The Diacylglycerol-dependent translocation of ras guanine nucleotide-releasing protein 4 inside a human mast cell line results in substantial phenotypic changes, including expression of interleukin 13 receptor alpha2.
Ras guanine nucleotide-releasing protein 4 (RasGRP4) is a mast cell (MC)-restricted guanine nucleotide exchange factor and diacylglycerol (DAG)/phorbol ester receptor. An RasGRP4-defective variant of the human MC line HMC-1 was used to create stable clones expressing green fluorescent protein-labeled RasGRP4 for monitoring the movement of this protein inside MCs after exposure to phorbol 12-myristate 13-acetate (PMA), and for evaluating the protein's ability to control gene expression. RasGRP4 resided primarily in the cytosol. After exposure to PMA, RasGRP4 quickly translocated to the inner leaflet of the cell's plasma membrane. 15-30 min later, this signaling protein translocated from the plasma membrane to other intracellular sites. The translocation of RasGRP4 from the cytosol to its varied membrane compartments was found to be highly dependent on Phe(548) in the protein's C1 DAG/PMA-binding domain. Extracellular signal-regulated kinases 1 and 2 were activated during this translocation process, and c-kit/CD117 was lost from the cell's surface. Transcript-profiling approaches revealed that RasGRP4 profoundly regulated the expression of hundreds of genes in HMC-1 cells. For example, the expression of the transcript that encodes the interleukin (IL) 13 receptor IL-13Ralpha2 increased 61- to 860-fold in RasGRP4-expressing HMC-1 cells. A marked increase in IL-13Ralpha2 protein levels also was found. The accumulated data suggest RasGRP4 translocates to varied intracellular compartments via its DAG/PMA-binding domain to regulate signaling pathways that control gene and protein expression in MCs, including the cell's ability to respond to IL-13.
Katsoulotos
Gregory P
GP
Department of Medicine, University of New South Wales, and Department of Immunology, Allergy, and Infectious Diseases, St. George Hospital, 2 South St. Centre, New South Wales 2217, Australia.
Qi
Miao
M
Department of Medicine, University of New South Wales, and Department of Immunology, Allergy, and Infectious Diseases, St. George Hospital, 2 South St. Centre, New South Wales 2217, Australia.
Qi
Jian Cheng
JC
Department of Medicine, University of New South Wales, and Department of Immunology, Allergy, and Infectious Diseases, St. George Hospital, 2 South St. Centre, New South Wales 2217, Australia.
Tanaka
Kumiko
K
Department of Medicine, University of New South Wales, and Department of Immunology, Allergy, and Infectious Diseases, St. George Hospital, 2 South St. Centre, New South Wales 2217, Australia.
Hughes
William E
WE
The Garvan Institute of Medical Research, 384 Victoria Street, Sydney, New South Wales 2010, Australia.
Molloy
Timothy J
TJ
Blood Diseases and Cancer Research Laboratory, St Vincent's Hospital, New South Wales 2010, Australia.
Adachi
Roberto
R
Department of Pulmonary Medicine, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030.
Stevens
Richard L
RL
Department of Medicine, Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts 02115.
Krilis
Steven A
SA
Department of Medicine, University of New South Wales, and Department of Immunology, Allergy, and Infectious Diseases, St. George Hospital, 2 South St. Centre, New South Wales 2217, Australia. Electronic address: S.Krilis@unsw.edu.au.
D002460:Cell Line; D004075:Diglycerides / Q000378:metabolism*; D004705:Endocytosis / Q000187:drug effects; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D018793:Interleukin-13 / Q000378:metabolism; D053663:Interleukin-13 Receptor alpha2 Subunit / Q000235:genetics / Q000378:metabolism*; D008407:Mast Cells / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3 / Q000378:metabolism; D010641:Phenotype; D010766:Phosphorylation / Q000187:drug effects; D021381:Protein Transport; D019009:Proto-Oncogene Proteins c-kit / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D050800:STAT6 Transcription Factor / Q000378:metabolism; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D020663:ras Guanine Nucleotide Exchange Factors / Q000235:genetics / Q000378:metabolism*
D004075:Diglycerides; D018793:Interleukin-13; D053663:Interleukin-13 Receptor alpha2 Subunit; C462507:RASGRP4 protein, human; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D050800:STAT6 Transcription Factor; D020663:ras Guanine Nucleotide Exchange Factors; D049452:Green Fluorescent Proteins; D019009:Proto-Oncogene Proteins c-kit; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D013755:Tetradecanoylphorbol Acetate