Identification of novel coreceptors in GPCRs harboring several tyrosines in the N'-terminal region
| Project/Area Number |
14370099
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Virology
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| Research Institution | Gunma University |
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Principal Investigator |
SHIMIZU Nobuaki Gunma University, Assistant Professor, 大学院・医学系研究科, 講師 (70261831)
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| Co-Investigator(Kenkyū-buntansha) |
TANAKA Atsushi Gunma University, Assistant, 大学院・医学系研究科, 教務員 (20321953)
HOSHINO Hiroo Gunma University, Assistant Professor, 大学院・医学系研究科, 教授 (00107434)
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| Project Period (FY) |
2002 – 2004
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| Project Status |
Completed (Fiscal Year 2004)
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| Budget Amount *help |
¥6,200,000 (Direct Cost: ¥6,200,000)
Fiscal Year 2004: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 2003: ¥2,000,000 (Direct Cost: ¥2,000,000)
Fiscal Year 2002: ¥2,200,000 (Direct Cost: ¥2,200,000)
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| Keywords | HIV / GPCR / Coreceptor / Cell tropism / Infection / AIDS / Diversity / Mutation / Envタンパク質 |
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| Research Abstract |
[Background]G protein-coupled receptors(GPCRs), which have capacity to act as coreceptors for human immunodeficiency virus type 1, commonly contain several tyrosine residues with asparagines or aspartic acids in the N'-terminal extracellular region(NTR). Sulfation of these tyrosines were demonstrated to enhance coreceptor activity. In this study, in order to know roles of the tyrosine residues in coreceptor activity, we constructed chimeras of extracellular domains among CCR5 and GPR1. GPR1 is an orphan GPCR, which had been identified to act as a coreceptor for HIV-1 strains with the cell tropism for pericytes in brain blood vessels. Moreover, several CCR5 mutants with amino acid substitutions in the NTR were also constructed. Coreceptor activity of a chemokine receptor, D6, which also contains several tyrosine residues in the NTR, was examined.
[Method] CCR5, D6, and GPR1 genes were cloned in the expression plasmids, pMX-puro or pCX-bsr. Using the PCR method, chimeric and amino acid substitution mutants were constructed and transduced into a human glioma-derived cell line, NP-2/CD4. NP-2/CD4 cells were strictly resistant to HIV-1 infection, although the CD4 gene was transduced and highly expressed. Susceptibilities of NP-2/CD4 cells transduced with D6 or CCR5 mutants to HIV-1 strains were determined.
[Results] A substitution of the tyrosine (the 3rd amino acid position) into alanine had no effect on the coreceptor activity of CCR5. On the other hand, the substitution of the tyrosine (the 15th a.a.) completely abolish the coreceptor activity. Importance of the other tyrosines in the activity was varied depending on viral strains. NP-2/CD4 cells transduced with D6 gene showed susceptibility to several HIV-1 strains with both CCR5 and CXCR4 uses.
[Discussion] There may be a conserved structure in NTRs of the GPCRs with coreceptor activities, which is critical for the interaction with the Env protein of HIV-1. This structure will be a clue to develop new anti-HIV-1 drugs.
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Report
(4 results)
Research Products
(5 results)
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[Book] 斎の舞へ2005
Author(s)
清水宣明, 甲野善紀
Total Pages
350
Publisher
仮立舎(印刷中)
Description
「研究成果報告書概要(和文)」より
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