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J. Biol. Chem., Vol. 283, Issue 3, 1610-1621, January 18, 2008

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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 2^*

Gregory P. Katsoulotos¹², Miao Qi¹, Jian Cheng Qi, Kumiko Tanaka, William E. Hughes, Timothy J. Molloy^¶, Roberto Adachi^||, Richard L. Stevens^**, and Steven A. Krilis³

From the 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, The Garvan Institute of Medical Research, 384 Victoria Street, Sydney, New South Wales 2010, Australia, the ^¶Blood Diseases and Cancer Research Laboratory, St Vincent's Hospital, New South Wales 2010, Australia, the ^||Department of Pulmonary Medicine, University of Texas M. D. Anderson Cancer Center, Houston, Texas 77030, and the ^**Department of Medicine, Harvard Medical School and Brigham and Women's Hospital, Boston, Massachusetts 02115

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 Phe⁵⁴⁸ 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-13R2 increased 61- to 860-fold in RasGRP4-expressing HMC-1 cells. A marked increase in IL-13R2 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.

Received for publication, August 22, 2007 , and in revised form, October 4, 2007.

^* This work was supported in part by a National Health and Medical Research Council (NHMRC) (Australia) Project Grant (to S. A. K.) and by National Institutes of Health Grants AI-54950 and HL-36110 (to R. L. S.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Tables S1-S10.

¹ Both authors contributed equally to this work.

² Supported by an NHMRC Medical Postgraduate Scholarship.

³ To whom correspondence should be addressed. Tel.: 61-29-350-2955; Fax: 61-29-350-3981; E-mail: S.Krilis{at}unsw.edu.au.

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