Retinal guanylate cyclases 1 and 2 (GC1 and GC2) are responsible for synthesis of cyclic GMP in rods and cones, but their individual contributions to phototransduction are unknown. We report here that the deletion of both GC1 and GC2 rendered rod and cone photoreceptors nonfunctional and unstable. In the rod outer segments of GC double knock-out mice, guanylate cyclase-activating proteins 1 and 2, and cyclic GMP phosphodiesterase were undetectable, although rhodopsin and transducin ␣-subunit were mostly unaffected. Outer segment membranes of GC1 ؊/؊ and GC double knock-out cones were destabilized and devoid of cone transducin (␣-and ␥-subunits), cone phosphodiesterase, and G protein-coupled receptor kinase 1, whereas cone pigments were present at reduced levels. Real time reverse transcription-PCR analyses demonstrated normal RNA transcript levels for the downregulated proteins, indicating that down-regulation is posttranslational. We interpret these results to demonstrate an intrinsic requirement of GCs for stability and/or transport of a set of membrane-associated phototransduction proteins.

Mouse photoreceptor function and survival critically depend on Ca2+-regulated retinal membrane guanylyl cyclase (RetGC), comprised of two isozymes, RetGC1 and RetGC2. We characterized the content, catalytic constants and regulation of native RetGC1 and RetGC2 isozymes using mice lacking guanylyl cyclase activating proteins GCAP1 and GCAP2 and deficient for either GUCY2F or GUCY2E genes, respectively. We found that the characteristics of both native RetGC isozymes were considerably different from other reported estimates made for mammalian RetGCs: the content of RetGC1 per mouse rod outer segments (ROS) was at least 3-fold lower, the molar ratio (RetGC2:RetGC1) 6-fold higher, and the catalytic constants of both GCAP-activated isozymes between 12 and 19-fold higher than previously measured in bovine ROS. The native RetGC isozymes had different basal activity and were accelerated 5 to 28-fold at physiological concentrations of GCAPs. RetGC2 alone was capable of contributing as much as 135-165 μM cGMP s−1 or almost 23-28% to the maximal cGMP synthesis rate in mouse ROS. At the maximal level of activation by GCAP, this isozyme alone could provide a significantly high rate of cGMP synthesis compared to what is expected for normal recovery of a mouse rod, and this can help explain some of the unresolved paradoxes of rod physiology. GCAP-activated native RetGC1 and RetGC2 were less sensitive to inhibition by Ca2+ in the presence of GCAP1 (EC50Ca ~132-139 nM) than GCAP2 (EC50Ca ~50-59 nM), thus arguing that Ca2+ sensor properties of GCAP in a functional RetGC/GCAP complex are defined not by a particular target isozyme but the intrinsic properties of GCAPs themselves.

Lecithin retinol acyl transferase (LRAT) and retinal pigment epithelium protein 65 (RPE65) are key enzymes of the retinoid cycle. In Lrat Ϫ/Ϫ and Rpe65 Ϫ/Ϫ mice, models of human Leber congenital amaurosis, the retinoid cycle is disrupted and 11-cis-retinal, the chromophore of visual pigments, is not produced. The Lrat Ϫ/Ϫ and Rpe65 Ϫ/Ϫ retina phenotype presents with rapid sectorial cone degeneration, and the visual pigments, S-opsin and M/L-opsin, fail to traffic to cone outer segments appropriately. In contrast, rod opsin traffics normally in mutant rods. Concomitantly, guanylate cyclase 1, cone T␣-subunit, cone phosphodiesterase 6␣Ј (PDE6␣Ј), and GRK1 (G-protein-coupled receptor kinase 1; opsin kinase) are not transported to Lrat Ϫ/Ϫ and Rpe65 Ϫ/Ϫ cone outer segments. Aberrant localization of these membrane-associated proteins was evident at postnatal day 15, before the onset of ventral and central cone degeneration. Protein levels of cone T␣ and cone PDE6␣Ј were reduced, whereas their transcript levels were unchanged, suggesting posttranslational degradation. In an Rpe65 Ϫ/Ϫ Rho Ϫ/Ϫ double knock-out model, trafficking of cone pigments and membrane-associated cone phototransduction polypeptides to the outer segments proceeded normally after 11-cis-retinal administration. These results suggest that ventral and central cone opsins must be regenerated with 11-cis-retinal to permit transport to the outer segments. Furthermore, the presence of 11-cis-retinal is essential for proper transport of several membrane-associated cone phototransduction polypeptides in these cones.

We discuss putative mechanisms of membrane protein transport in photoreceptors based on Pde6d and Gucy2e/Gucy2f knockout mice. Knockout of the Pde6d gene encoding PrBP/delta, a prenyl binding protein present in the retina at relatively high levels, was shown to impair transport of G-protein coupled receptor kinase 1 (GRK1) and cone phosphodiesterase alpha' subunit (PDE6alpha') to the rod and cone outer segments. Other prenylated proteins are minimally affected, suggesting some specificity of interaction. Knockout of the Gucy2e gene encoding guanylate cyclase 1 (GC1) disrupted transport of G-protein coupled receptor kinase 1 (GRK1), cone PDE6alpha', cone transducin alpha and gamma subunits (cTalpha and cTgamma) to the cone outer segments, while a GC1/GC2 double knockout prevented transport of rod PDE6, but not transducin, GRK1, or rhodopsin, to the rod outer segments. These knockout phenotypes suggest that PrBP/delta functions in extracting prenylated proteins from the endoplasmic reticulum (ER) where they dock after prenylation, and that GC-bearing membranes may co-transport peripheral membrane proteins in vesicles. We conclude that distinct pathways have evolved in rods and cones for transport of integral and peripherally membrane-associated proteins.

Mutations in GUCY2D are associated with recessive Leber congenital amaurosis-1 (LCA1). GUCY2D encodes photoreceptor-specific, retinal guanylate cyclase-1 (RetGC1). Reports of retinal degeneration in LCA1 are conflicting; some describe no obvious degeneration and others report loss of both rods and cones. Proof of concept studies in models representing the spectrum of phenotypes is warranted. We have previously demonstrated adeno-associated virus (AAV)-mediated RetGC1 is therapeutic in GC1ko mice, a model exhibiting loss of cones only. The purpose of this study was to characterize AAV-mediated gene therapy in the RetGC1/RetGC2 double knockout (GCdko) mouse, a model lacking rod and cone function and exhibiting progressive loss of both photoreceptor subclasses. Use of this model also allowed for the evaluation of the functional efficiency of transgenic RetGC1 isozyme. Subretinal delivery of AAV8(Y733F) vector containing the human rhodopsin kinase (hGRK1) promoter driving murine Gucy2e was performed in GCdko mice at various postnatal time points. Treatment resulted in restoration of rod and cone function at all treatment ages and preservation of retinal structure in GCdko mice treated as late as 7 weeks of age. Functional gains and structural preservation were stable for at least 1 year. Treatment also conferred cortical-and subcortical-based visually-guided behavior. Functional efficiency of transgenic RetGC1 was indistinguishable from that of endogenous isozyme in congenic wild-type (WT) mice. This study clearly demonstrates AAV-mediated RetGC1 expression restores function to and preserves structure of rod and cone photoreceptors in a degenerative model of retinal guanylate cyclase deficiency, further supporting development of an AAV-based vector for treatment of LCA1.

Targeted deletion of membrane guanylyl cyclases (GCs) has yielded new information concerning their function. Here, we summarize briefly recent results of laboratory generated non-photoreceptor GC knockouts characterized by complex phenotypes affecting the vasculature, heart, brain, kidney and other tissues. The main emphasis of the review, however, addresses the two GCs expressed in retinal photoreceptors, termed GC-E and GC-F. Naturally occurring GC-E (GUCY2D) null alleles in human and chicken are associated with an early onset blinding disorder, termed 'Leber Congenital Amaurosis type 1' (LCA-1), characterized by extinguished scotopic and photopic ERGs, and retina degeneration. In mouse, a GC-E null genotype produces a recessive cone dystrophy, while rods remain functional. Rod function is supported by the presence of GC-F (Gucy2f), a close relative of GC-E. Deletion of Gucy2f has very little effect on rod and cone physiology and survival. However, a GC-E/GC-F double knockout (GCdko) phenotypically resembles human LCA-1 with extinguished ERGs and rod/cone degneration. In GCdko rods, PDE6 and GCAPs are absent in outer segments. In contrast, GC-E -/-cones lack proteins of the entire phototransduction cascade. These results suggest that GC-E may participate in transport of peripheral membrane proteins from the endoplasmic reticulum (ER) to the outer segments. KeywordsMembrane guanylate cyclase; targeted deletions; rod and cone photoreceptors; photoreceptor membrane protein transport Soluble and membrane guanylate cyclasesGuanylate cyclases (GCs) synthesize cyclic GMP (cGMP), a secondary messenger in many pathways, in response to diverse signals, such as nitric oxide (NO), peptide ligands (hormones), and fluxes in intracellular Ca 2+ mediated by Ca 2+ -binding proteins ([Ca 2+ ] i ) [1,2]. These signals use specific guanylate cyclase receptors and cofactors to initiate the conversion of cytosolic GTP to cGMP. Intracellular cGMP regulates cellular physiology by activating protein kinases, directly gating specific ion channels, or altering intracellular cyclic nucleotide concentrations through regulation of phosphodiesterases (PDEs). Guanylate cyclases are classified as either soluble or membrane (particulate), based on both their cellular distribution and structural domains [2,3]. Soluble guanylate cyclases are heterodimeric proteins consisting *Corresponding author. John A. Moran Eye Center, 65 Mario Capecchi Dr., Salt Lake City UT 84132. wbaehr@hsc.utah.edu. Phone 801-585-6643. Membrane GC isozymes (GC-A to GC-G, Table 1) exhibit highly conserved domain structures, an extracellular domain (ECD) which comprises a large part of the N-terminal part of the molecule, a single transmembrane (TM) region, an intracellular protein kinase-like homology domain (KHD), a dimerization (hinge) domain (DD), and a C-terminal catalytic domain (CAT). NIH Public AccessBased on their ligand specificities, membrane GCs have been subdivided into natriuretic peptide receptors (GC-A, GC-B), intestinal peptide-binding receptors (...

The shaker rat is an X-linked recessive spontaneous model of progressive Purkinje cell (PC) degeneration exhibiting a shaking ataxia and wide stance. Generation of Wistar Furth (WF)/Brown Norwegian (BN) F1 hybrids and genetic mapping of F2 sib-sib offspring using polymorphic markers narrowed the candidate gene region to 26 Mbp denoted by the last recombinant genetic marker DXRat21 at 133 Mbp to qter (the end of the long arm). In the WF background, the shaker mutation has complete penetrance, results in a stereotypic phenotype and there is a narrow window for age of disease onset; by contrast, the F2 hybrid phenotype was more varied, with a later age of onset and likely non-penetrance of the mutation. By deep RNA-sequencing, five variants were found in the candidate region; four were novel without known annotation. One of the variants caused an arginine (R) to cysteine (C) change at codon 35 of the ATPase, Ca2+ transporting, plasma membrane 3 (Atp2b3) gene encoding PMCA3 that has high expression in the cerebellum. The variant was well supported by hundreds of overlapping reads, and was found in 100% of all affected replicas and 0% of the wild-type (WT) replicas. The mutation segregated with disease in all affected animals and the amino acid change was found in an evolutionarily conserved region of PMCA3. Despite strong genetic evidence for pathogenicity, in vitro analyses of PMCA3R35C function did not show any differences to WT PMCA3. Because Atp2b3 mutation leads to congenital ataxia in humans, the identified Atp2b3 missense change in the shaker rat presents a good candidate for the shaker rat phenotype based on genetic criteria, but cannot yet be considered a definite pathogenic variant owing to lack of functional changes.

Inactivation of the p53 tumor suppressor by mutation or overexpression of negative regulators occurs frequently in cancer. Since p53 plays a key role in regulating proliferation or apoptosis in response to DNA damaging chemotherapies, strategies aimed at reactivating p53 are increasingly being sought. Strategies to reactivate wild-type p53 include the use of small molecules capable of releasing wild-type p53 from key, cellular negative regulators, such as Hdm2 and HdmX. Derivatives of the Hdm2 antagonist Nutlin-3 are in clinical trials. However, Nutlin-3 specifically disrupts Hdm2-p53, leaving tumors harboring high levels of HdmX resistant to Nutlin-3 treatment. Here we identify CTX1, a novel small molecule that overcomes HdmX-mediated p53 repression. CTX1 binds directly to HdmX to prevent p53-HdmX complex formation, resulting in the rapidly induction of p53 in a DNA damage-independent manner. Treatment of a panel of cancer cells with CTX1 induced apoptosis or suppressed proliferation and importantly, CTX1 demonstrates promising activity as a single agent in a mouse model of circulating primary human leukemia. CTX1 is a small molecule HdmX inhibitor that demonstrates promise as a cancer therapeutic candidate.