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Mutagenesis and Functional Analysis of Ion Channels Heterologously Expressed in Mammalian Cells
Published on: October 1, 2010
Mutational analysis on the function of the SWAP-70 PH domain
Isamu Wakamatsu1, Sayoko Ihara, Yasuhisa Fukui
1Division of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan.
Molecular and Cellular Biochemistry
|June 21, 2006
Summary
SWAP-70 protein binding to phosphatidylinositol trisphosphate (PtdIns(3,4,5)P(3)) is not essential for membrane ruffling when Rac is highly expressed. Efficient SWAP-70 and Rac interaction can occur independently of PtdIns(3,4,5)P(3) binding.
Area of Science:
- Cell biology
- Molecular biology
- Biochemistry
Background:
- SWAP-70 is a phosphatidylinositol trisphosphate (PtdIns(3,4,5)P(3))-binding protein implicated in membrane ruffling.
- SWAP-70 is known to cooperate with activated Rac in cellular processes.
Purpose of the Study:
- To investigate the role of PtdIns(3,4,5)P(3) binding activity of SWAP-70 in its function.
- To determine if PtdIns(3,4,5)P(3) binding is essential for SWAP-70's effects on membrane ruffling, especially in the presence of Rac.
Main Methods:
- Site-directed mutagenesis was used to alter the PH domain of SWAP-70, specifically targeting positively charged amino acids.
- Experiments involved overexpressing SWAP-70 mutants and Rac to assess effects on membrane ruffling and protein translocation.
Main Results:
- Mutations in the PH domain that abolished PtdIns(3,4,5)P(3) binding also impaired SWAP-70 translocation and its ability to enhance membrane ruffling or exert dominant-negative effects.
- However, when Rac was overexpressed, these PtdIns(3,4,5)P(3) binding-deficient mutants could still translocate to the membrane and affect membrane ruffling.
Conclusions:
- PtdIns(3,4,5)P(3) binding activity of SWAP-70 is dispensable for its membrane translocation and effects on membrane ruffling when Rac is efficiently interacting with SWAP-70.
- PtdIns(3,4,5)P(3) binding may serve to facilitate SWAP-70 recruitment to activated Rac, but is not strictly required for the downstream events.
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