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J. Biol. Chem., Vol. 280, Issue 2, 1248-1256, January 14, 2005

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Evaluation of the 17-kDa Prenyl-binding Protein as a Regulatory Protein for Phototransduction in Retinal Photoreceptors^*

Angela W. Norton, Suzanne Hosier, Jennifer M. Terew, Ning Li, Anuradha Dhingra¶, Noga Vardi¶, Wolfgang Baehr, and Rick H. Cote||

From the Department of Biochemistry and Molecular Biology, University of New Hampshire, Durham, New Hampshire 03824-2617, the Moran Eye Center, University of Utah Health Center, Salt Lake City, Utah 84132, and the ^¶Department of Neuroscience, University of Pennsylvania, Philadelphia, Pennsylvania 19104

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

Received for publication, September 13, 2004 , and in revised form, October 20, 2004.

^* This work was supported by National Institutes of Health Grants EY05798 (to R. H. C.), EY08123 (to W. B.), and EY11105 (to N. V.) and is Scientific Contribution #2250 from the New Hampshire Agricultural Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank^TM/EBI Data Bank with accession number(s) AY044177.

^|| To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, University of New Hampshire, Durham, NH 03824-2617. Tel.: 603-862-2458; Fax: 603-862-4013; E-mail: Rick.Cote{at}unh.edu.

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