Fluorescopic evaluation of protein-lipid relations in cellular signalling

IntroductionCellular communication is partly mediated through the modulation of protein activity, structure and dynamics by lipids. In contrast to the biochemical aspects of lipid signalling, relatively little is known about the physical properties of the "signal" lipids (lipids involved in cellular signalling) in membranes and their interaction with membrane proteins. Knowledge about these properties contributes to the understanding of the molecular mechanism of intracellular communication. The main objective of this thesis is to evaluate organisational and motional aspects of protein-lipid systems that play a role in this signalling. Fluorescence spectroscopy was employed to distinguish between interacting elements of the protein-lipid binding equilibrium from the non-interacting ones and to reveal their motional properties and characteristics in organisation. The first chapter of this thesis summarises the functional relevance and fundamental mechanisms of protein-lipid interactions and introduces some fluorescopic tools which can be applied in these investigations. In the remaining chapters the biophysical properties of the phosphoinositides phosphatidylinositol (PI), phosphatidylinositol- 4-phosphate (PIP) and phosphatidylinositol-4, 5-biphosphate (PIP 2 ) and diacylglycerol (DG) and their relation to protein kinase C (PKC) and Band 3 (an abundant anion exchanging protein in erythrocyte membranes) form a central theme. Three types of binding are evaluated: 1) peripheral membrane binding of proteins, 2) lipids replacing other lipids at the protein surface and 3) lipids which bind to vacant cofactor sites at the protein. By combining complementary fluorescopic techniques, and by refinement of fluorescence data analysis, new aspects of protein-lipid relations have been elucidated which are summarised below. As a model study and introduction for the protein-lipid studies described in this thesis, Chapter 2 considers the association of lysozyme with acidic vesicles. From the experiments it could be concluded that the protein conformation and dynamics of lysozyme are effected when the protein interacts with acidic membranes. Apart from the general interest in protein-lipid interactions, this study demonstrates that a fractional analysis of time-resolved fluorescence and fluorescence anisotropy decay curves can provide accurate binding curves of protein molecules to lipid interfaces.The interaction of PKC with PS and phosphoinositides and DGVarious aspects of the application of fluorescence anisotropy in the evaluation of lipid motion are introduced in Chapter 3. In that Chapter the motional characteristics of three 1, 6- diphenyl-1, 3, 5-hexatriene (DPH) labelled phosphatidylcholines are compared.Based on the results of this study the effect of PKC on the reorientational properties of fluorescent analogues of DG and PC have been evaluated using a constrained model of analysis (Chapter 4). It appeared that in membranes DPH-DG experiences a larger motional freedom than DPH-PC, which indicates that DG induces a lipid packing irregularity. Defects in membrane organisation lead to a partial exposure of hydrophobic regions of phospholipids to aqueous environment and thereby increase the ground state energy of bilayers (Jain & Zakim, 1987). Therefore the activation energy associated with insertion of PKC into the hydrophobic membrane core could be reduced by DG. Three complementary observations provide evidence that PKC is able to interact with PS containing membranes in the absence of calcium:1) The isotropic rotation of PS containing micelles slows down upon addition of PKC (Chapter 4).2) Micelles and vesicles containing pyrene labelled lipids quench the tryptophan fluorescence of PKC (Chapter 5).3) PKC significantly reduces the collision frequency of pyrene labelled PS (Chapter 5).Addition of calcium results in the binding of PKC to DG (Chapter 6). For this DG-PKC interaction the presence of PS is a prerequisite (Chapters 4, 5, 6). The precise mechanism of this calcium dependence of DG binding remains unclear. Calcium enhances the binding of PKC to PS. As a result of this enhanced PS binding the probability of finding a DG molecule close to PKC may increase since PKC molecules retain longer at the membrane surface. Alternatively, calcium may induce changes in PKC that result in exposure of a shielded lipid cofactor site for DG. As a consequence of the interaction with PKC, the lateral and rotational diffusion and orientational freedom (Chapters 4 and 5) of labelled DG are reduced. The interaction with DG is highly specific, since the motional properties of labelled PC, PS and PI analogues are only moderately effected by PKC (Chapters 4 and 5) and these lipids are not able to quench PKC fluorescence at low concentrations (Chapter 6). However, labelled phosphoinositides PIP and PIP 2 partly mimic the properties of DG. They bind with high affinity to PKC and the acyl chain dynamics of (dipyr)PIP and (dipyr)DG are equally effected by PKC (Chapters 5 and 6). In addition, like is known for DG, only a very limited number of phosphoinositides bind to one PKC molecule (Chapter 6). Double labelling experiments and replacement studies suggest that PIP and DG can bind simultaneously to PKC while PIP 2 and DG cannot (Chapters 5 and 6). In addition, it appeared that only PIP 2 and DG effectively activate PKC. Combined, these results indicate a cofactor role of PIP 2 for PKC.Although the role of PIP 2 in the activation of PKC has to be established in vivo , its properties do fit excellently to a messenger function. Radioactive labelling studies with H-inositol and P-phosphate revealed that the turnover of PIP 2 is extremely rapid and in resting cells its level is low (reviewed by Hokin, 1985). The major part of PIP 2 present at resting conditions, may even be not available for PKC activation. Several studies have shown that the inositol lipids are heterogeneously distributed in the different cellular membranes and that only a small fraction of the total inositol lipid content is available for enzymes like phospholipase C (see for a review Downes & Michell, 1985). Like most other signalling lipids the PIP 2 concentration increases transiently to high levels within seconds after hormone stimulation of cells (Hansen et al., 1986). The dual role of PIP 2 as a DG-precursor and as a direct PKC-activator opens new avenues for the differentiation of signals via the various PKC families. The cell responses that induce the PLC catalysed hydrolysis of phosphoinositides always result in the co-appearance of DG and calcium. Several PKC-isoforms, however, do no need calcium for their activity. Other lipid cofactors like PIP 2 may function as cofactors for these calcium independent protein kinases. For the calcium dependent protein kinases an inositol triphosphate (IP 3 ) mediated calcium release would imply the production of DG.When calcium is provided via calcium channels in the plasma membrane or via sphingosine phosphate signalling (Dessai et al., 1992), no DG is released and other lipid activators like PIP 2 can take over the cofactor role. In this DG-independent activation, the temporal availability of calcium in the cytosol is the main switch for PKC activity. In this respect, it would be interesting to know if there are situations in the cell in which the PIP-kinase and (phosphoinositide-specific) phospholipase C activities are uncoupled. In that case the levels of PIP 2 can be regulated independently from those of DG, which would make the double role of PIP 2 in the regulation of PKC activity even more attractive (Figure 1).Heterogeneous distribution of phosphoinositides in membranesIn Chapter 7, the interaction of the phosphoinositides with human erythrocyte Band 3 protein was characterised using resonance energy transfer. The efficiency of quenching of the fluorescence from tryptophan residues in the protein by pyrene lipids in the membrane decreased in the order pyrPIP 2 , pyrPIP, pyrPI and pyrPC indicating different spatial distributions of these lipids with respect to Band 3. Global analysis of the quenched tryptophan fluorescence decays yielded indices for the spatial distributions of the different lipids. It appeared that the charged lipids PIP 2 and PIP spend longer time in the vicinity of Band 3 than PI and PC. In investigations complementary to these protein fluorescence observations, the monomer/excimer fluorescence of pyrene labelled phosphoinositides in DOPC membranes was evaluated in the absence and presence of Band 3 protein (Chapter 8). In combination with theoretical refinements of the model for lipid migration, these experiments revealed estimates for the lateral dynamics and lipid-lipid repulsion in membranes. In addition, the relative binding constants and the minimum number of Band 3 sites possessing affinity for the phosphoinositides could be roughly estimated. Consistent with intuitive expectations the lipid-lipid repulsion appeared highly dependent on the amount of negative charge of the lipid headgroups. Combining these experiments indicates that lipid-lipid repulsion and the Band 3-lipid interactions induce a non- random distribution of the Phosphoinositides. This non-random distribution will probably effect the accessibility of the inositol lipids for phospholipase C and for other phosphoinositide dependent proteins like PKC and may result in the functional heterogeneity of the phosphoinositides (Downes & Michell, 1985).