2002 Fiscal Year Final Research Report Summary
Functional analysis of the PDZ protein Pick1 during neural development in Drosophila
| Project/Area Number |
13680809
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Developmental biology
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| Research Institution | Kumamoto University |
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Principal Investigator |
GO Masahiro Graduate School of Medical Sciences, Research Associate, 医学研究科, 助手 (00304999)
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| Project Period (FY) |
2001 – 2002
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| Keywords | Drosophila / PDZ protein / neural development / synapse formation / UAS-GAL4 / yeast two-hybrid |
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| Research Abstract |
PICK1 and GRIP are synaptic PDZ proteins which have a similar binding activity as a PDZ protein. We have cloned and sequenced Drosophila homologs of PICK1 and GRIP. The domain structures were well conserved in both proteins between mammals and Drosophila. At a sequence level, PICK1 showed a high homology to PICK1 in other species, but GRIP showed an only low homology to GRIP in other species. In situ hybridization in embryos revealed that the PICK1 RNA is expressed exclusively in the nervous system, whereas the GRIP RNA is expressed almost specifically in muscle cells and visceral mesoderm cells. We have introduced myc tagged full length constructs of both proteins into flies for misexpression by the UAS-GAL4 system. As a result, the PICK1 protein was detected along the neurites including the tips, although the staining was not uniform. The GRIP protein was found to be localized to post-synaptic regions of terminal structures of motor neurons. These results suggest pre- and post-synaptic functions of the PICK1 and GRIP proteins, respectively. Antiserum against the GRIP protein also revealed that GRIP was expressed in almost all neurons in the central nervous system of third instar larvae. This observation was also confirmed by RT-PCR and the expression pattern from an enhancer trap line for GRIP. In order to see loss-of-function phenotypes, dsRNA for each protein was expressed by UAS-GAL4 system. RNAi resulted in pupal lethality for both PICK1 and GRIP. We also found a viable P element line whose insertion site is very close to the transcription start codon of GRIP. I am in the process of obtaining deletion mutants for GRIP by removing the P element insertion. In order to search for proteins that bind the PDZ domains of GRIP, we did a screen by yeast two-hybrid system. So far, the Neurexin-1 protein, which is thought to function at synapses, was found to bind the PDZ domains of GRIP strongly and specifically, and therefore is a good candidate as the target protein.
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Research Products
(2 results)
