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J. Biol. Chem., Vol. 278, Issue 46, 45160-45170, November 14, 2003
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Effect of Clathrin Heavy Chain- and 
-Adaptin-specific Small Inhibitory RNAs on Endocytic Accessory Proteins and Receptor Trafficking in HeLa Cells*
¶||
From the
Department of Cell Biology in the Center of Anatomy, Hannover Medical School, D-30623 Hannover, Germany and the Department of Biochemistry and Cell Biology of the Max-Planck-Institute for Biophysical Chemistry, D-37070 Göttingen, Germany
To assess the contribution of individual endocytic proteins to the assembly of clathrin coated pits, we depleted the clathrin heavy chain and the 
-adaptin subunit of AP-2 in HeLa-cells using RNA interference. 48 h after transfection with clathrin heavy chain-specific short interfering RNA both, the heavy and light chains were depleted by more than 80%. Residual clathrin was mainly membrane-associated, and an increase in shallow pits was noted. The membrane-association of adaptors, clathrin assembly lymphoid myeloid leukemia protein (CALM), epsin, dynamin, and Eps15 was only moderately affected by the knockdown and all proteins still displayed a punctate staining distribution. Clathrin depletion inhibited the uptake of transferrin but not that of the epidermal growth factor. However, efficient sorting of the epidermal growth factor into hepatocyte growth factor-regulated tyrosine kinase substrate-positive endosomes was impaired. Depletion of -adaptin abolished almost completely the plasma membrane association of clathrin. Binding of Eps15 to membranes was strongly and that of CALM moderately reduced. Whereas the uptake of transferrin was efficiently blocked in -adaptin knockdown cells, the internalization and sorting of the epidermal growth factor was not significantly impaired. Since neither clathrin nor AP-2 is essential for the internalization of EGF, we conclude that it is taken up by an alternative mechanism.
Received for publication, July 8, 2003 , and in revised form, September 3, 2003.
* This work was supported by the German Research Foundation and the Fond der Chemischen Industrie. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
¶ Current address: Alnylam Pharmaceuticals, 790 Memorial Dr., Suite 202, Cambridge, MA 02139.
|| To whom correspondence should be addressed. Fax: 49-511-532-3903; E-mail: ungewickell.ernst{at}mh-hannover.de.
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