Views on membrane protein clustering...
First I tell you what the textbook dogma is, and then how differently I imagine the phenomenon.
The widely accepted dogma:
receptors in the plasma membrane are monomeric and inactive in the absence of stimulation. Ligand binding induces receptor dimerization leading to transmembrane signaling.
- Receptors are usually clustered even in the absence of stimulation. We and others have presented several lines of evidence for the existence of preformed receptor clusters. The size of these clusters (i.e. the diameter of the cluster and the number of proteins/cluster) and the fraction of clustered proteins (i.e. how many % of a given protein species is present in clusters and as monomers) is determined by the protein and the lipid environment.
- Several classes of clusters exist.Clusters come in different sizes. Dimers, trimers, tetramers and even larger associations up to hundreds of molecules populate the cell membrane. As can be seen in the figure below these clusters differ in how they are held together and what their functions are. Investigators must keep in mind that the methods used for clustering measurements exhibit certain selectivity for cluster sizes.
- Clusters are dynamic. Clusters are in a constant, ligand-induced and ligand-independent (constitutive) turnover. This statement holds for (most) small- and large-scale clusters. The cell membrane is not in thermodynamic equilibrium; therefore the continuously changing landscape is not surprising.
A couple of my papers supporting the above:
- Activation-dependent clustering of the erbB2 receptor tyrosine kinase detected by scanning near-field optical microscopy. J. Cell Sci. 112: 1733-1741 (1999)
- Cell fusion experiments reveal distinctly different association characteristics of cell-surface receptors. J. Cell Sci., 114: 4063-4071 (2001)
- Lipid rafts and the local density of ErbB proteins influence the biological role of homo- and heteroassociations of ErbB2. J. Cell Sci. 115: 4251-4262 (2002)
- Quantitative characterization of the large-scale association of ErbB1 and ErbB2 by flow cytometric homo-FRET measurements, Biophys. J. 95:2086-96 (2008)
- Coclustering of ErbB1 and ErbB2 revealed by FRET-sensitized acceptor bleaching. Biophys. J. 99:105-114 (2010)
- Distribution of resting and ligand-bound ErbB1 and ErbB2 receptor tyrosine kinases in living cells using number and brightness analysis. Proc Natl. Acad. Sci. USA, 107: 16524-16529 (2010)
Receptor-oriented drugs can be divided into two groups: low molecular weight tyrosine kinase inhibitors and monoclonal antibodies. Both of them are available against ErbB2, the member of the ErbB family which is often overexpressed in breast cancer leading to poor prognosis. Antibodies against growth factor receptors can grossly exert their action by one of the two mechanisms below:
- Signaling hypothesis: they inhibit ligand binding to the receptor, alter the conformation of the receptor or block the interaction of the receptor with other signaling molecules in the membrane. Since ErbB2 has no ligand ("orphan" receptor), the first option doesn't apply to ErbB2.
- Immune hypothesis: the cancer cell-bound antibody activates the complement system (CDC - complement-dependent cytotoxicity) or recruits cells armed with Fc receptors (ADCC - antibody-dependent cellular cytotoxicity).
Trastuzumab (Herceptin®) is a humanized monoclonal antibody against ErbB2. It exerts its action by altering/inhibiting the signaling mediated by ErbB2 (mechanism 1), but it is also known to cause immune system-mediated attack against the cancer (mechanism 2). Unfortunately, the development of resistance against trastuzumab is practically inevitable. Several assumptions have been put forward to explain the development of resistance (summarized in the following papers: Nahta and Esteva, Cancer Lett, 232: 123 (2006); Mukohara, Cancer Sci.,102: 1 (2011); Pohlmann et al., Clin. Cancer Res., 15: 7479 (2009).
We identified epitope masking as a mechanism leading to trastuzumab resistance. The expression of the large cell surface mucin MUC4 was shown to inhibit the binding of the antibody to ErbB2-overexpressing cells.We showed that pericellular hyaluronan also blocks trastuzumab binding and inhibition of hyaluronan synthesis increased the efficiency of trastuzumab treatment in experimental animals. We extended these investigations to clinical samples of ErbB2-overexpressing patients and showed that binding of trastuzumab to ErbB2 was inhibited by a high pericellular density of hyaluronan. Hyaluronidase treatment of the samples significantly increased trastuzumab binding.
Our hypothesis: Trastuzumab
resistance is caused by a variety of factors. Among these, epitope
masking seems to be important. MUC4 and hyaluronan can sterically
block the access of antibodies (and effector cells) to the tumor.
Alternatively, (especially hyaluronan) is expected to increase the
interstitial pressure around tumor cells inhibiting the transport of
molecules from blood vessels to the tumor. Our results imply that
inhibition of hyarluronan synthesis or induction of its breakdown can
significantly increase the potency of trastuzumab, and potentially other
drugs, in the treatment of breast cancer.
My papers supporting the above claim:
- Decreased accessibility and lack of activation of ErbB2 in JIMT-1, a Herceptin-resistant, MUC4-epxressing breast cancer cell line. Cancer Res., 65: 473-482 (2005)
- Hyaluronan-induced masking of ErbB2 and CD44-enhanced trastuzumab internalisation in trastuzumab resistant breast cancer. Eur. J. Cancer, 43:2423-2433 (2007)
- Binding of trastuzumab to ErbB2 Is inhibited by a high pericellular density of hyaluronan. J. Histochem. Cytochem. 60: 567-575 (2012)
Methods developed, theories conceived...
Quantitative ratiometric FRET measurements in microscopy:
Although methods abound for the measurement of FRET, ratiometric measurements, i.e. the determination of the fluorescence intensities in the donor, FRET and acceptor channels, has its appeal, e.g. time course measurements are possible. We have developed a method which takes spectral overspills and the alpha factor (explained under the next point) into account to yield pixel-by-pixel FRET values.
Calibration of FRET values in flow cytometric measurements using fluorescent proteins:
In FRET an excited donor molecule is quenched by a nearby acceptor, i.e. an excited acceptor is "created" from an excited donor. Since the donor and acceptor fluorescence intensities are measured by different detectors, their fluorescence intensities aren't comparable, i.e. the intensity of one donor molecule in the donor channel isn't identical to the intensity of an acceptor in the acceptor (or FRET) channel. The ratio of the fluorescence intensity of the same number of acceptor and donor molecules is called the alpha factor. We have worked out a way to determine this parameter in flow cytometric measurements of FRET using fluorescent proteins.
Novel calibration method for flow cytometric fluorescence resonance energy transfer measurements between visible fluorescent proteins. Cytometry, 67A: 86-96 (2005)
- conventional hetero-FRET in which spectroscopically different molecules interact;
- homo-FRET in which the donor and the acceptor are molecules of the same species. In homo-FRET the excitation energy of the first donor is spread all across the cluster if the fluorophores are within FRET distance from each other.
- most of ErbB1 is monomeric in the absence of stimulation and the rest forms small clusters. After EGF stimulation ErbB1 clusters get larger.
- ErbB2 forms large-scale homoclusters in the absence of stimulation containing inactive receptors. ErbB2 is recruited to heteroclusters upon ligand stimulation. Therefore, the size of ErbB2 homoclusters decrease after stimulation.
Quantitative characterization of the large-scale association of ErbB1 and ErbB2 by flow cytometric homo-FRET measurements. Biophys J. 95:2086-96 (2008)
Problem: Homo-FRET isn't suitable for the analysis of heteroclusters. Conventional hetero-FRET measurements don't resolve the stoiciometry of clusters and the fraction of clustered molecules.
Solution: some of the acceptors are within FRET distance from donors, while others are free. If the acceptor is photolabile and the donor is photostable, the photolabile acceptors within FRET distance from donors will get photobleached by FRET if the donor is excited. Although theoretically all free acceptors should be preserved, a certain fraction of them will be photobleached. This is taken into consideration by the bleaching correction factor (BCF). With this method we have shown that the ErbB1:ErbB2 ratio in heteroclusters changes from 1:1.5 to 1:4 upon EGF stimulation of quiescent SKBR-3 cells.