Analysis of C5a-mediated chemotaxis by lentiviral delivery of small interfering RNA.
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.
Hwang
Jong-Ik
JI
Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.
Fraser
Iain D C
ID
Choi
Sangdun
S
Qin
Xiao-Feng
XF
Simon
Melvin I
MI
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002634:Chemotaxis, Leukocyte / Q000502:physiology*; D015936:Complement C5a / Q000502:physiology*; D017931:DNA Primers; D005822:Genetic Vectors; D007700:Kinetics; D016086:Lentivirus / Q000235:genetics*; D051379:Mice; D034741:RNA, Small Interfering / Q000008:administration & dosage*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D034741:RNA, Small Interfering; D015936:Complement C5a