Characterization of the interaction between RhoGDI and Cdc42Hs using fluorescence spectroscopy - PubMed (original) (raw)
. 1996 Apr 26;271(17):10004-9.
doi: 10.1074/jbc.271.17.10004.
Affiliations
- PMID: 8626553
- DOI: 10.1074/jbc.271.17.10004
Free article
Characterization of the interaction between RhoGDI and Cdc42Hs using fluorescence spectroscopy
T K Nomanbhoy et al. J Biol Chem. 1996.
Free article
Abstract
The GDP-dissociation-inhibitor (GDI) for Rho-like GTP-binding proteins is capable of three different biochemical activities. These are the inhibition of GDP dissociation, the inhibition of GTP hydrolysis, and the stimulation of the release of GTP-binding proteins from membranes. In order to better understand how GDI interactions with Rho-like proteins mediate these different effects, we have set out to develop a direct fluorescence spectroscopic assay for the binding of the GDI to the Rho-like protein, Cdc42Hs. We show here that when the GDI interacts with Cdc42Hs that contains bound N-methylanthraniloyl GDP (Mant-GDP), there is an approximately 20% quenching of the Mant fluorescence. The GDI-induced quenching is only observed when Mant-GDP is bound to Spodoptera frugiperda-expressed Cdc42Hs and is not detected when the Mant nucleotide is bound to Escherichia coli-expressed Cdc42Hs and thus shows the same requirement for isoprenylated GTP-binding protein as that observed when assaying GDI activity. A truncated Cdc42Hs mutant that lacks 8 amino acids from the carboxyl terminus and is insensitive to GDI regulation also does not show changes in the fluorescence of its bound Mant-GDP upon GDI addition. Thus, the GDI-induced quenching of Mant-GDP provides a direct read-out for the binding of the GDI to Cdc42Hs. Titration profiles of the GDI-induced quenching of the Mant-GDP fluorescence are saturable and are well fit to a simple 1:1 binding model for Cdc42Hs-GDI interactions with an apparent Kd value of 30 nM. A very similar Kd value (28 nM) is measured when titrating the GDI-induced quenching of the fluorescence of Mant-guanylyl imidotriphosphate, bound to Cdc42Hs. These results suggest that the GDI can bind to the GDP-bound and GTP-bound forms of Cdc42Hs equally well. We also have used the fluorescence assay for GDI interactions to demonstrate that the differences in functional potency observed between the GDI molecule and a related human leukemic protein, designated LD4, are due to differences in their binding affinities for Cdc42Hs. This, together with the results from studies using GDI/LD4 chimeras, allow us to conclude that a limit region within the carboxyl-terminal domain of the GDI molecule is responsible for its ability to bind with higher affinity (compared with LD4) to Cdc42Hs.
Similar articles
- The identification and characterization of a GDP-dissociation inhibitor (GDI) for the CDC42Hs protein.
Leonard D, Hart MJ, Platko JV, Eva A, Henzel W, Evans T, Cerione RA. Leonard D, et al. J Biol Chem. 1992 Nov 15;267(32):22860-8. J Biol Chem. 1992. PMID: 1429634 - Investigation of the GTP-binding/GTPase cycle of Cdc42Hs using fluorescence spectroscopy.
Leonard DA, Evans T, Hart M, Cerione RA, Manor D. Leonard DA, et al. Biochemistry. 1994 Oct 11;33(40):12323-8. doi: 10.1021/bi00206a040. Biochemistry. 1994. PMID: 7918454 - Interaction between Cdc42Hs and RhoGDI is mediated through the Rho insert region.
Wu WJ, Leonard DA, A-Cerione R, Manor D. Wu WJ, et al. J Biol Chem. 1997 Oct 17;272(42):26153-8. doi: 10.1074/jbc.272.42.26153. J Biol Chem. 1997. PMID: 9334181 - Rho guanine dissociation inhibitors: pivotal molecules in cellular signalling.
Olofsson B. Olofsson B. Cell Signal. 1999 Aug;11(8):545-54. doi: 10.1016/s0898-6568(98)00063-1. Cell Signal. 1999. PMID: 10433515 Review. - A GDI/GDF-like system for sorting and shuttling ciliary proteins.
Ismail S. Ismail S. Small GTPases. 2017 Oct 2;8(4):208-211. doi: 10.1080/21541248.2016.1213782. Epub 2016 Aug 3. Small GTPases. 2017. PMID: 27487321 Free PMC article. Review.
Cited by
- Transient Structural Properties of the Rho GDP-Dissociation Inhibitor.
Medina Gomez S, Visco I, Merino F, Bieling P, Linser R. Medina Gomez S, et al. Angew Chem Int Ed Engl. 2024 Aug 19;63(34):e202403941. doi: 10.1002/anie.202403941. Epub 2024 Jul 24. Angew Chem Int Ed Engl. 2024. PMID: 38853146 Free PMC article. - In vitro fluorescence assay to measure GDP/GTP exchange of guanine nucleotide exchange factors of Rho family GTPases.
Blaise AM, Corcoran EE, Wattenberg ES, Zhang YL, Cottrell JR, Koleske AJ. Blaise AM, et al. Biol Methods Protoc. 2021 Dec 31;7(1):bpab024. doi: 10.1093/biomethods/bpab024. eCollection 2022. Biol Methods Protoc. 2021. PMID: 35087952 Free PMC article. - Isolation and Characterization of Heparan Sulfate from Human Lung Tissues.
Derler R, Kitic N, Gerlza T, Kungl AJ. Derler R, et al. Molecules. 2021 Sep 10;26(18):5512. doi: 10.3390/molecules26185512. Molecules. 2021. PMID: 34576979 Free PMC article. - Post-Translational Modification and Subcellular Compartmentalization: Emerging Concepts on the Regulation and Physiopathological Relevance of RhoGTPases.
Navarro-Lérida I, Sánchez-Álvarez M, Del Pozo MÁ. Navarro-Lérida I, et al. Cells. 2021 Aug 5;10(8):1990. doi: 10.3390/cells10081990. Cells. 2021. PMID: 34440759 Free PMC article. Review. - A Complete Survey of RhoGDI Targets Reveals Novel Interactions with Atypical Small GTPases.
Ahmad Mokhtar AMB, Ahmed SBM, Darling NJ, Harris M, Mott HR, Owen D. Ahmad Mokhtar AMB, et al. Biochemistry. 2021 May 18;60(19):1533-1551. doi: 10.1021/acs.biochem.1c00120. Epub 2021 Apr 29. Biochemistry. 2021. PMID: 33913706 Free PMC article.