Servizio Comunitario di Informazione in materia di Ricerca e Sviluppo - CORDIS

1. FRET assay developed for in vitro and in vivo use to screen for inhibitors of interactions of G-protein activating proteins

In their active, GTP bound forms, Rac and Cdc42 can interact with a fusion protein in which residues 75-18 of PAK are inserted in between the donor GFP (EBFP) and acceptor GFP (EGFP) and thereby change the FRET between the GFP fluorophores. The work on this system using purified proteins, which was described in the first year report, was written up and published. A key aim of this work is to be able to use the FRET system within living cells. We are attempting to demonstrate its utility in both intact bacterial and in mammalian cells. The former has the advantage of being easier to manipulate and allowing very high expression levels. It was felt that this might aid development of the appropriate technology for measurement of FRET and to understand the limiting sensitivity. Although, a bacterial system could be used as a drug screen, because the cell permeability differs from that of mammalian cells a mammalian cell system would be more acceptable.

Accordingly, we have begun the cloning process to set up an intracellular bacterial assay to monitor the interaction between EGFP-PAK-EBFP and Rac via FRET. In order to allow independent regulation of the co-expression of Rac and EGFP-PAK-EBFP suitable compatible vectors must be selected. Two such vectors are the pPROTet and pPROLar vectors supplied by Clontech that are inducible using tetracycline and IPTG/arabinose, respectively. However, in our hands these low copy number vectors are difficult to handle and, despite numerous attempts, we have had problems cloning Rac and PAK into them and then obtaining significant expression. In contrast, using standard expression vectors, e.g. pGEX and pET, the expression of both proteins is very high. Hence, we have decided to rethink the strategy and to select a further two compatible vectors, probably based on pGEX and pBAD expression systems, which have a good track record for expression.

In parallel, we have been setting up the same FRET system in mammalian cells. EGFP-PAK-EBFP was cloned into the mammalian expression vector, pcDNA-4 (Invitrogen) to generate a His-tagged fusion. HEK293T cells were transiently transfected with this vector. 24-86h later cells were harvested and the lysate analysed by Western Blot analysis using an antibody against the His-Tag and by recording fluorescence emission spectra. The blots showed a single band of the expected mobility for the construct and no indication of any degradation. This is of significance as there was concern that the relatively unstructured linker region containing PAK might be particularly susceptible to proteolysis. The transfected cells were significantly fluorescent as compared to mock-transfected cells.

A key issue with the transiently transfected cells is the variable level of expression from one cell to another. A lot of time has been spent in optimising the transfection procedure, but it is felt that this is intrinsically too variable to allow effective cotransfection with Rac. Hence we have initiated work on expression using the BacMam system, based on viral transduction.

Work on a more artificial system based on the Q61L constitutively activated Rac was completed. Furthermore we have decided to use a construct in which the CAAX motif has been deleted; this will both serve to prevent translocation to the plasma membrane and to prevent prenylation and hence interaction with RhoGDI. This would likely prevent interaction with EGFP-PAK-EBFP. The Rac has been cloned into pcDNA-4 and expression trials will begin shortly. The same construct will also be put into the BacMam system for co-expression with EGFP-PAK-EBFP.

The final experiments involved the titration of Q61LRac.GTP into each of the four proteins and measuring the two "FRET" ratios. The data obtained with cPAK were very similar to that we obtained previously (Graham et. al., Anal. Biochem. (2001) 296, 208-217 giving the anticipated Kd. The R(509/444) decreased from 3.9 to a minimum value of 2.3 at a concentration of Rac of about 4mM (EC50 1mM), whereas thrombin cleavage caused a larger decrease to a ratio of 1.0. Thus, Rac binding does not result in a complete separation of the GFP fluorophores. Addition of up to 20mM Rac.GTP did not cause any effect on the fluorescence properties of either EGFP-PAK-Rac-TCS-EBFP or EGFP-PAK-EBFP-Rac. If the interaction between Rac and PAK on separate molecules (i.e. intermolecular) was the only protein: protein interaction present, simulation to a Competition model showed that 10mM Rac should be sufficient to block this homodimerization.

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Peter N. LOWE, (Dr)
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