance, localization, and potential binding partners of PrBP/δ in retina and in purified rod outer segment (ROS) suspensions whose physiological and biochemical properties are well characterized. The amphibian homologue of PrBP/δ was cloned and sequenced and found to have 82% amino acid sequence identity with mammalian PrBP/δ. In contrast to bovine ROS, all off the PDE6 in purified frog ROS is membrane-associated. However, addition of recombinant frog PrBP/δ can solubilize PDE6 and prevent its activation by transducin. PrBP/δ also binds other prenylated photoreceptor proteins in vitro, including opsin kinase (GRK1/GRK7) and rab8. Quantitative immunoblot analysis off the PrBP/δ content of purified ROS reveals insufficient amounts of PrBP/δ (<0.1 PrBP/δ per PDE6) to serve as a subunit of PDE6 in either mammalian or amphibian photoreceptors. The immunolocalization of PrBP/δ in frog and bovine retina shows greatest PrBP/δ immunolabeling outside the photoreceptor cell layer. Within photoreceptors, only the inner segments of frog double cones are strongly labeled, whereas bovine photoreceptors reveal more PrBP/δ labeling near the junction of the inner and outer segments (connecting cilium) of photoreceptors. Together, these results rule out PrBP/δ as a PDE6 subunit and implicate PrBP/δ in the transport and membrane targeting of prenylated proteins (including PDE6) from their site of synthesis in the inner segment to their final destination in the outer segment of rods and cones.