期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2004
卷号：101
期号：2
页码：488-493
DOI：10.1073/pnas.0307549100
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：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}i2, or G{alpha}i3. 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.
