Assay and functional properties of PrBP(PDEdelta), a prenyl-binding protein interacting with multiple partners.

A 17-kDa prenyl-binding protein, PrBP(PDEdelta), is highly conserved among various species from human to Caenorhabditis elegans. First identified as a putative regulatory delta subunit of the cyclic nucleotide phosphodiesterase (PDE6) purified from mammalian photoreceptor cells, PrBP(PDEdelta) has been hypothesized to reduce activation of PDE6 by the heterotrimeric G-protein, transducin, thereby desensitizing the photoresponse. However, recent work shows that PrBP(PDEdelta) interacts with numerous prenylated proteins at their farnesylated or geranylgeranylated C-termini, as well as with non-prenylated proteins. These polypeptides include small GTPases such as Rab13, Ras, Rap, and Rho6, as well as components involved in phototransduction (e.g., rod and cone PDE6, rod and cone opsin kinases). Expression of PrBP(PDEdelta) in tissues and organisms not expressing PDE6, the demonstration of multiple interacting partners with PrBP(PDEdelta), and its low abundance in rod outer segments all argue against it being a regulatory PDE6 subunit. This raises intriguing questions as to its physiological functions. In this chapter, we review the current status of PrBP(PDEdelta) and describe some of the assays used to determine these interactions in detail. In mammalian photoreceptors, the results are consistent with a role of PrBP(PDEdelta) in the transport of prenylated proteins from their site of synthesis in the inner segment to the outer segment where phototransduction occurs.

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

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 (PDEdelta) tightly bound. This protein, here termed PrBP/delta, has been hypothesized to reduce activation of PDE6 by transducin, thereby desensitizing the photoresponse. To test the potential role of PrBP/delta in regulating phototransduction, we examined the abundance, localization, and potential binding partners of PrBP/delta in retina and in purified rod outer segment (ROS) suspensions whose physiological and biochemical properties are well characterized. The amphibian homologue of PrBP/delta was cloned and sequenced and found to have 82% amino acid sequence identity with mammalian PrBP/delta. In contrast to bovine ROS, all of the PDE6 in purified frog ROS is membrane-associated. However, addition of recombinant frog PrBP/delta can solubilize PDE6 and prevent its activation by transducin. PrBP/delta also binds other prenylated photoreceptor proteins in vitro, including opsin kinase (GRK1/GRK7) and rab8. Quantitative immunoblot analysis of the PrBP/delta content of purified ROS reveals insufficient amounts of PrBP/delta (<0.1 PrBP/delta per PDE6) to serve as a subunit of PDE6 in either mammalian or amphibian photoreceptors. The immunolocalization of PrBP/delta in frog and bovine retina shows greatest PrBP/delta 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/delta labeling near the junction of the inner and outer segments (connecting cilium) of photoreceptors. Together, these results rule out PrBP/delta as a PDE6 subunit and implicate PrBP/delta 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.