Cloning and Characterization of GEF-H1, a Microtubule-associated Guanine Nucleotide Exchange Factor for Rac and Rho GTPases*
- From the ‡Department of Pharmacology, Baylor College of Medicine, Houston, Texas 77030 and the ¶Department of Biochemistry, University of Tennessee, Memphis, Tennessee 38163
Abstract
The Rho-related small GTPases are critical elements involved in regulation of signal transduction cascades from extracellular stimuli to cell nucleus and cytoskeleton. The Dbl-like guanine nucleotide exchange factors (GEF) have been implicated in direct activation of these GTPases. Here we have identified a new member of the Dbl family, GEF-H1, by screening a human HeLa cell cDNA library. GEF-H1 encodes a 100-kDa protein containing the conserved structural array of a Dbl homology domain in tandem with a pleckstrin homology domain and is most closely related to thelfc oncogene, but additionally it contains a unique coiled-coil domain at the carboxyl terminus. Biochemical analysis reveals that GEF-H1 is capable of stimulating guanine nucleotide exchange of Rac and Rho but is inactive toward Cdc42, TC10, or Ras. Moreover, GEF-H1 binds to Rac and Rho proteins in both the GDP- and guanosine 5′-3-O-(thio)triphosphate-bound states without detectable affinity for Cdc42 or Ras. Immunofluorescence reveals that GEF-H1 colocalizes with microtubules through the carboxyl-terminal coiled-coil domain. Overexpression of GEF-H1 in COS-7 cells results in induction of membrane ruffles. These results suggest that GEF-H1 may have a direct role in activation of Rac and/or Rho and in bringing the activated GTPase to specific target sites such as microtubules.
Footnotes
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↵* This work was supported by the Busch and DeBakey funds (to H. B.), National Institutes of Health Grant GM53943 (to Y. Z.), and a fund from the American Heart Association, Texas Affiliate (to Y. R.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank™/EMBL Data Bank with accession number(s) U72206.
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↵§ To whom correspondence should be addressed. Tel.: 713-798-7907; Fax: 713-798-3145; E-mail: yren{at}bcm.tmc.edu.
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↵2 Y. Ren and Y. Zheng, unpublished observations.
- Abbreviations:
- GEF
-
guanine nucleotide exchange factor
- DH
-
Dbl homology
- GST
-
glutathioneS-transferase
- ORF
-
open reading frame
- PH
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Pleckstrin homology
- PAGE
-
polyacrylamide gel electrophoresis
- Pipes
-
1,4-piperazinediethanesulfonic acid
- GTPγS
-
guanosine 5′-3-O-(thio)triphosphate.
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- Received August 10, 1998.
- Revision received October 15, 1998.
- The American Society for Biochemistry and Molecular Biology, Inc.
