Fluoride Activation of the Rho Family GTP-binding Protein Cdc42Hs*
- From the Departments of ‡Pharmacology and§Chemistry, Cornell University, Ithaca, New York 14853
Abstract
Aluminum tetrafluoride (AlF4 −) activation of heterotrimeric G-protein α-subunits is a well established aspect of the biochemistry of these proteins; however, until recently it has been thought that AlF4 −does not mediate effects on the Ras superfamily of low molecular weight GTP-binding proteins. Recent work demonstrating aluminum fluoride-induced complex formation between Ras and its GTPase-activating proteins (RasGAP and NF1) has provided important insights into the mechanism of GAP-stimulated GTP hydrolysis. We have characterized the AlF4 −-induced complex formation between the GDP-bound form of the Rho subfamily G-protein Cdc42Hs and a limit functional domain of the Cdc42-GAP using a variety of biochemical techniques. Our results indicate that the apparent affinity of GAP for the AlF4 −-mediated complex is similar to the affinity observed for the activated (GTP-bound) form of Cdc42 and that beryllium (Be) can replace aluminum in mediating fluoride-induced complex formation. Additionally, the AlF4 −-induced interaction is weakened significantly by the catalytically compromised GAP(R305A) mutant, indicating that this arginine is critical in transition state stabilization. Unlike Ras, we find that AlF4 −and BeF3 −mediate complex formation between Cdc42Hs·GDP and downstream target/effector molecules, indicating that there are important differences in the mechanism of effector binding between the Ras and Rho subfamily G-proteins.
Footnotes
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↵* These studies were supported by National Institutes of Health Grants EY06429 and GM47458 and by a grant from the Human Frontiers of Science Program.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.
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↵¶ To whom correspondence should be addressed. Tel.: 607-253-3650; Fax: 607-253-3659.
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↵1 The abbreviations used are: GAP(s), GTPase-activating protein(s); GTPγS, guanosine 5′-3-O-(thio)triphosphate; MOPS, 4-morpholinepropanesulfonic acid; PBD, PAK binding domain; PAGE, polyacrylamide gel electrophoresis; mant, N-methylanthraniloyl; GppNHp, guanosine 5′-(β,γ-imido)triphosphate; GMP-PNP, guanosine 5′-(β,γ-imino) triphosphate; GIP, GTPase inhibitory protein.
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↵2 D. A. Leonard, R. A. Cerione, and D. Manor, manuscript in preparation.
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- Received August 15, 1997.
- Revision received November 21, 1997.
- The American Society for Biochemistry and Molecular Biology, Inc.
