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Cell Surface Receptor Internalization

Cell-surface receptors can be internalized by both clathrin dependant and independent mechanisms depending on the amount of extracellular signal present. At high signal levels, these receptors are more likely to undergo clathrin-independent internalization, whereas at lower signal levels clathrin-dependant internalization will dominate (Sigismund, et al., 2005). For EGFR, clathrin-independent internalization is associated with receptor degradation whereas no corresponding degradation is noted in the case of clathrin-dependent internalization, likely stemming from a mechanism by which cells attenuate their responding to high levels of extracellular signal in order to conserve cellular resources and adapt to current conditions (Sigismund, et al., 2005). These results suggest that for a given cell-surface receptor, clathrin-dependent receptor internalization will result in receptor recycling whereas clathrin-independent receptor internalization will result in receptor degradation.

In order to test this prediction, a number of experiments can be conducted to assess rates of receptor recycling in various conditions. First, the ligand doses at which the receptor molecule undergoes clathrin-mediated and clathrin-independent internalization must be established. This can be done using the techniques of Sigismund et al. by treating cells expressing the receptor of interest with varying doses of a rhodamine-labeled form of the corresponding ligand, fixing/permeabilizing these cells, and exposing them to fluorescently-conjugated antibodies specific to clathrin or calveolin (Sigismund, et al., 2005). At low ligand doses, colocalization with clathrin but not calveolin is expected, whereas at high doses the opposite should be true. If desired, whole cell lyates of differentially treated cells can be immunoprecipitated with an antibody specific to the receptor of interest and blotted with an antibody specific to ubiquitin. Clathrin-independent internalization pathways tend to result in the ubiquitination of the receptor molecule, and thus at high doses of ligand ubiquitin should co-immunoprecipitate with the receptor (Sigismund, et al., 2005). These tests will allow for the establishment of optimal ligand doses necessary to achieve clathrin-dependent or independent internalization for further experimentation.

To assess receptor recycling in clathrin-dependent and clathrin-independent internalization conditions, an assay similar to one described by Cong et al. can be utilized. Briefly, a FLAG-tagged form of the receptor will be overexpressed in cells which will be treated with the previously established low or high doses of ligand so as to induce clathrin-dependent or clathrin-independent internalization and with cyclohexamide to prevent further receptor synthesis. Receptor recycling can then be approximated by fixing (but not permeabilizing) cells at various timepoints up to ~90 minutes and assessing surface levels of FLAG-tagged receptor using a fluorescently-tagged anti-FLAG antibody (Cong, et al., 2001).

Since recycling is a fairly rapid process of approximately 10 minutes, the net change in surface levels of receptor over a longer timeframe will be indicative of the average differences in recycling rates (Sharma, et al., 2004). In conditions of clathrin-dependent receptor internalization it is expected that there will be a significantly higher rate of average receptor recycling than during clathrin-independent receptor internalization; a result which would confirm this hypothesis. In order to confirm the validity of this result, this experiment can be repeated in cells which are additionally treated with inhibitors of clathrin-independent internalization such as filipin, nystatin, and genistein (Sigismund, et al., 2005). If the hypothesis is correct, then there will not be an appreciable decrease in surface receptor levels in cells which cannot undergo clathrin-independent receptor internalization due to inhibition compared to the control, demonstrating that the clathrin-dependent internalization pathway is more likely to function in receptor recycling.

A Note

A recent reviewer comment on this piece noted that when referring to the use of clathrin coated vesicle transport, this piece makes use of the terms “clathrin dependent” and “clathrin mediated”. There was concern that these two terms may be distinct and that “clathrin mediated” implies uncertainty about the mechanisms of transport. In molecular biology, these terms are effectively interchangeable for the purposes of the article. As it implies, “Clathrin dependent” transport can only occur with the help of clathrin proteins; in their absence this transport would not be possible and hence it is a dependent process. Similarly, “clathrin mediated” transport is a type of intracellular transport that occurs in a way that is mediated by the clathrin protein. Hence, if the clathrin protein is absent then there would not be the proper proteins present to mediate the transport and as a result the transport would not be able to occur. Now, in cellular biology it is often common for redundant mechanisms to exist in cells, and hence these types of transport may still occur at some level thus complicating the process even in the absence of clathrin. That being said, for the purposes of this article and common understanding the two terms are effectively interchangeable.

Sources

Cong M, et al. Binding of the β2 Adrenergic Receptor to N-Ethylmaleimide-sensitive Factor Regulates Receptor Recycling. J Biol Chem. 2001; 276: 45145-45152.

Jarosch E, et al. Protein dislocation from the ER requires polyubiquitination and the AAA-ATPase Cdc48. Nature Cell Biology. 2002; 134-139.

Katzmann DJ, et al. Ubiquitin-Dependent Sorting into the Multivesicular Body Pathway Requires the Function of a Conserved Endosomal Protein Sorting Complex, ESCRT-I. Cell. 2001; 106: 145-155.

Palacios F, et al. Lysosomal Targeting of E-Cadherin: a Unique Mechanism for the Down-Regulation of Cell-Cell Adhesion during Epithelial to Mesenchymal Transitions. Molecular and Cell Biology. 2005; 25(1): 389-402.

Sigismund S, et al. Clathrin-independent endocytosis of ubiquitinated cargos. PNAS. 2005; 102(8):2760-2765.

Ye Y, et al. A membrane protein complex mediates retro-translocation from the ER lumen into the cytosol. Nature. 2004; 429: 841-847.


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