Biochemical defects in ABCR protein variants associated with human retinopathies.

Mutations in the gene encoding ABCR (ABCA4), a photoreceptor-specific ATP-binding cassette (ABC) transporter, are responsible for autosomal recessive Stargardt disease (STGD), an early onset macular degeneration, and some forms of autosomal recessive cone-rod dystrophy and autosomal recessive retinitis pigmentosa. Heterozygosity for ABCA4 mutations may also represent a risk factor for age-related macular degeneration (AMD), although this idea is controversial. An ongoing challenge in the analysis of ABCA4-based retinopathies arises from the observation that most of the ABCA4 sequence variants identified so far are missense mutations that are rare in both patient and control populations. With the current sample size of most sequence variants, one cannot determine statistically whether a particular sequence variant is pathogenic or neutral. A related challenge is to determine the degree to which each pathogenic variant impairs ABCR function, as genotype-phenotype analyses indicate that age of onset and disease severity correlate with different ABCA4 alleles. To address these questions, we performed a functional analysis of human ABCR and its variants. These experiments reveal a wide spectrum of biochemical defects in these variants and provide insight into the transport mechanism of ABCR.

A photoreceptor-specific cadherin is essential for the structural integrity of the outer segment and for photoreceptor survival.

A cadherin family member, prCAD, was identified in retina cDNA by subtractive hybridization and high throughput sequencing. prCAD is expressed only in retinal photoreceptors, and the prCAD protein is localized to the base of the outer segment of both rods and cones. In prCAD(-/-) mice, outer segments are disorganized and fragmented, and there is progressive death of photoreceptor cells. prCAD is unlikely to be involved in protein trafficking between inner and outer segments, since phototransduction proteins appear to be correctly localized and the light responses of both rods and cones are only modestly compromised in prCAD(-/-) mice. These experiments imply a highly specialized cell biological function for prCAD and suggest that localized adhesion activity is essential for outer segment integrity.

Identification and characterization of all-trans-retinol dehydrogenase from photoreceptor outer segments, the visual cycle enzyme that reduces all-trans-retinal to all-trans-retinol.

Retinol dehydrogenase (RDH), the enzyme that catalyzes the reduction of all-trans-retinal to all-trans-retinol within the photoreceptor outer segment, was the first visual cycle enzymatic activity to be identified. Previous work has shown that this enzyme utilizes NADPH, shows a marked preference for all-trans-retinal over 11-cis-retinal, and is tightly associated with the outer segment membrane. This paper reports the identification of a novel member of the short chain dehydrogenase/reductase family, photoreceptor RDH (prRDH), using subtraction and normalization of retina cDNA, high throughput sequencing, and data base homology searches to detect retina-specific genes. Bovine and human prRDH are highly homologous and are most closely related to 17-beta-hydroxysteroid dehydrogenase 1. The enzymatic properties of recombinant bovine prRDH closely match those previously reported for RDH activity in crude bovine rod outer segment preparations. In situ hybridization and RNA blotting show that the PRRDH gene is expressed specifically in photoreceptor cells, and protein blotting and immunocytochemistry show that prRDH localizes exclusively to both rod and cone outer segments and that prRDH is tightly associated with outer segment membranes. Taken together, these data indicate that prRDH is the enzyme responsible for the reduction of all-trans-retinal to all-trans-retinol within the photoreceptor outer segment.

Isoform diversity among fibroblast growth factor homologous factors is generated by alternative promoter usage and differential splicing.

Fibroblast growth factor (FGF) homologous factors-1, -2, -3, and -4 (FHFs 1-4; also referred to as FGFs 11-14) comprise a separate branch of the FGF family and have been implicated in the development of the nervous system and limbs. We report here the characterization of multiple isoforms of FHF-1, -2, -3, and -4 which are generated through the use of alternative start sites of transcription and splicing of one or more of a series of alternative 5'-exons. Several isoforms show different subcellular distributions when expressed in transfected tissue culture cells, and the corresponding differentially spliced transcripts show distinct expression patterns in developing and adult mouse tissues. Together with the evolutionary conservation of the FHF isoforms among human, mouse, and chicken, these data indicate that alternative promoter use and differential splicing are important regulatory processes in controlling the activities of this subfamily of FGFs.

Biochemical characterization of Wnt-frizzled interactions using a soluble, biologically active vertebrate Wnt protein.

Biochemical studies of Wnt signaling have been hampered by difficulties in obtaining large quantities of soluble, biologically active Wnt proteins. In this paper, we report the production in Drosophila S2 cells of biologically active Xenopus Wnt8 (XWnt8). Epitope- or alkaline phosphatase-tagged XWnt8 proteins are secreted by concentrated S2 cells in a form that is suitable for quantitative biochemical experiments with yields of 5 and 0.5 mg per liter, respectively. Conditions also are described for the production in 293 cells of an IgG fusion of the cysteine-rich domain (CRD) of mouse Frizzled 8 with a yield of 20 mg/liter. We demonstrate the use of these proteins for studying the interactions between soluble XWnt8 and various Frizzled proteins, membrane anchored or secreted CRDs, and a set of insertion mutants in the CRD of Drosophila Frizzled 2. In a solid phase binding assay, the affinity of the XWnt8-alkaline phosphatase fusion for the purified mouse Frizzled 8-CRD-IgG fusion is approximately 9 nM.

Mutually exclusive expression of human red and green visual pigment-reporter transgenes occurs at high frequency in murine cone photoreceptors.

This study examines the mechanism of mutually exclusive expression of the human X-linked red and green visual pigment genes in their respective cone photoreceptors by asking whether this expression pattern can be produced in a mammal that normally carries only a single X-linked visual pigment gene. To address this question, we generated transgenic mice that carry a single copy of a minimal human X chromosome visual pigment gene array in which the red and green pigment gene transcription units were replaced, respectively, by alkaline phosphatase and beta-galactosidase reporters. As determined by histochemical staining, the reporters are expressed exclusively in cone photoreceptor cells. In 20 transgenic mice carrying any one of three independent transgene insertion events, an average of 63% of expressing cones have alkaline phosphatase activity, 10% have beta-galactosidase activity, and 27% have activity for both reporters. Thus, mutually exclusive expression of red and green pigment transgenes can be achieved in a large fraction of cones in a dichromat mammal, suggesting a facile evolutionary path for the development of trichromacy after visual pigment gene duplication. These observations are consistent with a model of visual pigment expression in which stochastic pairing occurs between a locus control region and either the red or the green pigment gene promotor.

Identification and characterization of a conserved family of protein serine/threonine phosphatases homologous to Drosophila retinal degeneration C.

The Drosophila retinal degeneration C (rdgC) gene encodes an unusual protein serine/threonine phosphatase in that it contains at least two EF-hand motifs at its carboxy terminus. By a combination of large-scale sequencing of human retina cDNA clones and searches of expressed sequence tag and genomic DNA databases, we have identified two sequences in mammals [Protein Phosphatase with EF-hands-1 and 2 (PPEF-1 and PPEF-2)] and one in Caenorhabditis elegans (PPEF) that closely resemble rdgC. In the adult, PPEF-2 is expressed specifically in retinal rod photoreceptors and the pineal. In the retina, several isoforms of PPEF-2 are predicted to arise from differential splicing. The isoform that most closely resembles rdgC is localized to rod inner segments. Together with the recently described localization of PPEF-1 transcripts to primary somatosensory neurons and inner ear cells in the developing mouse, these data suggest that the PPEF family of protein serine/threonine phosphatases plays a specific and conserved role in diverse sensory neurons.

Molecular cloning of beta 3 subunit, a third form of the G protein beta-subunit polypeptide.

The signal-transducing guanine nucleotide-binding regulatory (G) proteins are heterotrimers composed of three subunits--alpha, beta, and gamma. Although multiple distinctive forms of the alpha subunit have been described, only two forms of the beta subunits of the G proteins have been identified. To investigate further the structural diversity of the beta subunits, we screened bovine and human retina cDNA libraries and isolated clones encoding three distinct types of G protein beta subunit. One form was identical to previously isolated beta 1-subunit cDNA clones that encode the 36-kDa form of the beta subunit, whereas a second form was identical to previously described beta 2 cDNAs that encode the 35-kDa beta isoform. In addition, we identified another species, designated beta 3 subunit, which encodes a third distinct form of the beta subunit. The beta 3-subunit cDNA corresponds to a 2.0-kilobase mRNA expressed in all tissues and clonal cell lines examined. Nucleotide sequence analysis indicates that the encoded peptide consists of 340-amino acid residues with a Mr of 37,221. The amino acid sequences of the three beta subunits are closely related: 83% identity between beta 1 and beta 3 subunits and 81% identity between beta 2 and beta 3 subunits. By contrast, the 3'-untranslated regions of the three cDNAs show no significant homology. Our data support the hypothesis that a family of beta-subunit polypeptides exists and extend understanding of beta-subunit structure.

Fibroblast growth factor (FGF) homologous factors: new members of the FGF family implicated in nervous system development.

Four new members of the fibroblast growth factor (FGF) family, referred to as fibroblast growth factor homologous factors (FHFs), have been identified by a combination of random cDNA sequencing, data base searches, and degenerate PCR. Pairwise comparisons between the four FHFs show between 58% and 71% amino acid sequence identity, but each FHF shows less than 30% identity when compared with other FGFs. Like FGF-1 (acidic FGF) and FGF-2 (basic FGF), the FHFs lack a classical signal sequence and contain clusters of basic residues that can act as nuclear localization signals. In transiently transfected 293 cells FHF-1 accumulates in the nucleus and is not secreted. Each FHF is expressed in the developing and adult nervous systems, suggesting a role for this branch of the FGF family in nervous system development and function.

A family of secreted proteins contains homology to the cysteine-rich ligand-binding domain of frizzled receptors.

This paper describes the identification of a new family of mammalian genes that encode secreted proteins containing homology to the cysteine-rich ligand-binding domain found in the frizzled family of transmembrane receptors. The secreted frizzled-related proteins (sFRPs) are approximately 30 kDa in size, and each contains a putative signal sequence, a frizzled-like cysteine-rich domain, and a conserved hydrophilic carboxy-terminal domain. The sFRPs are not the products of differential splicing of the known frizzled genes. Glycosylphosphatidylinositol-anchored derivatives of sFRP-2 and sFRP-3 produced in transfected human embryonic kidney cells confer cell-surface binding by the Drosophila Wingless protein. These observations suggest that sFRPs may function in vivo to modulate Wnt signaling, or, alternatively, as novel ligands for as yet unidentified receptors.

A new secreted protein that binds to Wnt proteins and inhibits their activities.

The Wnt proteins constitute a large family of extracellular signalling molecules that are found throughout the animal kingdom and are important for a wide variety of normal and pathological developmental processes. Here we describe Wnt-inhibitory factor-1 (WIF-1), a secreted protein that binds to Wnt proteins and inhibits their activities. WIF-1 is present in fish, amphibia and mammals, and is expressed during Xenopus and zebrafish development in a complex pattern that includes paraxial presomitic mesoderm, notochord, branchial arches and neural crest derivatives. We use Xenopus embryos to show that WIF-1 overexpression affects somitogenesis (the generation of trunk mesoderm segments), in agreement with its normal expression in paraxial mesoderm. In vitro, WIF-1 binds to Drosophila Wingless and Xenopus Wnt8 produced by Drosophila S2 cells. Together with earlier results obtained with the secreted Frizzled-related proteins, our results indicate that Wnt proteins interact with structurally diverse extracellular inhibitors, presumably to fine-tune the spatial and temporal patterns of Wnt activity.

Genetic deficiency of the alpha subunit of the guanine nucleotide-binding protein Gs as the molecular basis for Albright hereditary osteodystrophy.

Patients who have pseudohypoparathyroidism type I associated with Albright hereditary osteodystrophy commonly have a genetic deficiency of the alpha subunit of the G protein that stimulates adenylyl cyclase (alpha Gs) (ATP pyrophosphate-lyase, EC 4.6.1.1). To discover the molecular mechanism that causes alpha Gs deficiency in these patients, we examined eight kindreds with one or more members affected with Albright hereditary osteodystrophy or pseudohypoparathyroidism and alpha Gs deficiency. In these families, alpha Gs deficiency and the Albright hereditary osteodystrophy phenotype were transmitted together in a dominant inheritance pattern. Using a cDNA hybridization probe for alpha Gs, restriction analysis with several endonucleases showed no abnormalities of restriction fragments or gene dosage. RNA blot and dot blot analysis of total RNA from cultured fibroblasts obtained from the patients revealed approximately equal to 50% reduced mRNA levels for alpha Gs in affected members of six of the pedigrees but normal levels in affected members of the two other pedigrees, compared to mRNA levels in fibroblasts from unaffected individuals. By contrast, mRNA levels encoding the alpha subunit of the G protein that inhibits adenylyl cyclase were not altered. Our findings suggest that several molecular mechanisms produce alpha Gs deficiency in patients with pseudohypoparathyroidism type Ia and that major gene rearrangements or deletions are not a common cause for alpha Gs deficiency in pseudohypoparathyroidism type I.

Influence of thyroid hormone status on expression of genes encoding G protein subunits in the rat heart.

We have studied the influence of thyroid hormone status in vivo on expression of the genes encoding guanine nucleotide-binding regulatory protein (G protein) alpha-subunits Gs alpha, Gi alpha(2), Gi alpha(3), and both the 36-kDa form (beta 1) and the 35-kDa form (beta 2) of the beta-subunit in rat ventricle. The relative amounts of immunoactive Gi alpha(2) and Gi alpha(3) were greater in ventricular membranes from hypothyroid animals than from euthyroid animals (1.9- and 2.6-fold, respectively). A corresponding 2.3-fold increase in Gi alpha(2) mRNA was observed as well as a 1.5-fold increase in Gi alpha(3) mRNA. The relative amounts of immunoactive beta 1 and beta 2 polypeptides were also increased (2.8- and 1.8-fold, respectively) in the hypothyroid state and corresponded with comparable increases in the relative levels of beta 1 and beta 2 mRNAs. No difference was seen between the amounts of Gi alpha(2), Gi alpha(3), beta 1, and beta 2 in the euthyroid state and the hyperthyroid state. In contrast to these effects of thyroid hormone status on Gi alpha and beta, the steady-state amounts of Gs alpha protein and mRNA were not altered by thyroid hormone status. Thyroid hormone status did not alter sensitivity of adenylyl cyclase to stimulation by sodium fluoride or guanyl-5'-yl imidodiphosphate (GppNHp), nor did it influence GppNHp-induced inhibition of forskolin-stimulated enzyme activity. These results demonstrate that thyroid hormone status in vivo can regulate expression of specific G protein subunits in rat myocardium. However, the physiological consequences of these changes remain unclear.

Mutation in the gene encoding the stimulatory G protein of adenylate cyclase in Albright's hereditary osteodystrophy.

Albright's hereditary osteodystrophy is an autosomal dominant disorder characterized by a short stature, brachydactyly, subcutaneous ossifications, and reduced expression or function of the alpha subunit of the stimulatory G protein (Gs alpha) of adenylate cyclase, which is necessary for the action of parathyroid and other hormones that use cyclic AMP as an intracellular second messenger. We identified a unique Gs alpha protein in erythrocytes from two related patients with Albright's hereditary osteodystrophy and reduced Gs alpha bioactivity. The Gs alpha variant was recognized by a carboxyl terminal-specific Gs alpha antiserum but not by polyclonal antiserums specific for the amino terminus of Gs alpha. To investigate the molecular basis for this structurally abnormal Gs alpha protein, we studied the Gs alpha gene by restriction-endonuclease analysis. DNA from the two patients had an abnormal restriction-fragment pattern when digested with Ncol, which was consistent with loss of an Ncol restriction site in exon 1 of one Gs alpha allele. Amplification of a 260-base-pair region that includes exon 1 of the Gs alpha gene and direct sequencing of the amplified DNA revealed an A-to-G transition at position +1 in one Gs alpha allele from each of the two patients. This mutation converts the initiator ATG (methionine) codon to GTG (valine), blocking initiation of translation at the normal site. Translation of the abnormal Gs alpha messenger RNA would result in the synthesis of a truncated Gs alpha molecule lacking the amino terminus. We conclude that in at least some patients with Albright's hereditary osteodystrophy, the disease is caused by a single-base substitution in the Gs alpha gene and is thus due to an inherited mutation in a human G protein.