The Putative cGMP Phosphodieserase Subunit ("PDE") Functions as a Prenyl Binding Protein (PBP)

Abstract

Abstract: : Purpose: To define the role and function of "PDEδ", a putative subunit of rod cGMP phosphodiesterase (PDE). Methods: Immunocytochemistry with bovine "PDEδ" specific polyclonal antibody. Yeast 2-hybrid (y2h) screening of a bovine retina cDNA expression library with a "PDEδ" bait construct cloned into pGBKT7. GST pull down assays to verify stable binding and interactions of y2h positive clones with "PDEδ". Fluorescence resonance energy transfer (FRET) with dansylated prenyl ligands to determine dissociation constants. Results: "PDEδ" was originally shown to copurify with rod PDE from bovine retina and thought to be a fourth subunit of PDE. However, other studies showed that "PDEδ" can interact with several small GTPases, including Rab13, Ras, Rap, and the retinitis pigmentosa GTPase regulator (RPGR), all of which are prenylated, as well as Arl2/Arl3 which are not known to be modified. Immunocytochemistry with a "PDEδ" specific antibody shows distribution of "PDEδ" strongly in cones, weaker in rods and other cell types of the bovine retina. We find by y2h screening with a "PDEδ" bait that it can interact with rhodopsin kinase (GRK1), and that prenylation (GRK1 is farnesylated) is essential for this interaction. When the C-terminal CAAX box motif signaling prenylation is mutated (C to S), the mutant GRK1 does not associate with "PDEδ". In-vitro binding assays (GST pull downs) indicate that both recombinant GRK1 and GRK7 (which is geranylgeranylated) co-precipitate with a GST-"PDEδ" fusion protein. Using fluorescence resonance energy transfer techniques exploiting the intrinsic tryptophan fluorescence of "PDEδ" and dansylated prenyl chains as fluorescent ligands, we show that "PDEδ" specifically binds geranylgeranyl and farnesyl moieties with a Kd of 19.0 µM and 0.70 µM, respectively. Conclusions: "PDEδ" can be detected at high levels in bovine cones, thus it is not an exclusive subunit of rod PDE. Our experiments establish that "PDEδ" can function as a prenyl binding protein (PBP) interacting with multiple prenylated proteins. Its precise function when interacting with hydrophobic tails in a GDI-like fashion is unknown. PDEδ’s function is not limited to prenyl binding since it also interacts with non-prenylated proteins. We propose to rename "PDEδ" into prenyl binding protein (PBP).

Keywords: photoreceptors • molecular biology • protein structure/function