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

Hwang, Jong-Ik and Fraser, Iain D. C. and Choi, Sangdun and Qin, Xiao-Feng and Simon, Melvin I. (2004) 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, 101 (2). pp. 488-493. ISSN 0027-8424. http://resolver.caltech.edu/CaltechAUTHORS:HWApnas04a

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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.


Item Type:Article
Additional Information:Copyright © 2004 by the National Academy of Sciences. From the cover. Contributed by Melvin I. Simon, November 14, 2003. Published online before print December 29, 2003, 10.1073/pnas.0307549100 This work was supported by National Institutes of Health Grants GM34236 (to M.I.S.) and GM 62114 (to I.D.C.F. and S.C. under the Alliance for Cellular Signaling Cooperative Agreement), and by Damon Runyon–Walter Winchell Fellowship DRG-1568 (to X.-F.Q.).
Subject Keywords:BETA-GAMMA-SUBUNITS, CULTURED-MAMMALIAN-CELLS, HUMAN MAST-CELLS, SIGNAL-TRANSDUCTION, PHOSPHOLIPASE-C, RECEPTORS, C5A, ACTIVATION, CHANNEL, ROLES
Record Number:CaltechAUTHORS:HWApnas04a
Persistent URL:http://resolver.caltech.edu/CaltechAUTHORS:HWApnas04a
Alternative URL:http://dx.doi.org/10.1073/pnas.0307549100
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:1373
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:13 Jan 2006
Last Modified:26 Dec 2012 08:44

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