ABS548 Sigma-AldrichAnti-Eps15 Antibody

Clathrin-mediated endocytosis occurs at multiple independent import sites on the plasma membrane, but how these positions are selected and how different cargo is simultaneously recognized is obscure. FCHO1 and FCHO2 are early-arriving proteins at surface clathrin assemblies and are speculated to act as compulsory coat nucleators, preceding the core clathrin adaptor AP-2. Here, we show that the μ-homology domain of FCHO1/2 represents an endocytic interaction hub. Translational silencing of fcho1 in zebrafish embryos causes strong dorsoventral patterning defects analogous to Bmp signal failure. The Fcho1 μ-homology domain interacts with the Bmp receptor Alk8, uncovering an endocytic component that positively modulates Bmp signal transmission. Still, the fcho1 morphant phenotype is distinct from severe embryonic defects apparent when AP-2 is depleted. Our data thus challenge the primacy of FCHO1/2 in coat initiation.

Several vectorial transport routes in mammalian cells involve clathrin and associated proteins. In kidney epithelia urine production requires numerous transport processes. However, only little is known about the distribution of clathrin and its associated proteins in this organ in situ. We now report on the presence and distribution of clathrin and its accessory proteins AP1, AP2, Eps15, Epsin, CALM and Clint/EpsinR in the epithelia of the rat kidney cortex using immunoblotting, immunofluorescence and immuno-electron microscopy. Our data show that all investigated proteins are ubiquitously present in rat kidney cortex epithelia, however, with distinct distribution patterns. In the renal corpuscle, podocytes showed the most conspicuous labelling. Clathrin, AP2 and CALM were highly expressed in foot processes, while AP1 was primarily localized in the cell body. In the proximal tubule all proteins were present in dots along the plasma membrane and most conspicuous below the brush border. However, clathrin and AP2 co-localized in vesicle subtypes distinct from those containing clathrin and AP1. In the distal tubule and in the cortical collecting duct all proteins were found in the apex of the cells; however, AP1 and Clint/EpsinR showed additional staining in perinuclear dots. The occurrence and distribution of the investigated proteins in kidney epithelia are discussed with respect to their possible involvement in the functions of the specific nephron segment.

To assess the contribution of individual endocytic proteins to the assembly of clathrin coated pits, we depleted the clathrin heavy chain and the alpha-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 alpha-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 alpha-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.

An expression cloning method which allows direct isolation of cDNAs encoding substrates for tyrosine kinases was applied to the study of the epidermal growth factor (EGF) receptor (EGFR) signaling pathway. A previously undescribed cDNA was isolated and designated eps15. The structural features of the predicted eps15 gene product allow its subdivision into three domains. Domain I contains signatures of a regulatory domain, including a candidate tyrosine phosphorylation site and EF-hand-type calcium-binding domains. Domain II presents the characteristic heptad repeats of coiled-coil rod-like proteins, and domain III displays a repeated aspartic acid-proline-phenylalanine motif similar to a consensus sequence of several methylases. Antibodies specific for the eps15 gene product recognize two proteins: a major species of 142 kDa and a minor component of 155 kDa, both of which are phosphorylated on tyrosine following EGFR activation by EGF in vivo. EGFR is also able to directly phosphorylate the eps15 product in vitro. In addition, phosphorylation of the eps15 gene product in vivo is relatively receptor specific, since the erbB-2 kinase phosphorylates it very inefficiently. Finally, overexpression of eps15 is sufficient to transform NIH 3T3 cells, thus suggesting that the eps15 gene product is involved in the regulation of mitogenic signals.