Solubilization of Membrane-bound Rod Phosphodiesterase by the Rod Phosphodiesterase Recombinant δ Subunit

Abstract

Retinal rod and cone phosphodiesterases are oligomeric enzymes that consist of a dimeric catalytic core (␣ 2 in cones and ␣␤ in rods) with inhibitory subunits (␥) that regulate their activity. In addition, a 17-kDa protein referred to as the ␦ subunit co-purifies with the rod soluble phosphodiesterase and the cone phosphodiesterase. We report here partial protein sequencing of the rod ␦ subunit and isolation of a cDNA clone encoding it. The predicted amino acid sequence is unrelated to any other known protein. Of eight bovine tissue mRNA preparations examined by Northern analysis, the strongest ␦ subunit-specific signal was present in the retina. A less intense signal was seen in the brain and adrenal mRNA. In bovine retinal sections, rod ␦ subunit anti-peptide antibodies label rod but not cone outer segments. ␦ subunit, added back to washed outer segment membranes, solubilizes a large fraction of the membrane-bound phosphodiesterase, indicating that this subunit binds to the classical membrane associated phosphodiesterase. The subunit forms a tight complex with native, but not trypsin-released phosphodiesterase, suggesting that the isoprenylated carboxyl termini of the catalytic subunits may be involved in binding of the ␦ subunit to the phosphodiesterase holoenzyme.Phototransduction in the retina involves a cascade of regulated biochemical processes (for recent reviews, see Refs. 1 and 2). Key components in this light-activated biochemical cascade are the retinal specific phosphodiesterases (PDE6s).1 These phosphodiesterases catalyze the conversion of cGMP to 5Ј-GMP when activated by the G protein, transducin. The retinal phosphodiesterases are multimeric proteins composed of catalytic and inhibitory subunits. The large subunits of the rod and cone phosphodiesterases (the ␣ and ␤ subunits in rods and the ␣Ј subunit in cones) dimerize to form the catalytic core of the isozymes. The smaller 11-and 13-kDa subunits (rod ␥ and cone ␥) serve as inhibitors of the rod and cone phosphodiesterases, respectively (3-5). Hurwitz et al. (6) identified a 17-kDa protein that immunoprecipitated with bovine retinal phosphodiesterases. Gillespie and Beavo (7) and Gillespie et al. (8) later demonstrated that a protein of this size co-purified with the isotonically soluble rod and cone phosphodiesterases, but not with the membrane-bound rod phosphodiesterase. A function for this 17-kDa protein subunit (also referred to as the ␦ or 15-kDa subunit) has not been described.The rod membrane-bound phosphodiesterase is loosely associated with the membrane at least in part due to C-terminal isoprenyl and carboxymethyl post-translational modifications. Several investigators (9, 10) have demonstrated that the ␣ subunit is modified by a farnesyl (C-15) group and the ␤ subunit is modified by a geranylgeranyl (C-20) group on their carboxyl termini. In addition both COOH termini are methylated as reported by Swanson and Applebury (11) and others (12). Mutation of the conserved cysteine residue to serine in the CAAX isoprenylation motif in the ...

Characteristics of Photoreceptor PDE (PDE6): similarities and differences to PDE5

“…27,28 A 17-kDa prenyl binding protein (PrBP), originally referred to as the 'delta' (d) subunit of PDE6, 29 can bind to PDE6 and release the holoenzyme from its membrane-associated state. 30 PrBP principally interacts with PDE6 at its prenylated C-terminus. 31 In contrast to the catalytic and inhibitory subunits of PDE6, PrBP is widely expressed in a variety of tissues.…”

“…31 In contrast to the catalytic and inhibitory subunits of PDE6, PrBP is widely expressed in a variety of tissues. 30,32 It is also highly conserved through evolution. 33 While PrBP has been shown to interact with many other binding partners, 34 neither PDE5 nor other phosphodiesterases have been reported to interact with this prenyl binding protein.…”

Mutations ofRPGR in X-linked retinitis pigmentosa (RP3)

“…Yeast two hybrid studies identified two proteins interact- ing with the RCC1 domain of RPGR: the phosphodiesterase delta subunit (PDE δ) [Linari et al, 1999a;Hong et al, 2001] and a novel protein called RPGRIP1 [Boylan and Wright, 2000;Roepman et al, 2000;Hong et al, 2001]. Interestingly, PDEδ not only interacts with retinal phosphodiesterase subunits [Florio et al, 1996], but also with the small GTPases Rab13 [Marzesco et al, 1998] and Arl3 [Linari et al, 1999b].…”

Sorting of lipidated cargo by the Arl2/Arl3 system

“…This was indeed verified by a number of biochemical and cellular studies which suggested that PDE6d is a general prenyl binding protein (therefore also called PrBP) without apparent selectivity. [33][34][35][36][37][38][39] In contrast to RhoGDI and RabGDI which are specific for the GDP-bound conformation of prenylated Rho or Rab proteins, 39,40 PDE6d binds Ras and Rheb independent of their nucleotidebound conformation. 34,41 The structure of the Arl2-PDE6d complex shows however that the hydrophobic pocket is partially occupied suggesting a mutually exclusive binding to prenylated cargo and/or Arl2.…”