Caracterizaci´ on de la fuente secundaria

An increasing number of articles report the application of FRET-based assays to measure biochemical activity in cells, by utilising one o f the measurement methods described above, or combinations thereof (reviewed in Wouters et al., 2001). Some applications of FRET-based assays are summarised here.

1.4.7.1 Detecting the functional interactions of proteins

The direct association of physically distinct proteins can be detected by measuring FRET between separate donor- and acceptor-labelled proteins. In most of these experiments the physical interaction of the proteins has been previously demonstrated by immunoprécipitation, or via the use of covalent cross-linking reagents, and so the application of FRET to measure the interaction provides new information regarding the nature of the interaction under physiological circumstances or demonstrates a spatio- temporal aspect in cells. Protein-protein interactions which have been demonstrated using FRET microscopy include the interactions of: Bcl-2 with Bax (Mahajan et al., 1998), PK C a with p i integrin (Ng et al., 1999b) and Grb2 with the EGF receptor (Sorkin et al., 2000). Clustering of molecules into homo-oligomers can also be demonstrated with FRET microscopy (Fig. 1.5 B). For example, the detection of FRET between fluorescently labelled G PI-anchored proteins suggests that they form discrete subdomains in the plasma membrane, providing some of the most convincing proof, thus far presented, for the existence of lipid rafts in living cells (Varma & Mayor, 1998; Jacobson & Dietrich, 1999).

Monitoring the loss of FRET upon physical separation of donor- and acceptor- labelled components has allowed the oligomeric status of proteins to be studied within intact cells. For example, this approach has been used to confirm that the A-subunit of

___________________________________________________________________ Chapter 1 cholera toxin separates from the B-subunit in an endosomal/ Golgi compartment after endocytosis (Bastiaens et al., 1996) and that dissociation of the PKC p i regulatory and catalytic domains, after phorbol ester treatment, allows the catalytic subunit to selectively enter the nucleus (Bastiaens & Jovin, 1996).

1.4.7.2 Biochemical ‘activity sensors’

Biochemical ‘activity sensors’ are designed so that a change in FRET efficiency gives some measure of the activity of a specific protein or the concentration of a specific metabolite or ion. M ost sensors so far reported consist of donor and acceptor fluorophores that are conjugated to either end of a protein domain or subunit which changes its conformation upon ligand binding and results in a change in FRET efficiency (Fig. 1.5 C). The donor and acceptor fluorophores are generally spectral variants of the GFP protein and thus these sensors are genetically encoded, which offers numerous advantages, including the fact that the construct can be targeted to a specific organelle. Sensors based on this principle have been designed to detect calcium (Miyawaki et al.,

1997), cAMP (Nagai et al., 2000), nitric oxide (Pearce et al., 2000) and tyrosine phosphorylation (Ting et a l, 2001) in living cells.

The activation state of a given protein determines whether or not it can associate with a binding partner and this provides the basis for another type of sensor. For example, GTPases cycle between a GTP- and GDP-bound state which dictates whether or not they can associate with binding partners. By labelling the GTPase with a donor fluorophore and its GTPase-state conditional binding partner with an acceptor fluorophore, FRET between donor and acceptor can be used to gauge the activity of the GTPase cascade (Fig. 1.5 A, D). This approach has been applied to study the activity of the small GTPases Rac (Kraynov et al., 2000) and Ras (Mochizuki et al., 2001) and heterodimeric G- proteins (Janetopoulos et al., 2001) in living cells.

Finally, the activity of proteolytic enzymes can be studied by fusing donor and acceptor fluorophores to either end of a sequence which encodes the protease sensitive sequence. Proteolytic activity is detected by monitoring the loss of FRET when the sequence undergoes cleavage (Fig. 1.5 E). Sensors based on this principle have been applied to study the activity of the caspase family of enzymes in cells (Mahajan et al., 1999; Harpur et al., 2001).

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1.4.7.3 Detecting post-translational modifications

Here, the term post-translational modification is applied to any stable or reversible covalent modification to an amino acid side chain which occurs after synthesis of the protein. This includes, but is not limited to: acylation, glycosylation, phosphorylation, acétylation, méthylation and nucleotide binding. A protein is labelled with a donor fluorophore and the post-translational modification of that protein is detected by an acceptor-labelled antibody which specifically binds to the post-translational modification. FRET is detected only when the acceptor-labelled antibody is bound to the donor- conjugated protein (Fig. 1.5 F). Under circumstances where the covalent modification is reversible and modulates the activity of the protein, this assay can also be regarded as an activity sensor. The approach has been applied to monitor the phosphorylation status of PK C a and the EGF receptor in cells (Ng et a l, 1999a; Wouters & Bastiaens, 2000).

1.4.8 Summary and perspective

A number of strategies can be employed to utilise FRET as an assay of biochemical activity in cells. As a result, new insights are being made into biochemical pathways. Much of the work presented in this thesis utilises a FRET-based assay to monitor the phosphorylation status of the EGF receptor (ErbB l) in cells. In the final section of this general introduction the biology of the ErbB receptor family is discussed.