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Identification of a GTP-bound Rho specific scFv molecular sensor by phage display selection
Marine Goffinet1, Patrick Chinestra, Isabelle Lajoie-Mazenc
1NSERM U563, CPTP, Signalisation cellulaire, GTPase Rho et cancers, F-31052, Toulouse, France. goffinet.marine@claudiusregaud.fr
Background:
The Rho GTPases A, B and C proteins, members of the Rho family whose activity is regulated by GDP/GTP cycling, function in many cellular pathways controlling proliferation and have recently been implicated in tumorigenesis. Although overexpression of Rho GTPases has been correlated with tumorigenesis, only their GTP-bound forms are able to activate the signalling pathways implicated in tumorigenesis. Thus, the focus of much recent research has been to identify biological tools capable of quantifying the level of cellular GTP-bound Rho, or determining the subcellular location of activation. However useful, these tools used to study the mechanism of Rho activation still have limitations. The aim of the present work was to employ phage display to identify a conformationally-specific single chain fragment variable (scFv) that recognizes the active, GTP-bound, form of Rho GTPases and is able to discriminate it from the inactive, GDP-bound, Rho in endogenous settings.
Results:
After five rounds of phage selection using a constitutively activated mutant of RhoB (RhoBQ63L), three scFvs (A8, C1 and D11) were selected for subsequent analysis. Further biochemical characterization was pursued for the single clone, C1, exhibiting an scFv structure. C1 was selective for the GTP-bound form of RhoA, RhoB, as well as RhoC, and failed to recognize GTP-loaded Rac1 or Cdc42, two other members of the Rho family. To enhance its production, soluble C1 was expressed in fusion with the N-terminal domain of phage protein pIII (scFv C1-N1N2), it appeared specifically associated with GTP-loaded recombinant RhoA and RhoB via immunoprecipitation, and endogenous activated Rho in HeLa cells as determined by immunofluorescence.
Conclusion:
We identified an antibody, C1-N1N2, specific for the GTP-bound form of RhoB from a phage library, and confirmed its specificity towards GTP-bound RhoA and RhoC, as well as RhoB. The success of C1-N1N2 in discriminating activated Rho in immunofluorescence studies implies that this new tool, in collaboration with currently used RhoA and B antibodies, has the potential to analyze Rho activation in cell function and tumor development.
Insights
Researchers developed a new antibody, C1-N1N2, that specifically detects the active, GTP-bound forms of Rho GTPases (RhoA, B, and C). This tool aids in studying cell proliferation and tumor development by identifying activated Rho proteins.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Rho GTPases regulate cell proliferation and are implicated in tumorigenesis.
- Only the GTP-bound active forms of Rho GTPases drive tumorigenesis.
- Existing tools for studying Rho activation have limitations.
Purpose of the Study:
- To identify a conformationally-specific single chain fragment variable (scFv) using phage display.
- To develop a tool that recognizes the active, GTP-bound form of Rho GTPases.
- To discriminate between GDP-bound (inactive) and GTP-bound (active) Rho proteins in endogenous settings.
Main Methods:
- Phage display selection using a constitutively activated RhoB mutant.
- Biochemical characterization of selected scFvs.
- Expression of scFv C1 in fusion with phage protein pIII (scFv C1-N1N2).
- Immunoprecipitation and immunofluorescence assays.
Main Results:
- Three scFvs (A8, C1, D11) were identified; clone C1 was further analyzed.
- scFv C1 specifically recognized GTP-bound RhoA, RhoB, and RhoC, but not Rac1 or Cdc42.
- scFv C1-N1N2 associated with GTP-loaded recombinant RhoA/RhoB and endogenous activated Rho in HeLa cells.
Conclusions:
- An antibody, C1-N1N2, specific for GTP-bound Rho GTPases was identified.
- C1-N1N2 successfully discriminated activated Rho in immunofluorescence studies.
- This new tool can aid in analyzing Rho activation in cell function and tumor development.
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