Organization and Function of Receptor Tyrosine Kinases is Linked to Lipid Microdomains

Receptor tyrosine kinases are prominent examples of the proteins involved in cell signaling that form molecular associations and superstructures in the cell membrane. Cooperative interactions between or among receptor tyrosine kinases play a pivotal role in signal transduction. This includes homo- and hetero-dimerizations as well as potentially higher-order associations, which may be either dependent or independent of ligands. In addition to protein-protein interactions, lipid microdomains also play an important role in the organization of superstructures. The EGF receptor, the insulin receptor, the PDGF receptor, vascular endothelial growth factor (VEGF) and nerve growth factor (NGF) receptors have been shown to be localized to low density, cholesterol-rich membrane domains. In all cases, signaling by these receptors can be modulated by changes in cellular cholesterol content. Thus, raft localization appears to be of functional importance to these receptors (for a review, see [129]).

One of the best-studied receptors in this respect is the EGF receptor which belongs to the type I family of transmembrane receptor tyrosine kinases. In addition to EGFR or ErbBl, the other members of the family are ErbB2 (HER2 or Neu), ErbB3 and ErbB4 [130-133]. Within a given tissue, these receptors are rarely expressed alone, but are found in various combinations. Members of the family display various degrees and combinations of homo- and hetero-associations at the cell surface, depending on their relative expression levels. ErbB2 is an orphan receptor: no soluble physiological ligand, specific to ErbB2, has been detected so far. Despite this fact, ErbB2 participates actively in ErbB receptor combinations, and receptor complexes including the ErbB2-ErbB3 dimer which appears to be more potent in mitogenic activity than any other combination [130-134]. Recently, a molecular model was built for the nearly full-length ErbB2 dimer based on the X-ray or nuclear magnetic resonance (NMR) structures of extracellular, transmembrane and intracellular domains, and intramolecular distances determined by FRET. Favorable dimerization interactions were predicted for the extracellular, transmembrane and protein kinase domains which may act in a coordinated fashion in ErbB2 homodimerization, or alternatively in ErbB heterodimeriza-tions [135].

Molecular-scale physical associations among ErbB family members have been studied using classical biochemical [136,137], molecular biological and biophysical methods [48,62,138,139]. When isolated from cells, members of the ErbB family self-associate (homoassociate) and associate with other family members (hetero-associate) [136]. However, experiments on isolated proteins are inherently unable to detect interactions in cellular environments in vivo and in situ, and cannot detect heterogeneity within or among cells. FRET measurements detected dimerization of ErbB1 receptors in fixed [138,140] and living cells [140]. FRET was also applied to monitor the association pattern of ErbB2 in breast tumor cells [48,62,138,139]. There was considerable homoassociation of ErbB2 and heteroassociation of ErbB2 with EGFR in quiescent breast tumor cells. ErbB2 homoassociation was enhanced by EGF treatment in SKBR-3 cells and in the BT474 subline BT474M1 with high tumorigenic potential, whereas the original BT474 line was resistant to this effect. These differences correlated well with EGFR expression. Since these measurements were performed with flow cytometry, one single FRET efficiency value was obtained for each cell analyzed. In order to reveal heterogeneity in the homo-association pattern of ErbB2 within a single cell, one of the microscopic FRET approaches had to be utilized. Donor photobleaching FRET microscopy was used to visualize FRET efficiency within single cells with spatial resolution limited only by diffraction in the optical microscope [48,50,141]. This allows detailed analysis of the spatial heterogeneity of molecular interactions. Donor pbFRET measurements showed that ErbB2 homoassociation was also heterogeneous in unstimulated breast tumor cells; and membrane domains with erbB2 homoassociation had mean diameters of less than 1 mm [62,142]. It was not clear whether the domain size was imposed by the optical resolution limit of wide-field microscopy in the X-Y plane or whether it originated from the actual size of ErbB2 aggregates.

In order to refine the size estimate of domains containing ErbB2 molecules, it was necessary to use SNOM [50,143,144]. This technique is not limited by diffraction optics, and can readily image objects in the 0.1 to 1 mm range, including sub-mm lipid and protein clusters in the plasma membrane [4] (see Section 7.2). ErbB2 was concentrated in irregular membrane patches with a mean diameter of approximately 500 nm, containing up to 1000 ErbB2 molecules in nonactivated SKBR-3 and MDA453 human breast tumor cells. The mean cluster diameter increased to 600-900 nm when SKBR-3 cells were treated with EGF, heregulin or a partially agonistic anti-erbB2 antibody. The increase in cluster size was inhibited by an EGFR-specific tyrosine kinase inhibitor, suggesting that EGFR was somehow involved in organizing this ErbB2 superstructure. Since the domain size was larger than the resolution limit of confocal microscopy (200-300 nm in the X-Y plane), we were able to confirm the SNOM results with confocal laser scanning microscopy (Fig. 7.6) [142].

The role of lipid rafts in the organization of these EGFR and ErbB2-containing superstructures has also been revealed in further experiments. The size of lipid rafts was investigated with dye-labeled cholera toxin B (CTX-B) subunit, which binds to the glycosphingolipid GM1 ganglioside. In order to study the role of lipid rafts in the homoassociation patterns of ErbB2, confocal microscopic studies were performed. The signal from one laser beam was used to monitor lipid rafts, and signals excited by the other two laser beams to reveal the homoassociation pattern of ErbB2. Observations suggest that similarly to ErbB1 [129], ErbB2 is localized mostly in GM1-enriched membrane domains, that are distinct from caveolae. However, there is a negative correlation between ErbB2 homoassociation and the local density of the lipid raft marker CTX-B. This environment could alter the association properties of ErbB2. Since stimulating ErbB2 increases the size of ErbB2 clusters [142] and lipid rafts [139], the amount of ErbB2 concentrated in rafts is very likely related to the function of the protein. Localization of ErbB2 in lipid rafts is dynamic, since it can be dislodged from rafts by cholera toxin-induced raft crosslinking [139]. Upon crosslinking with CTX-B, GM1 leaves ErbB protein clusters behind and migrates into caveolae. The association properties and biological activity of ErbB2 excluded from rafts differ from those inside rafts. For example, internalization of ErbB2 mediated by 4D5 (the parent murine version of trastuzumab, a monoclonal anti-ErbB2 antibody used in breast cancer therapy) is blocked in CTX-B-pretreated cells, while its antiproliferative effect is not. A role of lipid rafts in limiting autoactivation of the ErbB signaling system is supported by the increased tyrosine phosphorylation of Shc after removing ErbB proteins from rafts by CTX-B treatment. On the other hand, lipid rafts are also responsible for

Lipid Raft Detection Snom

Fig. 7.6 Scanning near-field optical microscopy (SNOM) and confocal laser scanning microscopy (CLSM) images of TAMRA-4D5-labeled ErbB2 receptors on SKBR-3 cells. SKBR-3 cells grown on glass coverslips were labeled on ice using TAMRA-conjugated 4D5 monoclonal anti-ErbB2 antibody Fab fragments. (A) For SNOM, a custom-built shared aperture instrument was used on formaldehyde-fixed, dehydrated, air-dried samples [142]. (B) 1-mm optical sections of live cells were obtained in CLSM with a Zeiss LSM 410 instrument. Fluorescence intensity is displayed in pseudocolor. (Scale bar = 2 mm.)

Fig. 7.6 Scanning near-field optical microscopy (SNOM) and confocal laser scanning microscopy (CLSM) images of TAMRA-4D5-labeled ErbB2 receptors on SKBR-3 cells. SKBR-3 cells grown on glass coverslips were labeled on ice using TAMRA-conjugated 4D5 monoclonal anti-ErbB2 antibody Fab fragments. (A) For SNOM, a custom-built shared aperture instrument was used on formaldehyde-fixed, dehydrated, air-dried samples [142]. (B) 1-mm optical sections of live cells were obtained in CLSM with a Zeiss LSM 410 instrument. Fluorescence intensity is displayed in pseudocolor. (Scale bar = 2 mm.)

maintaining ErbB proteins in a growth factor-responsive state. This is supported by the following results:

• Neither EGF, nor heregulin are able to activate Shc if ErbB proteins are removed from lipid rafts.

• The formation of heregulin-responsive ErbB2/ErbB3 heterodimers and here-gulin-induced ErbB2 tyrosine phosphorylation decrease if ErbB proteins are removed from lipid rafts.

These results emphasize that alterations in the local environment of ErbB2 strongly influence its association properties, which are reflected in its biological activity and in its behavior as a target for therapy [139].

The results of several studies have implied that integrins and growth factor receptors cooperate in tumor formation, and have demonstrated the existence of integrin-growth factor receptor complexes leading to a decreased threshold of transmembrane signaling [145-147]. Similar to ErbB2, integrins were shown to be raft-associated [148]. Cooperative signaling between ErbB proteins and integrins [146,149] is a common feature of invasive cancer cells, and association of b1-integrin with ErbB2 proteins could provide a framework in which tumor cell metastasis might be better understood. FRET and confocal microscopic measurements demonstrated an association between ErbB2 and b1-integrin in the nanometer range and on the scale of membrane microdomains, respectively. Lipid rafts showed a substantial overlap with both ErbB2- and p1-integrin-rich microdo-

mains [150]. These results corroborated the existence of molecular interactions between ErbB2 and p1-integrins and their association with lipid rafts. Interestingly, although ErbB2-positive but trastuzumab-resistant breast and gastric cancer cell lines were found to express a substantially higher amount of p1-integrin than corresponding trastuzumab lines, there was no significant difference among them in the trastuzumab-induced tyrosine phosphorylation of ErbB2, which was probably caused by the weak functional interaction between the two proteins.

In summary, there is ample evidence for the role of local factors, for example, the lipid environment, other receptor tyrosine kinases and integrins, in influencing receptor tyrosine kinase activity and consequential modulation of the ability of these molecules in driving cellular functions. In addition to a more thorough understanding of the complexity of the receptor tyrosine kinase signaling networks, experiments linking lipid rafts and receptor function may facilitate the development of more efficient therapeutic strategies targeting receptor tyrosine kina-ses [150].

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