Homer3 regulates the establishment of neutrophil polarity.
Most chemoattractants rely on activation of the heterotrimeric G-protein Gαi to regulate directional cell migration, but few links from Gαi to chemotactic effectors are known. Through affinity chromatography using primary neutrophil lysate, we identify Homer3 as a novel Gαi2-binding protein. RNA interference-mediated knockdown of Homer3 in neutrophil-like HL-60 cells impairs chemotaxis and the establishment of polarity of phosphatidylinositol 3,4,5-triphosphate (PIP3) and the actin cytoskeleton, as well as the persistence of the WAVE2 complex. Most previously characterized proteins that are required for cell polarity are needed for actin assembly or activation of core chemotactic effectors such as the Rac GTPase. In contrast, Homer3-knockdown cells show normal magnitude and kinetics of chemoattractant-induced activation of phosphoinositide 3-kinase and Rac effectors. Chemoattractant-stimulated Homer3-knockdown cells also exhibit a normal initial magnitude of actin polymerization but fail to polarize actin assembly and intracellular PIP3 and are defective in the initiation of cell polarity and motility. Our data suggest that Homer3 acts as a scaffold that spatially organizes actin assembly to support neutrophil polarity and motility downstream of GPCR activation.
Cardiovascular Research Institute and Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158.
Julie
J
Wu
Cardiovascular Research Institute and Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158.
Anne
A
Pipathsouk
Department of Cell Biochemistry, Groningen Biological Sciences and Biotechnology Institute, University of Groningen, 9700 AB Groningen, Netherlands.
A
A
Keizer-Gunnink
Department of Biochemistry and Netherlands Proteomics Centre, Groningen Biological Sciences and Biotechnology Institute, University of Groningen, 9700 AB Groningen, Netherlands.
F
F
Fusetti
NIZO Food Research, 6718 ZB Ede, Netherlands Centre for Molecular and Biomolecular Informatics, Radboud University Medical Center, Nijmegen, 6525 GA Nijmegen, Netherlands.
W
W
Alkema
Green Center for Systems Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390.
Shanshan
S
Liu
Green Center for Systems Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390.
Steven
S
Altschuler
Green Center for Systems Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390.
Lani
L
Wu
Department of Cell Biochemistry, Groningen Biological Sciences and Biotechnology Institute, University of Groningen, 9700 AB Groningen, Netherlands.
Arjan
A
Kortholt
Cardiovascular Research Institute and Department of Biochemistry and Biophysics, University of California, San Francisco, San Francisco, CA 94158 orion.weiner@ucsf.edu.
Orion D
OD
Weiner
D008841:Actin Cytoskeleton / Q000378:metabolism; D002352:Carrier Proteins / Q000502:physiology*; D016764:Cell Polarity; D002633:Chemotaxis; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D018922:HL-60 Cells; D000072233:Homer Scaffolding Proteins; D006801:Humans; D009504:Neutrophils / Q000502:physiology*; D034741:RNA, Small Interfering / Q000235:genetics; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism
D002352:Carrier Proteins; C000606616:HOMER3 protein, human; D000072233:Homer Scaffolding Proteins; D034741:RNA, Small Interfering; D043562:Receptors, G-Protein-Coupled