The transport of many proteins – transmembrane proteins as well as GPI-anchored proteins - has been shown to be facilitated by association with detergent resistant microdomains (DRMs), called “lipid rafts” or shortly “rafts”.
Especially the correct targeting of various apically sorted proteins such as SI or DPPIV is dependent on a composition with lipid rafts, whereas basal sorted proteins are normally not included in rafts (reviewed by Schuck and Simons, 2004).
DRMs are described as subvesicular structures, enriched by cholesterol and glycophingolipids, with a decreased fluidity compared with lipids of the cell membrane bilayer (Simons and Ikonen, 1997). As mentioned above, rafts are discriminated from other membranous components by their solubility to non-ionic detergents like Brij, Chaps, Lubrol, Tween, Triton X-100 etc. Solubilization with each detergent results in a distinct lipid composition pattern of the resistant microdomain (Alfalah et al., 2005). Hence we can principally distinguish different types of rafts.
Therefore it is more accurate to describe e.g. Brij-rafts or Triton X-100-rafts. The latter one is commonly used to determine if a protein “enters rafts or not”.
Furthermore, diverse raft-dependent delivery machineries are discriminable according to the proportion of the two major components of DRMs: with distinct inhibitors, the synthesis of sphingolipid (by fumonisin) or cholesterol (using β-cyclodextrin) can be inhibited, and thus their contribution to the establishment of DRMs can be estimated. For example it has been shown that for raft-mediated cell surface delivery of DPPIV, depletion of cholesterol had more severe affect than an inhibition of sphingolipid synthesis (Alfalah et al., 2002).
However, there are also apically sorted proteins described which are transported independently of DRMs. The LPH for example does not enter detergent insoluble microdomains (Jacob & Naim 2001). Here, basically different transport mechanisms are proposed for the correct targeting of this protein in comparison to the SI as a marker for DRM-dependent apical trafficking.
It has become quite evident that a correct membrane anchoring plays a key role in DRM-dependent transport: Deletion of the transmembrane domain abolished
association of the SI with lipid rafts (Jacob et al., 2000) and modifications in the membrane anchoring of the influenza virus proteins haemagglutinin and neuraminidase prevented directed transport via rafts (Kundu et al., 1996;Scheiffele et al., 1997).
Likewise, the influence of an intact glycosylation pattern has been shown for some proteins. For example, the association with lipid microdomains and thereby the correct targeting to the apical membrane of the SI requires O-linked glycans, which was proved by inhibition of O-glycosylation with benzyl-GalNAc (Alfalah et al., 1999).
Similar, DPPIV is delivered to the apical membrane by association with DRMs, mediated by correctly O- and N-linked sugar residues (Alfalah et al., 2002).
Beside the described examples, it has been also reported that glycoproteins which are involved in cell signalling or cell adhesion, such as growth factor receptors or intercellular contact molecules, associate with DRMs (Roepstorff et al., 2002;Niethammer et al., 2002). For the neural cell adhesion molecule (NCAM), a crucial role of rafts in neurite outgrowth was demonstrated (Niethammer et al., 2002).
Within the cadherin superfamily it has been found that all members – as far as investigated – are localized in lipid rafts (see for review Angst et al., 2001).
In some studies it has been reported, that cadherins colocalize with signalling proteins which might be involved in transferring extracellular signals generated by the cadherin molecules. T-cadherin for example colocalizes with small trimeric G-proteins and SRC family kinases in lipid rafts (Doyle et al., 1998;Philippova et al., 1998).
Furthermore, for N-cadherin it has been recently proved that DRMs play a key role not only in the transport but also in function of this cadherin (Causeret et al., 2005).
The authors showed that N-cadherin is localized in lipid rafts at sites of cell-cell contacts and that after depletion of these microdomains, cell-adhesion function of N-cadherin was abolished although the protein is still correctly transported to the plasma membrane and its association with intracellular signal pathway molecules (catenins) is not affected. Moreover, the association with DRMs is dependent on the formation of homologous N-cadherin dimers and their association with F-actin.
Therefore, it is proposed in this paper that rafts could act as new potential regulators of cadherin mediated cell adhesion.
In case of the protocadherin subgroup, reports of detergent resistant microdomains are rather rare. Although they are structurally closely related to cadherins of the classical type, it is not certain that they must be included in lipid rafts, too. In contrast, for association with DRMs, the cytoplasmic tail has been shown to play a crucial role in recruitment of transmembrane proteins into lipid microdomains. Whereas classical cadherins exhibit a highly conserved intracellular fragment (> 90 %), the cytosolic region of most protocadherins is unique and very specific for ligand-binding functions.
Nevertheless, some clusters of protocadherins have been reported to express multiple cysteine residues in the intracellular tail, which are potential sites for sacylation / palmitoylation, contributing to inclusion into lipid rafts (Angst et al., 2001).
Particularly for the eight cadherin related neuronal receptors (CNRs), a protocadherin subfamily which shares an identical cytoplasmic tail and exhibits six extracellular cadherin repeats, an association with lipid rafts has been demonstrated indirectly (Kohmura et al., 1998). However, the implication of DRMs in transport and function of the protocadherin subgroup have not yet been well elucidated.
For PLKC, the performed experiments showed that a fraction of the protein is transported by Triton X-100 rafts, whereas another fraction (the high mannose ER form and parts of the complex glycosylated fraction) is not rafts associated.
Weaker detergent like Brij 96, Lubrol and Tween 20 are not able to solubilize the mature form of PLKC, indicating that the protein is indeed masked in a distinct type of detergent insoluble lipid microdomains. Recently it was proposed for Tween 20 to determine a new sort of lipid microdomains which separate apical and basal directed proteins early in the protein pathway (Alfalah et al., 2005). The results of detergent solubility of PLKC support this theory, as the mannose rich band, representing an early precursor form localized in the ER or associated with vesicles transported between ER and golgi, is found in the Tween-20 insoluble fraction. This could define the fraction of PLKC targeted to the apical membrane.
In contrast to many glycoproteins mentioned above the inclusion of PLKC in lipid microdomains is not dependent on complex glycosylation, as assessed by evaluation the rafts-association of PLKC in the glycosylation deficient CHO/Lec cells.
Furthermore, studies of the deletion mutants provide more evidence about structural requirements of association with DRMs. A deletion of the transmembrane domain as well as a truncation of the cytosolic tail result both in an abolished inclusion into lipid rafts. However, the membrane-anchorless mutant is correctly targeted to the apical cell surface with the same efficiency as wild type PLKC, indicating that that the association with rafts is not inevitable in correct transport and targeting of PLKC. On the contrary, PLKC lacking the intracellular domain is not included into lipid rafts either but this mutant is randomly delivered to both membranes. Therefore, important determinants facilitating the association with detergent insoluble microdomains are composed of at least the transmembrane domain and the cytosolic tail. This is in line with experiments deleting the transmembrane of SI, haemagglutinin or neuraminidase described above.
The mistargeting of PLKC∆Cyt and especially the loss of lateral transport in respect of abolished association with DRMs raises the question whether a raft – mediated protein trafficking of PLKC targets molecules only to the lateral but not to the apical membrane. This hypothesis is supported by the observation that PLKC∆TM is delivered very efficiently to the apical cell surface without interaction with lipid microdomains.
Finally, further studies are necessary to investigate the role of rafts-association in cell-aggregation function of protocadherin LKC.
One requirement for cell contact molecules to be transported with DRMs could be a targeted and regulated transport into distinct areas of cell-cell contact sites or a decreased or enhanced endocytosis. Thereby, the sensitive association with lipid rafts is a powerful tool to up- or downregulate expression and thus adhesive function of cell adhesion molecules upon intra- or extracellular stimuli. Via this selective modification of cadherin surface expression, cell-cell adhesion, growth inhibition and cell migration might be modulated.
Surprisingly Causeret et al. were able to show that lipid rafts accomplish more functions than mere transport of proteins (Causeret et al., 2005). As mentioned above, the group was able to show that rafts are necessary for stabilization of N-cadherin mediated cell adhesion. However, it remains to be proved, if this effect is due to a specific depletion of rafts by the applied inhibitors (β-cyclodextrin and cholesterol oxidase) or rather caused by a general modification of the plasma membrane upon treatment with these reagents.
5.6 PLKC involved in cell-cell contacts