Constitutive Gαi Coupling Activity of Very Large G Protein-coupled Receptor 1 (VLGR1) and Its Regulation by PDZD7 Protein

The very large G protein-coupled receptor 1 (VLGR1) is a core component in inner ear hair cell development. Mutations in the vlgr1 gene cause Usher syndrome, the symptoms of which include congenital hearing loss and progressive retinitis pigmentosa. However, the mechanism of VLGR1-regulated intracellular signaling and its role in Usher syndrome remain elusive. Here, we show that VLGR1 is processed into two fragments after autocleavage at the G protein-coupled receptor proteolytic site. The cleaved VLGR1 β-subunit constitutively inhibited adenylate cyclase (AC) activity through Gα_i coupling. Co-expression of the Gα_iq chimera with the VLGR1 β-subunit changed its activity to the phospholipase C/nuclear factor of activated T cells signaling pathway, which demonstrates the Gα_i protein coupling specificity of this subunit. An R6002A mutation in intracellular loop 2 of VLGR1 abolished Gα_i coupling, but the pathogenic VLGR1 Y6236fsx1 mutant showed increased AC inhibition. Furthermore, overexpression of another Usher syndrome protein, PDZD7, decreased the AC inhibition of the VLGR1 β-subunit but showed no effect on the VLGR1 Y6236fsx1 mutant. Taken together, we identified an independent Gα_i signaling pathway of the VLGR1 β-subunit and its regulatory mechanisms that may have a role in the development of Usher syndrome.     

Mutations in the VLGR1 gene implicate G-protein signaling in the pathogenesis of Usher syndrome type II

Usher syndrome type II (USH2) is a genetically heterogeneous autosomal recessive disorder with at least three genetic subtypes (USH2A, USH2B, and USH2C) and is classified phenotypically as congenital hearing loss and progressive retinitis pigmentosa. The VLGR1 (MASS1) gene in the 5q14.3-q21.1 USH2C locus was considered a likely candidate on the basis of its protein motif structure and expressed-sequence-tag representation from both cochlear and retinal subtracted libraries. Denaturing high-performance liquid chromatography and direct sequencing of polymerase-chain-reaction products amplified from 10 genetically independent patients with USH2C and 156 other patients with USH2 identified four isoform-specific VLGR1 mutations (Q2301X, I2906FS, M2931FS, and T6244X) from three families with USH2C, as well as two sporadic cases. All patients with VLGR1 mutations are female, a significant deviation from random expectations. The ligand(s) for the VLGR1 protein is unknown, but on the basis of its potential extracellular and intracellular protein-protein interaction domains and its wide mRNA expression profile, it is probable that VLGR1 serves diverse cellular and signaling processes. VLGR1 mutations have been previously identified in both humans and mice and are associated with a reflex-seizure phenotype in both species. The identification of additional VLGR1 mutations to test whether a phenotype/genotype correlation exists, akin to that shown for other Usher syndrome disease genes, is warranted.     

A second CYP26 P450 in humans and zebrafish: CYP26B1.

A new P450 gene has been found in humans. It has 44% sequence identity to CYP26A1 from human and mouse, which places it in a new subfamily, CYP26B. There is only one human EST from a retinal library (AA012833) that matches the coding region. No homologous ESTs are found in mouse. A zebrafish EST AI721901 shows 68% identity to the human protein. This zebrafish EST is only 41% identical to the zebrafish CYP26A1 protein sequence, so it represents the homolog of the human CYP26B1 sequence. It is not known if this gene product will act on all-trans-retinoic acid like the CYP26A1 protein or if it might hydroxylate the 9-cis- or 13-cis-retinoic acid isoforms not recognized by CYP26A1. The importance of the CYP26A1 P450 in mouse and zebrafish development flags the CYP26B1 gene as a potential developmental gene.     

cDNA cloning and sequence analysis of preproendothelin-1 (PPET-1) from salmon, Oncorhynchus keta

The presence of endothelin (ET)-like immunoreactivity and the cardiovascular effects of mammalian ET-1 in fish have been reported. To identify ET-related peptides in fish, we screened the cDNA library of the salmon (Oncorhynchus keta) stomach by means of rapid amplification of cDNA ends, and we cloned cDNAs encoding an ET-related peptide. The salmon ET-related sequence of 21 amino acids is identical to the trout ET-1 peptide recently purified from kidney specimens of Oncorhynchus mykiss. The deduced amino acid sequence of salmon pre-proET-1 (PPET-1) comprises 244 amino acids, including a putative signal sequence and mature ET-1, as well as big ET-1 and ET-1-like sequences. This precursor, the first reported PPET-1 sequence for Salmoniformes, Teleostei, has low homology with the sequences of human, mouse, frog (Xenopus laevis), and zebrafish (Danio rerio) PPET-1 (26%, 29%, 24%, and 39%, respectively).     

The Fanconi anemia gene network is conserved from zebrafish to human

Fanconi anemia (FA) is a complex disease involving nine identified and two unidentified loci that define a network essential for maintaining genomic stability. To test the hypothesis that the FA network is conserved in vertebrate genomes, we cloned and sequenced zebrafish (Danio rerio) cDNAs and/or genomic BAC clones orthologous to all nine cloned FA genes (FANCA, FANCB, FANCC, FANCD1, FANCD2, FANCE, FANCF, FANCG, and FANCL), and identified orthologs in the genome database for the pufferfish Tetraodon nigroviridis. Genomic organization of exons and introns was nearly identical between zebrafish and human for all genes examined. Hydrophobicity plots revealed conservation of FA protein structure. Evolutionarily conserved regions identified functionally important domains, since many amino acid residues mutated in human disease alleles or shown to be critical in targeted mutagenesis studies are identical in zebrafish and human. Comparative genomic analysis demonstrated conserved syntenies for all FA genes. We conclude that the FA gene network has remained intact since the last common ancestor of zebrafish and human lineages. The application of powerful genetic, cellular, and embryological methodologies make zebrafish a useful model for discovering FA gene functions, identifying new genes in the network, and identifying therapeutic compounds.     

Sulfation of hydroxychlorobiphenyls. Molecular cloning, expression, and functional characterization of zebrafish SULT1 sulfotransferases.

As a first step toward developing a zebrafish model for investigating the role of sulfation in counteracting environmental estrogenic chemicals, we have embarked on the identification and characterization of cytosolic sulfotransferases (STs) in zebrafish. By searching the zebrafish expressed sequence tag database, we have identified two cDNA clones encoding putative cytosolic STs. These two zebrafish ST cDNAs were isolated and subjected to nucleotide sequencing. Sequence data revealed that the two zebrafish STs are highly homologous, being approximately 82% identical in their amino acid sequences. Both of them display approximately 50% amino acid sequence identity to human SULT1A1, rat SULT1A1, and mouse SULT1C1 ST. These two zebrafish STs therefore appear to belong to the SULT1 cytosolic ST gene family. Recombinant zebrafish STs (designated SULT1 STs 1 and 2), expressed using the pGEX-2TK prokaryotic expression system and purified from transformed Escherichia coli cells, migrated as approximately 35 kDa proteins on SDS/PAGE. Purified zebrafish SULT1 STs 1 and 2 displayed differential sulfating activities toward a number of endogenous compounds and xenobiotics including hydroxychlorobiphenyls. Kinetic constants of the two enzymes toward two representative hydroxychlorobiphenyls, 3-chloro-4-biphenylol and 3,3',5,5'-tetrachloro-4,4'-biphenyldiol, and 3,3',5-triiodo-l-thyronine were determined. A thermostability experiment revealed the two enzymes to be relatively stable over the range 20-43 degrees C. Among 10 different divalent metal cations tested, Co2+, Zn2+, Cd2+, and Pb2+ exhibited considerable inhibitory effects, while Hg2+ and Cu2+ rendered both enzymes virtually inactive.     

Sequence similarities between a novel putative G protein-coupled receptor and Na+/Ca2+ exchangers define a cation binding domain

cDNA clones encoding a novel putative G protein-coupled receptor have been characterized. The receptor is widely expressed in normal solid tissues. Consisting of 1967 amino acid residues, this receptor is one of the largest known and is therefore referred to as a very large G protein-coupled receptor, or VLGR1. It is most closely related to the secretin family of G protein-coupled receptors based on similarity of the sequences of its transmembrane segments. As demonstrated by cell surface labeling with a biotin derivative, the recombinant protein is expressed on the surface of transfected mammalian cells. Whereas several other recently described receptors in this family also have large extracellular domains, the large extracellular domain of VLGR1 has a unique structure. It has nine imperfectly repeated units that are rich in acidic residues and are spaced at intervals of approximately 120 amino acid residues. These repeats resemble the regulatory domains of Na+/Ca2+ exchangers as well as a component of an extracellular aggregation factor of marine sponges. Bacterial fusion proteins containing two or four repeats specifically bind 45Ca in overlay experiments; binding is competed poorly by Mg2+ but competed well by neomycin, Al3+, and Gd3+. These results define a consensus cation binding motif employed in several widely divergent types of proteins. The ligand for VLGR1, its function, and the signaling pathway(s) it employs remain to be defined.     

A chicken mRNA similar to heterogeneous nuclear ribonucleoprotein H1

Heterogeneous nuclear ribonucleoproteins are predominantly nuclear RNA-binding proteins that function in a variety of cellular activities. The objective of these experiments was to clone a cDNA for a chicken protein similar to other previously reported heterogeneous ribonucleoproteins for other species. The 5' and 3' ends of the chicken mRNA were cloned using Rapid Amplification of cDNA Ends (RACE). Subsequently, the expression of the mRNA sequence was confirmed via Northern analysis. The deduced amino acid sequence was approximately 86% identical to corresponding regions of human, mouse, or zebrafish proteins similar to heterogeneous nuclear ribonucleoprotein H1. The expression data confirmed the size of the predicted mRNA sequence. The newly identified sequence may be employed in future studies aimed at understanding the role of heterogeneous nuclear ribonucleoproteins in avian species.     

The proliferation associated nuclear element (PANE1) is conserved between mammals and fish and preferentially expressed in activated lymphoid cells

The proliferation associated nuclear element (PANE1) had been identified in a screen for genes activated in mouse mammary epithelium transformed by stabilized beta-catenin. We have now cloned the human and zebrafish orthologs, analyzed their expression and expressed them ectopically in tissue culture cell lines. PANE1 consists of 180 amino acids and displays 38% conservation between man and zebrafish. Expression of the human PANE1 gene was detected preferentially in immune cells including leukemias and lymphomas, tumor tissues and tumor derived cell lines. In B- and T-cells PANE1 RNA was only detected after the respective cell types were activated either in vivo or in vitro.     

Homology-modeled ligand-binding domains of zebrafish estrogen receptors alpha, beta1, and beta2: from in silico to in vivo studies of estrogen interactions in Danio rerio as a model system

Homology models were constructed for the ligand-binding domains of zebrafish estrogen receptors (zfERs) alpha, beta(1), and beta(2). Estradiol-binding sites are nearly identical in zfERs and their human homologs, suggesting that zebrafish will serve as a good model system for studying human ER-binding drugs. Conversely, studies of endocrine disruptor effects on zebrafish will benefit from the wealth of data available on xenoestrogen interactions with human ERs. Compounds flagged by the Interagency Coordinating Committee on the Validation of Alternative Methods for endocrine disruptor screening were docked into our zfER homology models. Ideally, these in silico docking studies would be complemented with in vivo binding studies. To this end, fluorescently tagged estradiol was docked into zfERalpha and found to bind in the same manner as in human ERalpha, with fluorescein preferentially occupying a region between helices 11 and 12. Fluorescently tagged estradiol was synthesized and was found to localize along the path of primordial germ cell migration in the developing zebrafish embryo 3 d after fertilization, consistent with previous reports of 1) a role for estradiol in sex determination, and 2) the first appearance of ERs 2 d after fertilization. These data provide a foundation for future in silico and in vivo binding studies of estrogen agonists and antagonists with zebrafish ERs.     

Identification of a novel thyroid hormone-sulfating cytosolic sulfotransferase, SULT1 ST5, from zebrafish

By employing RT-PCR in conjunction with 3'-RACE, a full-length cDNA encoding a novel zebrafish cytosolic sulfotransferase (SULT) was cloned and sequenced. Sequence analysis revealed that this zebrafish SULT (designated SULT1 ST5) is, at the amino acid sequence level, close to 50% identical to human and dog SULT1B1 (thyroid hormone SULT). A recombinant form of zebrafish SULT1 ST5 was expressed using the pGEX-2TK bacterial expression system and purified from transformed BL21 (DE3) cells. Purified zebrafish SULT1 ST5 migrated as a 34 kDa protein and displayed substrate specificity for thyroid hormones and their metabolites among various endogenous compounds tested. The enzyme also exhibited sulfating activities toward some xenobiotic phenolic compounds. Its pH optima were 6.0 and 9.0 with 3,3',5-triiodo-l-thyronine (l-T3) as substrate and 6.0 with beta-naphthol as substrate. Kinetic constants of the enzyme with thyroid hormones and their metabolites as substrates were determined. Quantitative evaluation of the regulatory effects of divalent metal cations on the l-T3-sulfating activity of SULT1 ST5 revealed that Fe2+, Hg2+, Co2+, Zn2+, Cu2+, Cd2+ and Pb2+ exhibited dramatic inhibitory effects, whereas Mn2+ showed a significant stimulation. Developmental stage-dependent expression experiments revealed a significant level of expression of this novel zebrafish thyroid hormone-sulfating SULT at the beginning of the hatching period during embryogenesis, which gradually increased to a high level of expression throughout the larval stage into maturity.