EPS15 IS CONSTITUTIVELY OLIGOMERIZED DUE TO HEMOPHILIC INTERACTION OFITS 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 c om plex AP2 and its localization in clathrin-coated pits have implicat ed Eps15 in the regulation of vesicle trafficking, Here we show that E ps15 exists in several multimeric states in, vivo, When purified recom binant Eps15 or lysates of NIH 3T3 cells were treated with cross-linki ng reagents, covalent dimers of Eps15 and larger covalent multimers we re detected in high yield, Large Eps15 oligomers co-immunoprecipitated with AP-2 at an efficiency higher than that of Eps15 dimers, Furtherm ore, cross-linking of the membrane-bound fraction of Eps15 in mildly p ermeabilized 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 rat io of the monomers versus the aggregated forms of Eps15. These experim ents showed that essentially all the Eps15 was aggregated, whereas mon omers of Eps15 could be obtained only under strong denaturing conditio ns. To map the region of Eps15 responsible for dimerization, fusion pr oteins corresponding to the three structural domains of Eps15 were pre pared, Cross linking analysis revealed that the central portion of Eps 15, which possesses a coiled-coil region (residues 321-520), serves as the interacting interface, The possibility that hetero-oligomeric com plexes of Eps15 dimers and AP-2 function during the recruitment of pro teins into coated pits is discussed.