Binding of guanylyl cyclase activating protein 1 (GCAP1) to retinal guanylyl cyclase (RetGC1). The role of individual EF-hands

Guanylyl cyclase activating protein 1 (GCAP1), after substitution of Ca(2+) by Mg(2+) in its EF-hands, stimulates photoreceptor guanylyl cyclase, RetGC1, in response to light. We inactivated metal binding in individual EF-hands of GCAP1 tagged with green fluorescent protein to assess their role in GCAP1 binding to RetGC1 in co-transfected HEK293 cells. When expressed alone, GCAP1 was uniformly distributed throughout the cytoplasm and the nuclei of the cells, but when co-expressed with either fluorescently tagged or non-tagged RetGC1, it co-localized with the cyclase in the membranes. The co-localization did not occur when the C-terminal portion of RetGC1, containing its regulatory and catalytic domains, was removed. Mutations that preserved Mg(2+) binding in all three metal-binding EF-hands did not affect GCAP1 association with the cyclase in live cells. Locking EF-hand 4 in its apo-conformation, incapable of binding either Ca(2+) or Mg(2+), had no effect on GCAP1 association with the cyclase. In contrast to EF-hand 4, inactivation of EF-hand 3 reduced the efficiency of the co-localization, and inactivation of EF-hand 2 drastically suppressed GCAP1 binding to the cyclase. These results directly demonstrate that metal binding in EF-hand 2 is crucial for GCAP1 attachment to RetGC1, and that in EF-hand 3 it is less critical, although it enhances the efficiency of the GCAP1 docking on the target enzyme. Metal binding in EF-hand 4 has no role in the primary attachment of GCAP1 to the cyclase, and it only triggers the activator-to-inhibitor functional switch in GCAP1.     

Gene replacement therapy restores RCBTB1 expression and cilium length in patient-derived retinal pigment epithelium

Biallelic mutations in the RCBTB1 gene cause retinal dystrophy. Here, we characterized the effects of RCBTB1 gene deficiency in retinal pigment epithelial (RPE) cells derived from a patient with RCBTB1-associated retinopathy and restored RCBTB1 expression in these cells using adeno-associated viral (AAV) vectors. Induced pluripotent stem cells derived from a patient with compound heterozygous RCBTB1 mutations (c.170delG and c.707delA) and healthy control subjects were differentiated into RPE cells. RPE cells were treated with AAV vectors carrying a RCBTB1 transgene. Patient-derived RPE cells showed reduced expression of RCBTB1. Expression of NFE2L2 showed a non-significant reduction in patient RPE cells compared with controls, while expression of its target genes (RXRA, IDH1 and SLC25A25) was significantly reduced. Trans-epithelial electrical resistance, surface microvillus densities and primary cilium lengths were reduced in patient-derived RPE cells, compared with controls. Treatment of patient RPE with AAV vectors significantly increased RCBTB1, NFE2L2 and RXRA expression and cilium lengths. Our study provides the first report examining the phenotype of RPE cells derived from a patient with RCBTB1-associated retinopathy. Furthermore, treatment of patient-derived RPE with AAV-RCBTB1 vectors corrected deficits in gene expression and RPE ultrastructure, supporting the use of gene replacement therapy for treating this inherited retinal disease.     

Evaluating the Role of Retinal Membrane Guanylyl Cyclase 1 (RetGC1) Domains in Binding Guanylyl Cyclase-activating Proteins (GCAPs)

Retinal membrane guanylyl cyclase 1 (RetGC1) regulated by guanylyl cyclase-activating proteins (GCAPs) controls photoreceptor recovery and when mutated causes blinding disorders. We evaluated the principal models of how GCAP1 and GCAP2 bind RetGC1: through a shared docking interface versus independent binding sites formed by distant portions of the cyclase intracellular domain. At near-saturating concentrations, GCAP1 and GCAP2 activated RetGC1 from HEK293 cells and RetGC2^−/−GCAPs1,2^−/− mouse retinas in a non-additive fashion. The M26R GCAP1, which binds but does not activate RetGC1, suppressed activation of recombinant and native RetGC1 by competing with both GCAP1 and GCAP2. Untagged GCAP1 displaced both GCAP1-GFP and GCAP2-GFP from the complex with RetGC1 in HEK293 cells. The intracellular segment of a natriuretic peptide receptor A guanylyl cyclase failed to bind GCAPs, but replacing its kinase homology and dimerization domains with those from RetGC1 restored GCAP1 and GCAP2 binding by the hybrid cyclase and its GCAP-dependent regulation. Deletion of the Tyr¹⁰¹⁶–Ser¹¹⁰³ fragment in RetGC1 did not block GCAP2 binding to the cyclase. In contrast, substitutions in the kinase homology domain, W708R and I734T, linked to Leber congenital amaurosis prevented binding of both GCAP1-GFP and GCAP2-GFP. Our results demonstrate that GCAPs cannot regulate RetGC1 using independent primary binding sites. Instead, GCAP1 and GCAP2 bind with the cyclase molecule in a mutually exclusive manner using a common or overlapping binding site(s) in the Arg⁴⁸⁸–Arg⁸⁵¹ portion of RetGC1, and mutations in that region causing Leber congenital amaurosis blindness disrupt activation of the cyclase by both GCAP1 and GCAP2.     

Enzymatic properties and regulation of the native isozymes of retinal membrane guanylyl cyclase (RetGC) from mouse photoreceptors

Mouse photoreceptor function and survival critically depend on Ca(2+)-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 5-28-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 Ca(2+) in the presence of GCAP1 (EC(50Ca) ～132-139 nM) than GCAP2 (EC(50Ca) ～50-59 nM), thus arguing that Ca(2+) 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.     

Dimerization Domain of Retinal Membrane Guanylyl Cyclase 1 (RetGC1) Is an Essential Part of Guanylyl Cyclase-activating Protein (GCAP) Binding Interface

The photoreceptor-specific proteins guanylyl cyclase-activating proteins (GCAPs) bind and regulate retinal membrane guanylyl cyclase 1 (RetGC1) but not natriuretic peptide receptor A (NPRA). Study of RetGC1 regulation in vitro and its association with fluorescently tagged GCAP in transfected cells showed that R822P substitution in the cyclase dimerization domain causing congenital early onset blindness disrupted RetGC1 ability to bind GCAP but did not eliminate its affinity for another photoreceptor-specific protein, retinal degeneration 3 (RD3). Likewise, the presence of the NPRA dimerization domain in RetGC1/NPRA chimera specifically disabled binding of GCAPs but not of RD3. In subsequent mapping using hybrid dimerization domains in RetGC1/NPRA chimera, multiple RetGC1-specific residues contributed to GCAP binding by the cyclase, but the region around Met⁸²³ was the most crucial. Either positively or negatively charged residues in that position completely blocked GCAP1 and GCAP2 but not RD3 binding similarly to the disease-causing mutation in the neighboring Arg⁸²². The specificity of GCAP binding imparted by RetGC1 dimerization domain was not directly related to promoting dimerization of the cyclase. The probability of coiled coil dimer formation computed for RetGC1/NPRA chimeras, even those incapable of binding GCAP, remained high, and functional complementation tests showed that the RetGC1 active site, which requires dimerization of the cyclase, was formed even when Met⁸²³ or Arg⁸²² was mutated. These results directly demonstrate that the interface for GCAP binding on RetGC1 requires not only the kinase homology region but also directly involves the dimerization domain and especially its portion containing Arg⁸²² and Met⁸²³.     

CXCR3 antagonism of SDF-1(5-67) restores trabecular function and prevents retinal neurodegeneration in a rat model of ocular hypertension

Glaucoma, the most common cause of irreversible blindness, is a neuropathy commonly initiated by pathological ocular hypertension due to unknown mechanisms of trabecular meshwork degeneration. Current antiglaucoma therapy does not target the causal trabecular pathology, which may explain why treatment failure is often observed. Here we show that the chemokine CXCL12, its truncated form SDF-1(5-67), and the receptors CXCR4 and CXCR3 are expressed in human glaucomatous trabecular tissue and a human trabecular cell line. SDF-1(5-67) is produced under the control of matrix metallo-proteinases, TNF-α, and TGF-β2, factors known to be involved in glaucoma. CXCL12 protects in vitro trabecular cells from apoptotic death via CXCR4 whereas SDF-1(5-67) induces apoptosis through CXCR3 and caspase activation. Ocular administration of SDF-1(5-67) in the rat increases intraocular pressure. In contrast, administration of a selective CXCR3 antagonist in a rat model of ocular hypertension decreases intraocular pressure, prevents retinal neurodegeneration, and preserves visual function. The protective effect of CXCR3 antagonism is related to restoration of the trabecular function. These data demonstrate that proteolytic cleavage of CXCL12 is involved in trabecular pathophysiology, and that local administration of a selective CXCR3 antagonist may be a beneficial therapeutic strategy for treating ocular hypertension and subsequent retinal degeneration.     

Retinal degeneration 3 (RD3) protein inhibits catalytic activity of retinal membrane guanylyl cyclase (RetGC) and its stimulation by activating proteins

Retinal membrane guanylyl cyclase (RetGC) in the outer segments of vertebrate photoreceptors is controlled by guanylyl cyclase activating proteins (GCAPs), responding to light-dependent changes of the intracellular Ca(2+) concentrations. We present evidence that a different RetGC binding protein, retinal degeneration 3 protein (RD3), is a high-affinity allosteric modulator of the cyclase which inhibits RetGC activity at submicromolar concentrations. It suppresses the basal activity of RetGC in the absence of GCAPs in a noncompetitive manner, and it inhibits the GCAP-stimulated RetGC at low intracellular Ca(2+) levels. RD3 opposes the allosteric activation of the cyclase by GCAP but does not significantly change Ca(2+) sensitivity of the GCAP-dependent regulation. We have tested a number of mutations in RD3 implicated in human retinal degenerative disorders and have found that several mutations prevent the stable expression of RD3 in HEK293 cells and decrease the affinity of RD3 for RetGC1. The RD3 mutant lacking the carboxy-terminal half of the protein and associated with Leber congenital amaurosis type 12 (LCA12) is unable to suppress the activity of the RetGC1/GCAP complex. Furthermore, the inhibitory activity of the G57V mutant implicated in cone-rod degeneration is strongly reduced. Our results suggest that inhibition of RetGC by RD3 may be utilized by photoreceptors to block RetGC activity during its maturation and/or incorporation into the photoreceptor outer segment rather than participate in dynamic regulation of the cyclase by Ca(2+) and GCAPs.     

Differential expression of D(1A) and D(1B) dopamine receptor mRNAs in the developing avian retina

In the chick retina, the D1 dopaminergic system differentiates very early, as shown by receptor-mediated increases in intracellular cyclic AMP concentration and the presence of [(3)H]SCH23390-specific binding sites. Here, we characterized, by RT-PCR, the expression of defined D1 receptor subtypes D(1A), D(1B), and D(1D) during the development of the chick retina. Total RNA was extracted from retinas of 6-day-old embryos (E6) to 1-day-old hatched chickens and reverse-transcribed. The resulting cDNA was amplified using D(1A)-, D(1B)-, or D(1D)-specific primers, and the PCR-amplified products were analyzed by electrophoresis. The fragment corresponding to D(1A) receptor was detected in developing retina as early as E7, whereas the fragment corresponding to D(1B) was observed starting around E10. No PCR product corresponding to D(1D) was observed in the retina, although it was detected in chick brain. As synaptogenesis in chick retina begins after E11 and [(3)H]SCH 23390 D1 binding sites increase after this stage, the present results show that expression of D(1B) receptor increases during synaptogenesis, whereas D(1A) is the receptor subtype associated with the D1-like actions of dopamine early in retina development.     

CRX controls retinal expression of the X-linked juvenile retinoschisis (RS1) gene

X-linked juvenile retinoschisis is a heritable condition of the retina in males caused by mutations in the RS1 gene. Still, the cellular function and retina-specific expression of RS1 are poorly understood. To address the latter issue, we characterized the minimal promoter driving expression of RS1 in the retina. Binding site prediction, site-directed mutagenesis, and reporter assays suggest an essential role of two nearby cone-rod homeobox (CRX)-responsive elements (CRE) in the proximal −177/+32 RS1 promoter. Chromatin immunoprecipitation associates the RS1 promoter in vivo with CRX, the coactivators CBP, P300, GCN5 and acetylated histone H3. Transgenic Xenopus laevis expressing a green fluorescent protein (GFP) reporter under the control of RS1 promoter sequences show that the −177/+32 fragment drives GFP expression in photoreceptors and bipolar cells. Mutating either of the two conserved CRX binding sites results in strongly decreased RS1 expression. Despite the presence of sequence motifs in the promoter, NRL and NR2E3 appear not to be essential for RS1 expression. Together, our in vitro and in vivo results indicate that two CRE sites in the minimal RS1 promoter region control retinal RS1 expression and establish CRX as a key factor driving this expression.     

Involvement of rhodopsin and ATP in the activation of membranous guanylate cyclase in retinal photoreceptor outer segments (ROS-GC) by GC-activating proteins (GCAPs): a new model for ROS-GC activation and its link to retinal diseases

Membranous guanylate cyclase in retinal photoreceptor outer segments (ROS-GC), a key enzyme for the recovery of photoreceptors to the dark state, has a topology identical to and cytoplasmic domains homologous to those of peptide-regulated GCs. However, under the prevailing concept, its activation mechanism is significantly different from those of peptide-regulated GCs: GC-activating proteins (GCAPs) function as the sole activator of ROS-GC in a Ca²⁺-sensitive manner, and neither reception of an outside signal by the extracellular domain (ECD) nor ATP binding to the kinase homology domain (KHD) is required for its activation. We have recently shown that ATP pre-binding to the KHD in ROS-GC drastically enhances its GCAP-stimulated activity, and that rhodopsin illumination, as the outside signal, is required for the ATP pre-binding. These results indicate that illuminated rhodopsin is involved in ROS-GC activation in two ways: to initiate ATP binding to ROS-GC for preparation of its activation and to reduce [Ca²⁺] through activation of cGMP phosphodiesterase. These two signal pathways are activated in a parallel and proportional manner and finally converge for strong activation of ROS-GC by Ca²⁺-free GCAPs. These results also suggest that the ECD receives the signal for ATP binding from illuminated rhodopsin. The ECD is projected into the intradiscal space, i.e., an intradiscal domain(s) of rhodopsin is also involved in the signal transfer. Many retinal disease-linked mutations are found in these intradiscal domains; however, their consequences are often unclear. This model will also provide novel insights into causal relationship between these mutations and certain retinal diseases.     

Lentiviral vector expression of tumour antigens in dendritic cells as an immunotherapeutic strategy

Therapeutic cancer vaccines need to stimulate a refractory immune system to make an effective anti-tumour response. We have explored the use of lentiviral vectors to deliver tumour antigen genes to dendritic cells (DC) as a possible mechanism of immune stimulation. Direct injection of a lentiviral vector encoding the melanoma antigen NY-ESO-1 in HLA-A2 transgenic mice primed NY-ESO-1-specific CD8+ cells that could be expanded by boosting with an NY-ESO-1 vaccinia virus. The expanded cells could kill NY-ESO-1_157–165 peptide-pulsed targets in vivo. In order to examine the priming step directly, we constructed another lentiviral vector expressing the melanoma antigen Melan-A (MART-1). Here we show that Melan-A protein is also efficiently expressed after transduction of human DC cultured from peripheral blood mononuclear cells. When these transduced DC are co-cultured with autologous naïve T cells, they cause the expansion of cells that recognise the HLA-A2 restricted Melan-A_27–35 epitope. The expanded cells are functional in that they release IFN-γ upon antigen stimulation. Melan-A lentiviral vector transduced DC caused a similar level of naïve T-cell expansion to Melan-A_27–35 peptide-pulsed DC in four experiments using different HLA-A2 positive donors. These data suggest that a vaccine based either on DC transduced with a lentiviral vector ex vivo, or on direct lentiviral vector injection, should be assessed in a phase I clinical trial.This article is a symposium paper from the “Robert Baldwin Symposium: 50 years of Cancer Immunotherapy”, held in Nottingham, Great Britain, on 30th June 2005.     

Phenotypic change regulates monocyte chemoattractant protein-1 (MCP-1) gene expression in human retinal pigment epithelial cells

We investigated the expression profile of monocyte chemoattractant protein-1 (MCP-1) in human retinal pigment epithelial (RPE) cells under different culture conditions and evaluated the molecular mechanism responsible for MCP-1 gene expression in RPE cells. After cellular confluence, total RNA was extracted and used for RT-PCR. Medium conditioned by RPE was used for ELISA and Western blotting. The result showed that RPE cells cultured on plastic expressed MCP-1 constitutively in the absence of any stimuli. On the other hand, growing human RPE on laminin-coated flasks instead of plastic reduced the production of MCP-1. In the RPE cells cultured on plastic, IkappaB was degraded and A20 protein increased concomitantly. MCP-1 upregulation in RPE cells on plastic was attenuated by the addition of MG-132, a proteasome inhibitor. Also, the addition of pyrolidine dithiocarbonate (PDTC) and hypoxic conditions (0.5% O2) decreased MCP-1 production in these cells. These findings suggested that the expression profile of MCP-1 is regulated by phenotypic alterations of the RPE cells. And the increased MCP-1 expression in RPE cells cultured on plastic is caused via spontaneous activation of NFkappaB induced by susceptibility to oxidative stress.     

Changes in retinal aquaporin-9 (AQP9) expression in glaucoma

The eye contains numerous water channel proteins and the roles of AQPs (aquaporins) in the retina are blurred, especially under disease conditions. The purpose of this study was to investigate the expression of AQP9 gene and proteins affected by elevated IOP (intraocular pressure) in a rat model of glaucoma induced by intravitreous injection of hypertonic saline into the episcleral veins. The gene and protein expressions of AQP9 were investigated by real-time PCR and Western blotting. The immunoreactive expression of AQP9, AQP4 and GFAP (glial fibrillary acidic protein) in the optic nerve of rats exposed to experimentally elevated IOP was detected by immunofluorescence microscopy. The mRNA and protein expression levels of AQP9 were up-regulated in the retina of an animal model of glaucoma. The immunoreactivities of the AQP9, AQP4 and GFAP were also detected and increased in the optic nerve region. The expression of AQP9 was up-regulated in this glaucoma model and the immunoreactivities of the AQP4 and GFAP were also detected as co-localizing with AQP9 in the optic nerve region, indicating retina ganglion cells were surrounded by activated astrocytes. This may indicate that the injured neurons may rely on the astrocytes. The alterations of AQP expression may compensate the glaucomatous damage.     

Impact of diazepam on pineal-adrenal axis in an avian model

Diazepam, a benzodiazepine derivative, better known as a melatonin blocker in mammals, was injected into pigeons at a dose of 3 mg/kg body weight/day for 1 h, 1 day, 7 days and 15 days. This was done to investigate whether diazepam-induced changes in the pineal gland were reflected in the functioning of the adrenal gland. The results indicated that diazepam caused inhibition of pineal function and the degree of inhibition was very much time dependent. In addition, the pineal gland was unable to modulate the adrenomedullary hormonal titre yet it considerably influenced the physiology of the adrenal cortex.     

PGH(2)-TxA(2)-receptor blockade restores vasoreactivity in a new rodent model of genetic hypertension.

The purpose of this study was to determine whether activation of prostaglandin H(2)-thromboxane A(2) (PGH(2)-TxA(2)) receptors impedes vasodilation in the in situ peripheral microcirculation of spontaneously hypertensive hamsters, a new rodent model of high-renin genetic hypertension. Using intravital microscopy, we found that vasodilation elicited by suffusion of acetylcholine and vasoactive intestinal peptide (VIP), two neurotransmitters localized in perivascular nerves in the peripheral circulation, on the in situ cheek pouch was significantly attenuated in spontaneously hypertensive hamsters relative to age- and genetically matched normotensive hamsters (P < 0.05). However, nitroglycerin-induced vasodilation was similar in both groups. Pretreatment with SQ-29548, a selective and potent PGH(2)-TxA(2)-receptor antagonist, restored acetylcholine- and VIP-induced vasodilation in spontaneously hypertensive hamsters. SQ-29548 had no significant effects on resting arteriolar diameter and on nitroglycerin-induced vasodilation in both groups. SQ-29548 slightly but significantly potentiated VIP- but not acetylcholine-induced vasodilation in normotensive hamsters. Collectively, these data indicate that activation of PGH(2)-TxA(2) receptors impedes agonist-induced vasodilation in the in situ cheek pouch of spontaneously hypertensive hamsters. We suggest that this model is suitable for studying the role of prostanoids in mediating vasomotor dysfunction observed in genetic hypertension.