Eps15 is constitutively oligomerized due to homophilic interaction of its coiled-coil region.
Eps15 is a member of an emerging family of proteins containing a novel protein/protein interaction domain, the EH domain, of as yet unknown function. Recent findings of Eps15 association with clathrin adaptor complex AP-2 and its localization in clathrin-coated pits have implicated Eps15 in the regulation of vesicle trafficking. Here we show that Eps15 exists in several multimeric states in vivo. When purified recombinant Eps15 or lysates of NIH 3T3 cells were treated with cross-linking reagents, covalent dimers of Eps15 and larger covalent multimers were detected in high yield. Large Eps15 oligomers co-immunoprecipitated with AP-2 at an efficiency higher than that of Eps15 dimers. Furthermore, cross-linking of the membrane-bound fraction of Eps15 in mildly permeabilized cells was as efficient as that of the cytosolic fraction. Size-exclusion column chromatography of recombinantly produced Eps15 and of total cell lysates was performed to examine the equilibrium ratio of the monomers versus the aggregated forms of Eps15. These experiments showed that essentially all the Eps15 was aggregated, whereas monomers of Eps15 could be obtained only under strong denaturing conditions. To map the region of Eps15 responsible for dimerization, fusion proteins corresponding to the three structural domains of Eps15 were prepared. Cross-linking analysis revealed that the central portion of Eps15, which possesses a coiled-coil region (residues 321-520), serves as the interacting interface. The possibility that hetero-oligomeric complexes of Eps15 dimers and AP-2 function during the recruitment of proteins into coated pits is discussed.
Tebar
F
F
Department of Pharmacology, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
Confalonieri
S
S
Carter
R E
RE
Di Fiore
P P
PP
Sorkin
A
A
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D000818:Animals; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D002850:Chromatography, Gel; D003432:Cross-Linking Reagents / Q000737:chemistry; D019281:Dimerization; D047908:Intracellular Signaling Peptides and Proteins; D051379:Mice; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D015398:Signal Transduction
D048868:Adaptor Proteins, Signal Transducing; D002135:Calcium-Binding Proteins; D003432:Cross-Linking Reagents; C082729:Eps15 protein, mouse; D047908:Intracellular Signaling Peptides and Proteins; D010750:Phosphoproteins