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

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 Gbeta(2) but is unaffected in cells lacking Gbeta(1), Galphai(2), or Galphai(3). Similarly, the C5a-mediated calcium response of single cells is either absent or significantly delayed and weakened by Gbeta(2) knockdown. Assessment of Akt1 phosphorylation levels in response to C5a shows rapid and sustained phosphorylation in both wild-type cells and cells lacking Gbeta(1). Cells lacking Gbeta(2) retain the rapid response but cannot sustain phospho-Akt1 levels. The phenotype of cells lacking Gbeta(2) can be reversed by overexpression of either human Gbeta(2) or mouse Gbeta(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, Gbeta(2)-derived Gbetagamma is the most effective mediator of chemotaxis to C5a.