In vitro interaction of tubulin with the photoreceptor cGMP phosphodiesterase gamma-subunit.
ABSTRACT: The alpha and beta tubulins compose the microtubule cytoskeleton which is involved in many cellular processes such as vesicular transport. The photoreceptor cells in the retina are neurons specialized for phototransduction. Here we report a novel interaction between tubulin and the photoreceptor cGMP phosphodiesterase (PDE6) gamma subunit (PDE gamma). The specificity and molecular details of the PDE gamma:tubulin interaction were analyzed through the experiments of pull down, microtubule co-sedimentation, and NMR spectroscopy. The tubulin-interacting site was identified to be in the PDE gamma C-terminal I67-G85 region, and the interaction interface appeared to be distinct from those with the other PDE gamma targets in phototransduction. We also observed that PDE gamma interacted with tubulin in a GTP-dependent manner. Our findings offer implications for non-phototransduction role(s) of PDE gamma in the photoreceptor neurons.
Project description:The inhibitory subunit (PDE gamma) of the cGMP phosphodiesterase (PDE alpha beta gamma 2) in rod outer segments (ROS) realizes its regulatory role in phototransduction by inhibition of PDE alpha beta catalytic activity. The photoreceptor G-protein, transducin, serves as a transducer from the receptor (rhodopsin) to the effector (PDE) and eliminates the inhibitory effect of PDE gamma by direct interaction with PDE gamma. Our previous study [Udovichenko, Cunnick, Gonzalez and Takemoto (1994) J: Biol. Chem. 269, 9850-9856] has shown that PDE gamma is a substrate for protein kinase C (PKC) from ROS and that phosphorylation by PKC increases the ability of PDE gamma to inhibit PDE alpha beta catalytic activity. Here we report that transducin is less effective in activation of PDE alpha beta (gamma p)2 (a complex of PDE alpha beta with phosphorylated PDE gamma, PDE gamma p) than PDE alpha beta gamma 2. PDE gamma p also increases the rate constant of GTP hydrolysis of transducin (from 0.16 S-1 for non-phosphorylated PDE gamma to 0.21 s-1 for PDE gamma p). These data suggest that phosphorylation of the inhibitory subunit of PDE by PKC may regulate the visual transduction cascade by decreasing the photoresponse.
Project description:The interaction of phosphodiesterase 6 (PDE6) with its inhibitory Pgamma-subunits (Pgamma) is unparalleled among PDE families and is central to vertebrate phototransduction. The C-terminus of Pgamma occludes the active site of PDE6, thereby preventing hydrolysis of cGMP. In this study, we examine the determinants of this critical interaction using structure-based loss-of-function mutagenesis of a chimeric PDE5/PDE6 catalytic domain and gain-of-function mutagenesis of the PDE5 catalytic domain. This analysis revealed the key role of PDE6-specific residues within the catalytic domain M-loop-alpha-helix 15 region and suggested an important contribution of the H-loop-M-loop interface to the PDE6 inhibition by the Pgamma C-terminus. Identification of the determinants for the PDE6-Pgamma interaction offers insights into the evolution of the visual effector enzyme.
Project description:The vertebrate rod photoreceptor undergoes daily growth and shedding to renew the rod outer segment (ROS), a modified cilium that contains the phototransduction machinery. It has been demonstrated that ROS shedding is regulated by the light-dark cycle; however, we do not yet have a satisfactory understanding of the molecular mechanisms that underlie this regulation. Given that phototransduction relies on the hydrolysis of cGMP via phosphodiesterase 6 (PDE6), we examined ROS growth and shedding in zebrafish treated with cGMP-specific PDE inhibitors.We used transgenic zebrafish that express an inducible, transmembrane-bound mCherry protein, which forms a stripe in the ROS following a heat shock pulse and serves as a marker of ROS renewal. Zebrafish were reared in constant darkness or treated with PDE inhibitors following heat shock. Measurements of growth and shedding were analyzed in confocal z-stacks collected from treated retinas.As in dark-reared zebrafish, shedding was reduced in larvae and adults treated with the PDE5/6 inhibitors sildenafil and vardenafil but not with the PDE5 inhibitor tadalafil. In addition, vardenafil noticeably affected rod inner segment morphology. The inhibitory effect of sildenafil on shedding was reversible with drug removal. Finally, cones were more sensitive than rods to the toxic effects of sildenafil and vardenafil.We show that pharmacologic inhibition of PDE6 mimics the inhibition of shedding by prolonged constant darkness. The data show that the influence of the light-dark cycle on ROS renewal is regulated, in part, by initiating the shedding process through activation of the phototransduction machinery.
Project description:BACKGROUND:Phosphodiesterase 6 (PDE6) is a protein complex that hydrolyses cGMP and acts as the effector of the vertebrate phototransduction cascade. The PDE6 holoenzyme consists of catalytic and inhibitory subunits belonging to two unrelated gene families. Rods and cones express distinct genes from both families: PDE6A and PDE6B code for the catalytic and PDE6G the inhibitory subunits in rods while PDE6C codes for the catalytic and PDE6H the inhibitory subunits in cones. We performed phylogenetic and comparative synteny analyses for both gene families in genomes from a broad range of animals. Furthermore, gene expression was investigated in zebrafish. RESULTS:We found that both gene families expanded from one to three members in the two rounds of genome doubling (2R) that occurred at the base of vertebrate evolution. The PDE6 inhibitory subunit gene family appears to be unique to vertebrates and expanded further after the teleost-specific genome doubling (3R). We also describe a new family member that originated in 2R and has been lost in amniotes, which we have named pde6i. Zebrafish has retained two additional copies of the PDE6 inhibitory subunit genes after 3R that are highly conserved, have high amino acid sequence identity, are coexpressed in the same photoreceptor type as their amniote orthologs and, interestingly, show strikingly different daily oscillation in gene expression levels. CONCLUSIONS:Together, these data suggest specialisation related to the adaptation to different light intensities during the day-night cycle, most likely maintaining the regulatory function of the PDE inhibitory subunits in the phototransduction cascade.
Project description:Rod and cone photoreceptor neurons utilize discrete PDE6 enzymes that are crucial for phototransduction. Rod PDE6 is composed of heterodimeric catalytic subunits (??), while the catalytic core of cone PDE6 (?') is a homodimer. It is not known if variations between PDE6 subunits preclude rod PDE6 catalytic subunits from coupling to the cone phototransduction pathway. To study this issue, we generated a cone-dominated mouse model lacking cone PDE6 (Nrl(-/-) cpfl1). In this animal model, using several independent experimental approaches, we demonstrated the expression of rod PDE6 (??) and the absence of cone PDE6 (?') catalytic subunits. The rod PDE6 enzyme expressed in cone cells is active and contributes to the hydrolysis of cGMP in response to light. In addition, rod PDE6 expressed in cone cells couples to the light signaling pathway to produce S-cone responses. However, S-cone responses and light-dependent cGMP hydrolysis were eliminated when the ?-subunit of rod PDE6 was removed (Nrl(-/-) cpfl1 rd). We conclude that either rod or cone PDE6 can effectively couple to the cone phototransduction pathway to mediate visual signaling. Interestingly, we also found that functional PDE6 is required for trafficking of M-opsin to cone outer segments.
Project description:Membrane-bound phosphodiesterase 6 (PDE6) plays an important role in visual signal transduction by regulating cGMP levels in rod photoreceptor cells. Our understanding of PDE6 catalysis and structure suffers from inadequate characterization of the ? and ? subunit catalytic core, interactions of the core with two intrinsically disordered, proteolysis-prone inhibitory PDE? (P?) subunits, and binding of two types of isoprenyl-binding protein ?, called PrBP/?, to the isoprenylated C-termini of the catalytic core. Structural studies of native PDE6 have been also been hampered by the lack of a heterologous expression system for the holoenzyme. In this work, we purified PDE6 in the presence of PrBP/? and screened for additives and detergents that selectively suppress PDE6 basal activity while sparing that of the trypsin-activated enzyme. Some detergents removed PrBP/? from the PDE complex, separating it from the holoenzyme after PDE6 purification. Additionally, selected detergents also significantly reduced the level of dissociation of PDE6 subunits, increasing their homogeneity and stabilizing the holoenzyme by substituting for its native membrane environment.
Project description:The retinal phosphodiesterase (PDE6) inhibitory gamma-subunit (PDEgamma) plays a central role in vertebrate phototransduction through alternate interactions with the catalytic alphabeta-subunits of PDE6 and the alpha-subunit of transducin (alpha(t)). Detailed structural analysis of PDEgamma has been hampered by its intrinsic disorder. We present here the NMR solution structure of PDEgamma, which reveals a loose fold with transient structural features resembling those seen previously in the x-ray structure of PDEgamma(46-87) when bound to alpha(t) in the transition-state complex. NMR mapping of the interaction between PDEgamma(46-87) and the chimeric PDE5/6 catalytic domain confirmed that C-terminal residues 74-87 of PDEgamma are involved in the association and demonstrated that its W70 indole group, which is critical for subsequent binding to alpha(t), is left free at this stage. These results indicate that the interaction between PDEgamma and alpha(t) during the phototransduction cascade involves the selection of preconfigured transient conformations.
Project description:The cGMP phosphodiesterase of rod photoreceptor cells, PDE6, is the key effector enzyme in phototransduction. Two large catalytic subunits, PDE6? and -?, each contain one catalytic domain and two non-catalytic GAF domains, whereas two small inhibitory PDE6? subunits allow tight regulation by the G protein transducin. The structure of holo-PDE6 in complex with the ROS-1 antibody Fab fragment was determined by cryo-electron microscopy. The ?11 Å map revealed previously unseen features of PDE6, and each domain was readily fit with high resolution structures. A structure of PDE6 in complex with prenyl-binding protein (PrBP/?) indicated the location of the PDE6 C-terminal prenylations. Reconstructions of complexes with Fab fragments bound to N or C termini of PDE6? revealed that PDE6? stretches from the catalytic domain at one end of the holoenzyme to the GAF-A domain at the other. Removal of PDE6? caused dramatic structural rearrangements, which were reversed upon its restoration.
Project description:This lecture details the elucidation of cGMP phosphodiesterase (PDE?), discovered 25 years ago by Joe Beavo at the University of Washington. PDE?, once identified as a fourth PDE6 subunit, is now regarded as a promiscuous prenyl-binding protein and important chaperone of prenylated small G proteins of the Ras superfamily and prenylated proteins of phototransduction. Alfred Wittinghofer's group in Germany showed that PDE? forms an immunoglobulin-like ?-sandwich fold that is closely related in structure to other lipid-binding proteins, for example, Uncoordinated 119 (UNC119) and RhoGDI. His group cocrystallized PDE? with ARL (Arf-like) 2(GTP), and later with farnesylated Rheb (ras homolog expressed in brain). PDE? specifically accommodates farnesyl and geranylgeranyl moieties in the absence of bound protein. Germline deletion of the Pde6d gene encoding PDE? impeded transport of rhodopsin kinase (GRK1) and PDE6 to outer segments, causing slowly progressing, recessive retinitis pigmentosa. A rare PDE6D null allele in human patients, discovered by Tania Attié-Bitach in France, specifically impeded trafficking of farnesylated phosphatidylinositol 3,4,5-trisphosphate (PIP3) 5-phosphatase (INPP5E) to cilia, causing severe syndromic ciliopathy (Joubert syndrome). Binding of cargo to PDE? is controlled by Arf-like proteins, ARL2 and ARL3, charged with guanosine-5'-triphosphate (GTP). Arf-like proteins 2 and 3 are unprenylated small GTPases that serve as cargo displacement factors. The lifetime of ARL3(GTP) is controlled by its GTPase-activating protein, retinitis pigmentosa protein 2 (RP2), which accelerates GTPase activity up to 90,000-fold. RP2 null alleles in human patients are associated with severe X-linked retinitis pigmentosa (XLRP). Germline deletion of RP2 in mouse, however, causes only a mild form of XLRP. Absence of RP2 prolongs the activity of ARL3(GTP) that, in turn, impedes PDE6?-cargo interactions and trafficking of prenylated protein to the outer segments. Hyperactive ARL3(GTP), acting as a hyperactive cargo displacement factor, is predicted to be key in the pathobiology of RP2-XLRP.
Project description:Light-activated cyclic GMP-phosphodiesterase (PDE) is the key effector enzyme of vertebrate photoreceptor cells which regulates the level of the internal transmitter cyclic GMP. PDE consists of catalytic P alpha and P beta subunits, and two copies of inhibitory P gamma subunit. The two P gamma subunits block the enzyme's activity in the dark and are removed by the alpha-subunit of transducin (alpha 1) upon light-activation of photoreceptor cells. Here we have examined the role of various regions of P gamma, the N-terminal, the central cationic and the C-terminal regions, in interaction with the catalytic subunits of PDE. N-Terminal truncation of P gamma (12-87-P gamma) did not change the potency of PDE inhibition, and thus we conclude that the P gamma N-terminal region is not critical for P gamma-P alpha beta interaction. The central region, 24-46-P gamma, participates in interaction with the catalytic P alpha beta subunits. A synthetic peptide corresponding to this site inhibited approximately 50% of trypsin-activated PDE (tPDE) (Ki approximately 15 microM) and competed with P gamma for inhibition of tPDE. We demonstrated, by using h.p.l.c. gel filtration, that 125I-Tyr-24-46-P gamma peptide bound with high affinity to tPDE, but not to P alpha beta gamma 2. The C-terminal region of 46-87-P gamma was found to be the major region involved in inhibition of PDE. It fully inhibited tPDE with a Ki of approximately 0.8 microM. It also bound to tPDE, but not P alpha beta gamma 2, in h.p.l.c. gel-filtration experiments. In addition, P gamma was cross-linked by p-phenylenedimaleimide to both P alpha and P beta, as was shown by using subunit-specific anti-P alpha, -P beta and -P gamma antibodies. Cys68 of P gamma, which presumably participates in cross-linking, is located near the P gamma C-terminus. These data provide evidence for two regions of P gamma that interact with, and inhibit, P alpha beta. The central region, 24-46 P gamma, is important in binding, but inhibits PDE only weakly, whereas the C-terminal region is most important for PDE inhibition. These results help to explain the well-known fact that P gamma trypsin-activation and C-terminal truncation both lead to PDE activation. Furthermore, our findings on the mechanism of PDE inhibition of P gamma are relevant for understanding the mechanism of PDE activation by transducin.