Discussion

the GFP signals, altering the resulting BRET-ratios. All this, together with the points discussed in the next section, made interpretation of the data unreliable. Experiments in this direction were therefore temporarily discontinued.

Figure 3.9: Effect of stimulation on BRET-Ratio- HEK cells were transfected with either the EGFRwE4 BRET pair (a) or EGFRwE4-Luc alone (b). The BRET-ratio was measured at the indicated time after EGF addition and is plotted without subtraction of the background BRET-ratio, to allow visualisation of the results of the donor only samples. A decrease in BRET-ratio in the first minutes after stimulation is visible ina, but variability in the same magnitude was detected in cells expressing the donor only (b). n = 5.

the RLuc signal can only be improved by increasing the transfection amounts or the number of cells per sample. Unfortunately, none of these options was applicable with the experimental conditions of this project.

Competition experiments, in which unlabelled protein is coexpressed with the BRET-pair, are often used as a control of specificity⁴⁹. Yet, for my system the control was unsatisfying for two main reasons. First, I could transfect the cells with only very limited amount of untagged protein DNA and, second, transfection with up to four different plasmids lead to variable ratio of RLuc- and GFP-protein expression. The use of cells stably expressing the donor and acceptor proteins would minimize these prob-lems. Possibly, higher expression of the unlabelled protein could be achieved and, at the same time, the expression ratio of the two BRET-proteins should be less variable.

Because the establishment of stable cell lines is particularly work intensive, this would be an interesting option only if further experiments are planned with the cell line.

As a second strategy, an indirect interaction analysis was performed. In this set up, donor and acceptor proteins were both receptor chimeras and the effect of ARNO overexpression was monitored. In both the EGFR-Luc/GFP and HER2-Luc/HER3-GFP expressing cells BRET-ratios higher as for the controls were measured. Interest-ingly, the EGFR BRET-pair gave a BRET signal almost 3-times higher than that of the HER2/HER3 pair (Fig. 3.4) and similar to that of the constitutive dimeric IGFR (Fig. 3.6). Even if the absolute BRET-signal is usually not comparable between differ-ent BRET-pairs (since energy transfer is dependdiffer-ent also on the relative position and orientation of the fluorophores dipoles, which vary from pair to pair) the structural similarity of the receptors studied is tempting to speculation. Indeed, the finding that the EGFR isoform used can actually not be stimulated (Fig. 3.8) because it lacks the exon 4, which is involved in EGF binding, suggests that the energy transfer observed could be a measure of dimerised EGF receptor. This hypotesis is supported by a very recent publication which reports identification of an exon 4-deletion variant of EGFR in gliomas, ovarian cancer tissues and prostate cancer tissues⁵². This mutant displayed minimal EGF binding activity and underwent ligand-independent autophosphorylation and self-dimerisation.

Founding on these results, it is interesting to note that overexpression of ARNO increased BRET between the EGFR pair. This implicate an effect of ARNO on already dimerised receptors, as we could show later with other methods⁵⁰. Nevertheless, these

results must be taken with caution, since they are only valid under the assumption that the expression ratio of RLuc and GFP labelled receptor is constant within a single experiment. In fact, this experiment again requires transfection of at least three differ-ent plasmids, a condition which can lead to variations in expression ratio. Moreover, the activity of ARNO itself affect EGFR expression, since activation of the receptor by ARNO overexpression enhances endocytosis.

Experiments were performed to analyse the effect of EGFR stimulation on BRET between EGFR-Luc and EGFR-GFP. With the current knowledge about the charac-teristics of the exon-4 deletion variant of the EGFR (Fig. 3.8 and Ref. 52), it is clear that no stimulation influence could be detected. With respect to the new constructs EGFRwE4, which were shown to be stimulatable, the analysis is more complicated.

Indeed, at first glance, one could interpret the results in Figure 3.9a as stimulation dependent change of BRET-ratio. The fact that the ratio is decreasing and not in-creasing, as one would intuitively expect, can be explained by conformational changes in the EGFR C-terminus, and consequent changes in the fluorophores relative orienta-tion, which overwhelm the effect of dimerisation (a similar result was shown by Yang et al. in a luciferase fragment complementation assay⁶⁰). In fact, analysis of the raw data showed a high variability in the luciferase and GFP signals upon EGF stimulation without detectable trends. Again, having to deal with very low signals increased the gravity of these random variations. Additionally, it was observed that the RLuc sig-nal observed for aliquots of the same cell population decreased rapidly with the time.

This was caused by deterioration of the DeepBlueeC solution in the instrument. Thus, for comparable luminescence, repeated priming of the instrument was needed during a measuring series. Still, the BRET² system used was not adequate for the analysis of stimulation. Ideally, one would monitor the changes of BRET after stimulation in a single sample. Unfortunately, luminescence induced by DeepBlueC is very short lived and allows only a single measurement per sample. The use of BRET¹ or eBRET, in which the substrates Coelenterazine h or EnduRen, respectively, allow detection for up to one to several hours⁴⁹, would therefore be more appropriate.

Surface plasmon resonance

As an alternative method to investigate a possible interaction of cytohesins and ErbB receptors, surface plasmon resonance (SPR) bioanalysis was chosen because it offers the chance to follow binding events in real time and thus determine a range of interaction characteristics, like association and dissociation rates. These experiments are described in Section 4.3.1.

The small molecule SecinH3 allowed the identification of new roles of cytohesins^{17, 50}, which, in turn, generated interest in SecinH3 derivatives with improved activity and solubility. In Section 4.3.2, the establishment of an SPR based platform for the analysis of the binding properties of these new compounds is described.

Although SPR has been used since the early Nineties for the analysis of biomolecular interactions, still various misconceptions are diffused. Thus, I will start with a detailed introduction to the theory of SPR (Section 4.1) and its application in affinity biosensors (Section 4.2).

4.1 Physics of surface plasmon resonance

Surface plasmon resonance is a phenomenon that occurs in planar metal-dielectric waveguides, as for example a metal/water interface⁶¹. Surface plasmons are electrons oscillations which propagate parallel to the interface and are usually generated by means of a prism coupler and the attenuated total reflection method⁶¹. Our device is using the Kretschmann geometry, in which the metal film is evaporated directly onto the prism^{61, 62}. When illuminated, the metal film reflects part of the light back into the

prism while a part of the light propagates in the metal as a so-called evanescent wave (an inhomogeneous electromagnetic wave which decays exponentially in the direction perpendicular to the prism-metal interface)⁶¹. If the metal film is sufficiently thin, the evanescent wave penetrates through it. For each wavelength, a single angle of incidence leads to excitation of surface plasmons at the outer boundary of the metal film via the evanescent wave field. As a result, SPR is seen as a drop in the intensity of the reflected light (Fig. 4.1)^{61, 62}.

Figure 4.1: The SPR angle - At a certain combination of wavelength and angle, the incident light excites plasmons in the gold film. As a result, a characteristic absorption of energy via the evanescent wave field occurs and SPR is seen as a drop in the intensity of the reflected light.⁶²Reprinted fromBiacore - Sensor surface handbook(Ref. 62), copyright 2005-2007 GE Healthcare Bio-Sciences AB.

Application of the perturbation theory to the electromagnetic theory of optical waveguides demonstrate that the propagation of surface plasmons is highly sensitive to changes in the refractive index at the boundary⁶¹, such as those resulting from adsorption of molecules to the metal surface. This is the property which is exploited in optical sensors based on surface plasmons⁶³, such as Biacore⁶².

Our instrument, a Biacore 3000, is an SPR sensor with angular modulation. That is, it uses monochromatic light for excitation and monitors the reflected light intensity at multiple angles of incidence. Resonance is thus detected as a dip in the intensity of the reflected light (Fig. 4.1a) and the sensor output is the angle of incidence yielding the lowest intensity (Fig. 4.1b)^{62, 63}. Changes in this angle are expressed in Resonance Units (RU).