CSF-1 receptor structure/function in MacCsf1r-/- macrophages: regulation of proliferation, differentiation, and morphology.
CSF-1 is the major regulator of tissue macrophage development and function. A GM-CSF-dependent, CSF-1 receptor (CSF-1R)-deficient F4/80(hi)Mac-1(+)Gr1(-)CD11c(+) bone marrow macrophage (BMM) line (MacCsf1r-/-) was developed to study the roles of the eight intracellular CSF-1R tyrosines phosphorylated upon receptor activation. Retroviral expression of the wild-type CSF-1R rescued the CSF-1-induced survival, proliferation, differentiation, and morphological characteristics of primary BMM. Mutation of all eight tyrosines failed to rescue, whereas the individual Y --> F mutants (544, 559, 697, 706, 721, 807, 921, 974) rescued these CSF-1-inducible phenotypes to varying degrees. The juxtamembrane domain Y559F and activation loop Y807F mutations severely compromised proliferation and differentiation, whereas Y706, Y721F, and Y974F mutations altered morphological responses, and Y706F increased differentiation. Despite their retention of significant in vitro tyrosine kinase activity, Y559F and Y807F mutants exhibited severely impaired in vivo receptor tyrosine phosphorylation, consistent with the existence of cellular mechanisms inhibiting CSF-1R tyrosine phosphorylation that are relieved by phosphorylation of these two sites. The MacCsf1r-/- macrophage line will facilitate genetic and proteomic approaches to CSF-1R structure/function studies in the major disease-related CSF-1R-expressing cell type.
Yu
Wenfeng
W
Department of Developmental and Molecular Biology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461, USA.
Chen
Jian
J
Xiong
Ying
Y
Pixley
Fiona J
FJ
Dai
Xu-Ming
XM
Yeung
Yee-Guide
YG
Stanley
E Richard
ER
D000818:Animals; D015153:Blotting, Western; D001854:Bone Marrow Cells / Q000378:metabolism; D002454:Cell Differentiation / Q000502:physiology*; D049109:Cell Proliferation; D002470:Cell Survival / Q000502:physiology; D002478:Cells, Cultured; D017077:Culture Media, Conditioned / Q000494:pharmacology; D007074:Immunoglobulin G / Q000276:immunology; D047468:Immunoprecipitation; D016173:Macrophage Colony-Stimulating Factor / Q000494:pharmacology; D008264:Macrophages / Q000166:cytology* / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D016297:Mutagenesis, Site-Directed; D009154:Mutation / Q000235:genetics; D010446:Peptide Fragments / Q000276:immunology; D010641:Phenotype; D010766:Phosphorylation; D011817:Rabbits; D016186:Receptor, Macrophage Colony-Stimulating Factor / Q000502:physiology*; D012190:Retroviridae / Q000235:genetics; D015398:Signal Transduction; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism
D017077:Culture Media, Conditioned; D007074:Immunoglobulin G; D010446:Peptide Fragments; D014443:Tyrosine; D016173:Macrophage Colony-Stimulating Factor; D016186:Receptor, Macrophage Colony-Stimulating Factor