Partial reconstitution of photoreceptor cGMP phosphodiesterase characteristics in cGMP phosphodiesterase-5. (17/192)

Photoreceptor cGMP phosphodiesterases (PDE6) are uniquely qualified to serve as effector enzymes in the vertebrate visual transduction cascade. In the dark-adapted photoreceptors, the activity of PDE6 is blocked via tight association with the inhibitory gamma-subunits (Pgamma). The Pgamma block is removed in the light-activated PDE6 by the visual G protein, transducin. Transducin-activated PDE6 exhibits an exceptionally high catalytic rate of cGMP hydrolysis ensuring high signal amplification. To identify the structural determinants for the inhibitory interaction with Pgamma and the remarkable cGMP hydrolytic ability, we sought to reproduce the PDE6 characteristics by mutagenesis of PDE5, a related cyclic GMP-specific, cGMP-binding PDE. PDE5 is insensitive to Pgamma and has a more than 100-fold lower k(cat) for cGMP hydrolysis. Our mutational analysis of chimeric PDE5/PDE6alpha' enzymes revealed that the inhibitory interaction of cone PDE6 catalytic subunits (PDE6alpha') with Pgamma is mediated primarily by three hydrophobic residues at the entry to the catalytic pocket, Met(758), Phe(777), and Phe(781). The maximal catalytic rate of PDE5 was enhanced by at least 10-fold with substitutions of PDE6alpha'-specific glycine residues for the corresponding PDE5 alanine residues, Ala(608) and Ala(612). The Gly residues are adjacent to the highly conserved metal binding motif His-Asn-X-X-His, which is essential for cGMP hydrolysis. Our results suggest that the unique Gly residues allow the PDE6 metal binding site to adopt a more favorable conformation for cGMP hydrolysis.  (+info)

Nrl and Sp nuclear proteins mediate transcription of rod-specific cGMP-phosphodiesterase beta-subunit gene: involvement of multiple response elements. (18/192)

cGMP-phosphodiesterase (PDE) is the key effector in rod photoreceptor signal transduction. Mutations in the gene encoding its catalytic beta-subunit (beta-PDE) cause retinal degenerations leading to blindness. We report that the short -93 to +53 sequence in the upstream region of this gene is sufficient for beta-PDE transcription in both Y79 human retinoblastoma cells and Xenopus embryo heads maintained ex vivo. This sequence also functions as a minimal rod-specific promoter in transgenic Xenopus tadpoles. The Nrl transcription factor binds in vitro to the betaAp1/NRE regulatory element located within this region and transactivates it when overexpressed in nonretinal 293 embryonic kidney cells. We also found a G/C-rich activator element, beta/GC, important for promoter activity in Y79 retinoblastoma cells and Xenopus embryos. Both the ubiquitous Sp1 and the central nervous system-specific Sp4 transcription factors are expressed in retina and interact with this element in vitro. Electrophoretic mobilities of beta/GC-Y79 nuclear protein complexes are altered by antibodies against Sp1 and Sp4. Thus, our results implicate Nrl, Sp1, and Sp4 in transcriptional regulation of the rod-specific minimal beta-PDE promoter. We also conclude that Xenopus laevis is an efficient system for analyzing the human beta-PDE promoter and may be used to study other human retinal genes ex vivo and in vivo.  (+info)

RGS9-G beta 5 substrate selectivity in photoreceptors. Opposing effects of constituent domains yield high affinity of RGS interaction with the G protein-effector complex. (19/192)

RGS proteins regulate the duration of G protein signaling by increasing the rate of GTP hydrolysis on G protein alpha subunits. The complex of RGS9 with type 5 G protein beta subunit (G beta 5) is abundant in photoreceptors, where it stimulates the GTPase activity of transducin. An important functional feature of RGS9-G beta 5 is its ability to activate transducin GTPase much more efficiently after transducin binds to its effector, cGMP phosphodiesterase. Here we show that different domains of RGS9-G beta 5 make opposite contributions toward this selectivity. G beta 5 bound to the G protein gamma subunit-like domain of RGS9 acts to reduce RGS9 affinity for transducin, whereas other structures restore this affinity specifically for the transducin-phosphodiesterase complex. We suggest that this mechanism may serve as a general principle conferring specificity of RGS protein action.  (+info)

Regulation of photoreceptor phosphodiesterase (PDE6) by phosphorylation of its inhibitory gamma subunit re-evaluated. (20/192)

Phosphorylation of the inhibitory gamma subunit (Pgamma) of rod cGMP phosphodiesterase (PDE6) has been reported to turn off visual excitation without the requirement for inactivation of the photoreceptor G-protein transducin. We evaluated the significance of Pgamma phosphorylation for PDE6 regulation by preparing Pgamma stoichiometrically phosphorylated at Thr(22) or at Thr(35). Phosphorylation of Pgamma at either residue caused a minor decrease--not the previously reported increase--in the ability of Pgamma to inhibit catalysis at the active site of purified PDE6 catalytic dimers. Likewise, Pgamma phosphorylation had little effect on its potency to inhibit transducin-activated PDE6 depleted of its endogenous Pgamma subunits. The strength of Pgamma interaction with the regulatory GAF domain of PDE6 was reduced severalfold upon Pgamma phosphorylation at Thr(22) (but not Thr(35)), as judged by allosteric changes in cGMP binding to these noncatalytic sites on the enzyme (Mou, H., and Cote, R. H. (2001) J. Biol. Chem. 276, 27527-27534). In contrast, the effects of Pgamma phosphorylation on its interactions with activated transducin were much more pronounced. Phosphorylation of Pgamma at either Thr(22) or Thr(35) greatly diminished its ability to bind activated transducin, consistent with earlier work. In situ phosphorylation of Pgamma by endogenous rod outer segment kinases was enhanced severalfold upon light activation, but only approximately 10% of the endogenous Pgamma was phosphorylated. This is attributed to Pgamma being a poor substrate for protein kinases when associated with the PDE6 holoenzyme. We conclude that, contrary to previous reports, Pgamma phosphorylation at either Thr(22) or Thr(35) modestly weakens its direct interactions with PDE6. However, Pgamma phosphorylation subsequent to its dissociation from PDE6 is likely to abolish its binding to activated transducin and may serve to make phosphorylated Pgamma available to regulate other signal transduction pathways (e.g. mitogen-activated protein kinase; Wan, K. F., Sambi, B. S., Frame, M., Tate, R., and Pyne, N. J. (2001) J. Biol. Chem. 276, 37802-37808) in photoreceptor cells.  (+info)

The rod cGMP-phosphodiesterase beta-subunit promoter is a specific target for Sp4 and is not activated by other Sp proteins or CRX. (21/192)

The beta-subunit of cGMP-phosphodiesterase (beta-PDE) is a key protein in phototransduction expressed exclusively in rod photoreceptors. It is necessary for visual function and for structural integrity of the retina. beta-PDE promoter deletions showed that the -45/-23 region containing a consensus Crx-response element (CRE) was necessary for low level transcriptional activity. Overexpressed Crx modestly transactivated this promoter in 293 human embryonic kidney cells; however, mutation of CRE had no significant effect on transcription either in transfected Y79 retinoblastoma cells or Xenopus embryonic heads. Thus, Crx is unlikely to be a critical beta-PDE transcriptional regulator in vivo. Interestingly, although the beta/GC element (-59/-49) binds multiple Sp transcription factors in vitro, only Sp4, but not Sp1 or Sp3, significantly enhanced beta-PDE promoter activity. Thus, the Sp4-mediated differential activation of the beta-PDE transcription defines the first specific Sp4 target gene reported to date and implies the importance of Sp4 for retinal function. Further extensive mutagenesis of the beta-PDE upstream sequences showed no additional regulatory elements. Although this promoter lacks a canonical TATA box or Inr element, it has the (T/A)-rich beta/TA sequence located within the -45/-23 region. We found that it binds purified TBP and TFIIB in gel mobility shift assays with cooperative enhancement of binding affinity.  (+info)

The complex of Arl2-GTP and PDE delta: from structure to function. (22/192)

Arf-like (Arl) proteins are close relatives of the Arf regulators of vesicular transport, but their function is unknown. Here, we present the crystal structure of full-length Arl2-GTP in complex with its effector PDE delta solved in two crystal forms (Protein Data Bank codes 1KSG, 1KSH and 1KSJ). Arl2 shows a dramatic conformational change from the GDP-bound form, which suggests that it is reversibly membrane associated. PDE delta is structurally closely related to RhoGDI and contains a deep empty hydrophobic pocket. Further experiments show that H-Ras, Rheb, Rho6 and G alpha(i1) interact with PDE delta and that, at least for H-Ras, the intact C-terminus is required. We suggest PDE delta to be a specific soluble transport factor for certain prenylated proteins and Arl2-GTP a regulator of PDE delta-mediated transport.  (+info)

Absence of photoreceptor rescue with D-cis-diltiazem in the rd mouse. (23/192)

PURPOSE: Because of a previous report suggesting that D-cis-diltiazem slows retinal degeneration in rd mice, this study was undertaken to examine the effect of D-cis-diltiazem on photoreceptor structure and function in this line of mice. METHODS: Mice were randomly assigned to daily intraperitoneal injections of D-cis-diltiazem or saline between postnatal days 9 and 24. On postnatal day 26 or 27, retinal function was assessed by recording dark-adapted bright-flash ERGs in all animals. Retinal morphology was examined in fixed sections and in immunolabeled frozen sections. Examiners were masked to the treatment group assignment. RESULTS: On postnatal days 26 and 27, diltiazem- and saline-treated mice had only one row of remaining photoreceptor cells throughout most of the central retina. Cone cells in the periphery had remnants of inner segments. Total cell counts and separate counts of rod and cone photoreceptor cells by immunostaining were similar in the diltiazem- versus saline-treated mice. Both groups of mice had, on average, comparable subnormal ERG amplitudes. CONCLUSIONS: D-cis-Diltiazem had no detectable effect on preservation of photoreceptor structure and function in rd mice.  (+info)

Cone neurite sprouting: an early onset abnormality of the cone photoreceptors in the retinal degeneration mouse. (24/192)

PURPOSE: Mutations in many rod genes can cause inherited blinding neurodegeneration in the retina characterized by sequential death of rod and cone photoreceptors. This study was to examine the morphological changes of the cone photoreceptors in retinal degeneration (rd1) mice caused by rod-specific cGMP phosphodiesterase beta-subunit gene mutation and to gain insights into the early cellular events underlying the secondary cone death. METHODS: Transgenic mice that have their living cones labeled by the green fluorescent protein (GFP) transgene and carry the homozygous rd1 mutation were generated, and identified by PCR analysis of the mouse tail DNA and PCR coupled Dde I digestion. The morphology of cone cells in live and fixed retinas from developing and adult mice was examined with fluorescence and scanning laser confocal microscopy. Some fixed mouse retinas were also examined by immunocytochemical staining. Volume images from the confocal three-dimensional (3D) data sets were processed with IMARIS software for 3D view of the detailed cone cell morphology. RESULTS: The cone photoreceptors in the rd1 retinas exhibited a novel process of neurite sprouting, in addition to the general pathological changes of cone degeneration such as shortening and loss of cone outer and inner segments, and loss and death of the cones. The cones gave rise to prominent neurite outgrowth from their axons and synaptic pedicles as well. Most neurites had beaded varicosities along their length and some terminated as bulbous structures. Some cone pedicles showed abnormally elongating and branching processes. The degenerating cones were disorganized, and migrated into the inner nuclear layer. Some cone neurites extended horizontally and appeared to contact the rod bipolar cells, while others projected into the inner plexiform layer. The aberrant cone sprouting started from P8 when rod degeneration generally began, and became evident by P10. In contrast, this abnormal cone neurite sprouting was not observed in the examined control mice that did not carry the rd1 mutation. Double-labeling with cone cell-specific peanut agglutinin confirmed that the fluorescent cells expressing the GFP in the rd1 retinas were indeed the cone photoreceptors. CONCLUSIONS: Cone photoreceptors in the rd1 mice underwent a remarkable process of neurite sprouting that appeared to start before the onset of cone cell death and persisted throughout the course of cone degeneration. This novel process of cone neurite sprouting may be a part of the early cellular events leading to the cone photoreceptor death in retinal degeneration of the rd1 mice.  (+info)