Binding of the Delta Subunit to Rod Phosphodiesterase Catalytic Subunits Requires Methylated, Prenylated C-Termini of the Catalytic Subunits

Cook, Terry A.; Ghomashchi, Farideh; Gelb, Michael H.; Florio, Stephanie K.; Beavo, Joseph A.

doi:10.1021/bi001070l

Cited by 56 publications

(52 citation statements)

References 31 publications

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“…The slow lateral diffusion and the absence of any significant interdisc transfer of EGFP-PDE6C both indicate that it is predominantly membranebound in Xenopus rods. This notion differs from the hypothesized existence of soluble PDE6 in photoreceptor cells (36,37). The hypothesis is based on the observation that upon extraction from bovine retina, a significant fraction of PDE6 is found in a soluble complex with the 17-kDa prenyl-binding protein PrBP/␦, also known as the ␦-subunit of PDE6 (36).…”

Section: Discussionmentioning

confidence: 98%

“…The hypothesis is based on the observation that upon extraction from bovine retina, a significant fraction of PDE6 is found in a soluble complex with the 17-kDa prenyl-binding protein PrBP/␦, also known as the ␦-subunit of PDE6 (36). PrBP/␦ was shown to interact with the methylated prenylated C termini of PDE6A and PDE6B and solubilize the enzyme from the membrane in vitro (37). However, immunohistochemical analysis has shown that PrBP/␦ localizes to the IS or near the RIS/ROS junction and does not co-localize with PDE6 (38).…”

Section: Discussionmentioning

confidence: 99%

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Characterization of Human Cone Phosphodiesterase-6 Ectopically Expressed in Xenopus laevis Rods

Muradov

Boyd

Haeri

et al. 2009

Journal of Biological Chemistry

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PDE6 (phosphodiesterase-6) is the effector molecule in the vertebrate phototransduction cascade. Progress in understanding the structure and function of PDE6 has been hindered by lack of an expression system of the enzyme. Here we report ectopic expression and analysis of compartmentalization and membrane dynamics of the enhanced green fluorescent protein (EGFP) fusion protein of human cone PDE6C in rods of transgenic Xenopus laevis. EGFP-PDE6C is correctly targeted to the rod outer segments in transgenic Xenopus, where it displayed a characteristic striated pattern of EGFP fluorescence. Immunofluorescence labeling indicated significant and light-independent co-localization of EGFP-PDE6C with the disc rim marker peripherin-2 and endogenous frog PDE6. The diffusion of EGFP-PDE6C on disc membranes investigated with fluorescence recovery after photobleaching was markedly slower than theoretically predicted. The enzymatic characteristics of immunoprecipitated recombinant PDE6C were similar to known properties of the native bovine PDE6C. PDE6C was potently inhibited by the cone-and rod-specific PDE6 ␥-subunits. Thus, transgenic Xenopus laevis is a unique expression system for PDE6 well suited for analysis of the mechanisms of visual diseases linked to PDE6 mutations.Phosphodiesterases of cyclic nucleotides (PDEs) 2 are essential enzymes controlling cellular levels of cAMP and cGMP. Eleven families of PDEs have been identified in mammals on the grounds of sequence homology, substrate selectivity, and regulation (1). Photoreceptor-specific PDEs in rods and cones comprise the sixth PDE family (PDE6) and serve as the effector enzymes in the vertebrate phototransduction cascade (1-5). Rod PDE6 is composed of homologous catalytic ␣-subunit (PDE6A) and ␤-subunit (PDE6B) and two copies of a small inhibitory ␥-subunit (P␥) (3). Cone PDE6 is a catalytic dimer of two identical ␣Ј-subunits (PDE6C) (3). A cone-specific inhibitory P␥-subunit is highly homologous to the rod P␥ (6). In rod photoreceptors, PDE6 is located in the specialized compartments called rod outer segments (ROS), where it associates with disc membranes. The membrane attachment of PDE6 is mediated by farnesylation of the PDE6A C terminus and geranylgeranylation of the PDE6B C terminus (7). In cones, geranylgeranylated PDE6C resides on infoldings of the cone outer segment plasma membrane (8). Following photoexcitation of rod or cone photoreceptor cells, PDE6 is activated by the GTPbound transducin ␣-subunit (G␣ t GTP) that relieves the P␥ inhibition of the enzyme. cGMP hydrolysis by active PDE6 leads to a cellular response due to a closure of cGMP-gated channels in the photoreceptor plasma membrane (2-5).Although PDE6 plays a prominent role in vertebrate vision, the structure-function relationships of PDE6 are poorly understood in comparison with other key phototransduction proteins. The lack of an expression system for PDE6 has become a major impediment for PDE6 research. Importantly, an expression system for PDE6 is required to elucidate the mechanisms of visua...

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Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Characterization of Human Cone Phosphodiesterase-6 Ectopically Expressed in Xenopus laevis Rods

Muradov

Boyd

Haeri

et al. 2009

Journal of Biological Chemistry

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“…Previous studies have shown that a large repertoire of prenyl proteins, such as subunits of PDE6, GRK1, GRK7, Rab13, Rap1a, Rap2b, RhoA, RhoB, Rnd1, Rap2c, RasD2, and Reb-L1, bind to PDEδ (20,22,27,(31)(32)(33). Among RAS proteins, in addition to KRAS4b, NRAS and HRAS have also been shown to bind to PDEδ (20,27).…”

Section: Identification Of a Conserved Sequence Motif In Kras4b And Imentioning

confidence: 99%

Structural basis of recognition of farnesylated and methylated KRAS4b by PDEδ

Dharmaiah

Bindu

Tran

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

150

197

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Farnesylation and carboxymethylation of KRAS4b (Kirsten rat sarcoma isoform 4b) are essential for its interaction with the plasma membrane where KRAS-mediated signaling events occur. Phosphodiesterase-δ (PDEδ) binds to KRAS4b and plays an important role in targeting it to cellular membranes. We solved structures of human farnesylated–methylated KRAS4b in complex with PDEδ in two different crystal forms. In these structures, the interaction is driven by the C-terminal amino acids together with the farnesylated and methylated C185 of KRAS4b that binds tightly in the central hydrophobic pocket present in PDEδ. In crystal form II, we see the full-length structure of farnesylated–methylated KRAS4b, including the hypervariable region. Crystal form I reveals structural details of farnesylated–methylated KRAS4b binding to PDEδ, and crystal form II suggests the potential binding mode of geranylgeranylated–methylated KRAS4b to PDEδ. We identified a 5-aa-long sequence motif (Lys-Ser-Lys-Thr-Lys) in KRAS4b that may enable PDEδ to bind both forms of prenylated KRAS4b. Structure and sequence analysis of various prenylated proteins that have been previously tested for binding to PDEδ provides a rationale for why some prenylated proteins, such as KRAS4a, RalA, RalB, and Rac1, do not bind to PDEδ. Comparison of all four available structures of PDEδ complexed with various prenylated proteins/peptides shows the presence of additional interactions due to a larger protein–protein interaction interface in KRAS4b–PDEδ complex. This interface might be exploited for designing an inhibitor with minimal off-target effects.

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“…The 17-kDa prenyl-binding protein (PrBP/␦), originally described as the ␦-subunit of PDE6 (33), is able to solubilize membrane-associated rod PDE6 in vitro (23,34) by binding to the hydrophobic prenyl groups attached to the C termini of the PDE6 catalytic subunits (35). Because hydrophobic interactions may stabilize GARP2-PDE6 interactions (see previous section), we wondered whether PrBP/␦ binding to PDE6 would solubilize the enzyme as a complex with GARP2 or, alternatively, compete with GARP2 for binding to a hydrophobic region on PDE6.…”

Section: The 17-kda Prenyl-binding Protein (Prbp/␦) Releases Pde6 Bumentioning

confidence: 99%

The Glutamic Acid-rich Protein-2 (GARP2) Is a High Affinity Rod Photoreceptor Phosphodiesterase (PDE6)-binding Protein That Modulates Its Catalytic Properties

Pentia

Hosier

Cote

2006

Journal of Biological Chemistry

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The glutamic acid-rich protein-2 (GARP2) is a splice variant of the ␤-subunit of the cGMP-gated ion channel of rod photoreceptors. GARP2 is believed to interact with several membrane-associated phototransduction proteins in rod photoreceptors. In this study, we demonstrated that GARP2 is a high affinity PDE6-binding protein and that PDE6 co-purifies with GARP2 during several stages of chromatographic purification. We found that hydrophobic interaction chromatography succeeds in quantitatively separating GARP2 from the PDE6 holoenzyme. Furthermore, the 17-kDa prenyl-binding protein, abundant in retinal cells, selectively released PDE6 (but not GARP2) from rod outer segment membranes, demonstrating the specificity of the interaction between GARP2 and PDE6. Purified GARP2 was able to suppress 80% of the basal activity of the nonactivated, membrane-bound PDE6 holoenzyme at concentrations equivalent to its endogenous concentration in rod outer segment membranes. However, GARP2 was unable to reverse the transducin activation of PDE6 (in contrast to a previous study) nor did it significantly alter catalysis of the fully activated PDE6 catalytic dimer. The high binding affinity of GARP2 for PDE6 and its ability to regulate PDE6 activity in its dark-adapted state suggest a novel role for GARP2 as a regulator of spontaneous activation of rod PDE6, thereby serving to lower rod photoreceptor "dark noise" and allowing these sensory cells to operate at the single photon detection limit.The visual transduction pathway in vertebrate photoreceptors is remarkable in many respects, including single photon detection capability (in rod photoreceptors), photoresponse kinetics on the millisecond time scale, and the ability to adapt to background illumination levels ranging from very dim illuminance levels (scotopic vision in rods) to bright sunlight (photopic vision in cones) (1). The very first steps in vision occur in the photoreceptor outer segment when photo-isomerized rhodopsin activates the heterotrimeric G-protein transducin, which proceeds to bind to and displace the inhibitory ␥-subunit (P␥) 2 of the photoreceptor phosphodiesterase (PDE6). Activated PDE6 rapidly lowers the cGMP concentration, resulting in closure of cGMP-gated channels in the plasma membrane and cell hyperpolarization (2-4). Several feedback mechanisms operate to actively terminate the photoresponse and restore the dark-adapted state, of which regulation of the lifetime of activated transducin is considered rate-limiting (2, 3). Rebinding of P␥ to the PDE6 catalytic subunits following transducin deactivation returns PDE6 to its nonactivated state and allows cGMP levels to return to their dark-adapted levels.Electrophysiological evidence supports the hypothesis that factors in addition to transducin deactivation are involved in regulating the lifetime of light-activated PDE6 during light adaptation of rod photoreceptors (5, 6). Several potential feedback mechanisms for modulating activated PDE6 have been proposed (7-9) but have not been explored in sufficient ...

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Binding of the Delta Subunit to Rod Phosphodiesterase Catalytic Subunits Requires Methylated, Prenylated C-Termini of the Catalytic Subunits

Cited by 56 publications

References 31 publications

Characterization of Human Cone Phosphodiesterase-6 Ectopically Expressed in Xenopus laevis Rods

Characterization of Human Cone Phosphodiesterase-6 Ectopically Expressed in Xenopus laevis Rods

Structural basis of recognition of farnesylated and methylated KRAS4b by PDEδ

The Glutamic Acid-rich Protein-2 (GARP2) Is a High Affinity Rod Photoreceptor Phosphodiesterase (PDE6)-binding Protein That Modulates Its Catalytic Properties

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