RESUMO
GPR177 is an evolutionarily conserved transmembrane protein necessary for Wnt protein secretion. Little is currently known, however, regarding expression of GPR177, especially in vertebrate species. We have developed an antiserum against GPR177, and used it to examine expression of GPR177 in human tissue culture cells, adult mouse, and rat tissues, as well as developing zebrafish embryos. In rodents, GPR177 is expressed in virtually all tissue types and brain regions examined. In zebrafish, GPR177 polypeptides are expressed throughout embryogenesis, and are detectable as early as 1 hr post-fertilization. In situ hybridization analysis reveals that gpr177 mRNA expression is prominent in embryonic zebrafish brain and ear. Structural studies suggest that GPR177 is modified by N-linked sugars, and that the protein contains an even number of transmembrane segments. The relatively ubiquitous expression of GPR177 suggests that this protein may serve to regulate Wnt secretion in a variety of embryonic and adult tissue types.
Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteínas Wnt/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Células Cultivadas , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Camundongos , Ratos , Ratos Sprague-Dawley , Receptores Acoplados a Proteínas G/genética , Distribuição Tecidual , Proteínas Wnt/genética , Peixe-Zebra/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
BACKGROUND: The semicircular canals, a subdivision of the vestibular system of the vertebrate inner ear, function as sensors of angular acceleration. Little is currently known, however, regarding the underlying molecular mechanisms that govern the development of this intricate structure. Zebrafish represent a particularly tractable model system for the study of inner ear development. This is because the ear can be easily visualized during early embryogenesis, and both forward and reverse genetic techniques are available that can be applied to the discovery of novel genes that contribute to proper ear development. We have previously shown that in zebrafish, the calcium sensing molecule neuronal calcium sensor-1 (NCS-1) is required for semicircular canal formation. The function of NCS-1 in regulating semicircular canal formation has not yet been elucidated. RESULTS: We initiated a multistep functional proteomic strategy to identify neuronal calcium sensor-1 (NCS-1) binding partners (NBPs) that contribute to inner ear development in zebrafish. By performing a Y2H screen in combination with literature and database searches, we identified 10 human NBPs. BLAST searches of the zebrafish EST and genomic databases allowed us to clone zebrafish orthologs of each of the human NBPs. By investigating the expression profiles of zebrafish NBP mRNAs, we identified seven that were expressed in the developing inner ear and overlapped with the ncs-1a expression profile. GST pulldown experiments confirmed that selected NBPs interacted with NCS-1, while morpholino-mediated knockdown experiments demonstrated an essential role for arf1, pi4kbeta, dan, and pink1 in semicircular canal formation. CONCLUSION: Based on their functional profiles, the hypothesis is presented that Ncs-1a/Pi4kbeta/Arf1 form a signaling pathway that regulates secretion of molecular components, including Dan and Bmp4, that are required for development of the vestibular apparatus. A second set of NBPs, consisting of Pink1, Hint2, and Slc25a25, are destined for localization in mitochondria. Our findings reveal a novel signalling pathway involved in development of the semicircular canal system, and suggest a previously unrecognized role for NCS-1 in mitochondrial function via its association with several mitochondrial proteins.
Assuntos
Orelha Interna/embriologia , Proteínas Sensoras de Cálcio Neuronal/genética , Neuropeptídeos/genética , Organogênese/genética , Proteínas de Peixe-Zebra/genética , Peixe-Zebra/genética , Animais , Proteínas de Transporte/classificação , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Orelha Interna/metabolismo , Orelha Interna/ultraestrutura , Embrião não Mamífero , Exocitose/genética , Exocitose/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Técnicas de Silenciamento de Genes , Proteínas Sensoras de Cálcio Neuronal/metabolismo , Neuropeptídeos/metabolismo , Organogênese/fisiologia , Proteômica/métodos , RNA Mensageiro/análise , Peixe-Zebra/embriologia , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/metabolismoRESUMO
The A2A adenosine receptor (AdR) subtype has emerged as an attractive target in the pursuit of improved therapy for Parkinson's disease (PD). This report focuses on characterization of zebrafish a2 AdRs. By mining the zebrafish EST and genomic sequence databases, we identified two zebrafish a2a (adora2a.1 and adora2a.2) genes and one a2b (adora2b) AdR gene. Sequence comparisons indicate that the predicted zebrafish A2 AdR polypeptides share 62-74% amino acid identity to mammalian A2 AdRs. We mapped the adora2a.1 gene to chromosome 8, the adora2a.2 gene to chromosome 21, and the adora2b gene to chromosome 5. Whole mount in situ hybridization analysis indicates zebrafish a2 AdR genes are expressed primarily within the central nervous system (CNS). Zebrafish are known to be sensitive to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a neurotoxin that causes selective loss of dopaminergic neurons and PD-like symptoms in humans as well as in animal models. Here we show that caffeine, an A2A AdR antagonist, is neuroprotective against the adverse effects of MPTP in zebrafish embryos. These results suggest that zebrafish AdRs may serve as useful targets for testing novel therapeutic strategies for the treatment of PD.