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PMID: 14699051 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

Analysis of C5a-mediated chemotaxis by lentiviral delivery of small interfering RNA.

Proceedings of the National Academy of Sciences of the United States of America ·Vol. 101 ·No. 2 ·2004-01-13 ·Pages 488-93

Hwang JI, Fraser ID, Choi S, Qin XF, Simon MI

Abstract

Immune cells respond to chemotactic signals by means of G protein-coupled receptors. Attempts to elucidate the function of specific G protein family members in these responses is complicated by redundancy among the different G protein isoforms. We have used lentiviral-based RNA interference to eliminate expression of specific G protein subunits selectively in J774A.1 mouse macrophages. The chemotactic response to the complement factors C5a and C3a is ablated in cells lacking G beta(2) but is unaffected in cells lacking G beta(1), G alpha i(2), or G alpha i(3). Similarly, the C5a-mediated calcium response of single cells is either absent or significantly delayed and weakened by G beta(2) knockdown. Assessment of Akt1 phosphorylation levels in response to C5a shows rapid and sustained phosphorylation in both wild-type cells and cells lacking G beta(1). Cells lacking G beta(2) retain the rapid response but cannot sustain phospho-Akt1 levels. The phenotype of cells lacking G beta(2) can be reversed by overexpression of either human G beta(2) or mouse G beta(1). These data demonstrate the usefulness of lentiviral-based RNA interference in the systematic analysis of a signaling pathway, and they suggest that in J774A.1 cells, G beta(2)-derived G beta gamma is the most effective mediator of chemotaxis to C5a.

MeSH Terms
Animals Base Sequence Cell Line Chemotaxis, Leukocyte/physiology Complement C5a/physiology DNA Primers Genetic Vectors Kinetics Lentivirus/genetics Mice RNA, Small Interfering/administration & dosage Reverse Transcriptase Polymerase Chain Reaction
Chemicals
DNA Primers RNA, Small Interfering Complement C5a
Authors & Affiliations
5 authors, click to expand affiliations / ORCID
Hwang Jong-Ik
Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
Fraser Iain D C
Choi Sangdun
Qin Xiao-Feng
Simon Melvin I
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Article Info
Journal
Proceedings of the National Academy of Sciences of the United States of America
Abbr.
Proc Natl Acad Sci U S A
ISSN
0027-8424
DOI
10.1073/pnas.0307549100
Published
2004-01-13
Epub
2003-00-29
Pages
488-93
Language
English
Region
United States
NLM ID
7505876
PMCID
PMC327174
Subset
IM
Grants
NIGMS NIH HHS · R37 GM034236 · United States
NIGMS NIH HHS · U54 GM062114 · United States
NIGMS NIH HHS · GM 62114 · United States
NIGMS NIH HHS · GM34236 · United States
External Links
PubMed source DOI: 10.1073/pnas.0307549100 PMC Full Text View journal details
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