To identify cellular components necessary for the intracellular trafficking and/or function of cell surface βPP we screened for proteins that interact with the cytoplasmic domain of βPP using the ''interaction trap''. Employing this strategy, we identified a novel human homologue of the rat FE65 gene, hFE65L. Given the high degree of conservation between βPP and the βPP homologues, APLP1 and APLP2, we also tested the cytoplasmic domains of the APLPs for interaction with hFE65L. APLP2, but not APLP1, was found to interact with hFE65L. These interactions were confirmed in vivo by successfully co-immunoprecipitating endogenous βPP and APLP2 from mammalian cells overexpressing a hemaglutinin-tagged fusion of the C-terminal region of hFE65L. Furthermore, the co-immunoprecipitation experiments show that the C-terminal antibody, 369W, recognizes both full-length βPP and a 14 kda immunoreactive band presumably corresponding to the C-terminal fragment obtained following secretase cleavage of βPP.Sequence analysis of the largest cDNA clone we isolated for the FE65L gene codes for a predicted protein of 730 amino acids which is 57% identical and 74% similar to the overlapping 499 amino acids of the rat FE65 protein. Comparisons of the hFE65L sequence with the Genbank database allowed us to identify human sequences similar to the rat FE65 protein. The Expressed Sequence Tags identified were assembled into open reading frames for two additional FE65-like human genes. One of these, which we have named hFE65, is 95% identical and 96% similar to the 366 overlapping amino acids of rat FE65. The other, hFE65L2, is 39% identical and 59% similar to the 129 overlapping amino acids of rat FE65.Amino acid comparisons of members of this novel human gene family reveals the presence of two phosphotyrosine interaction (PI) domains. The PI domain of the protein shc is known to interact with the NPXYp motif found in the cytoplasmic domain of a number of different growth factor receptors. Given that the NPXY motif is conserved in βPP and the APLPs it is conceivable that association of hFE65L with βPP and APLP2 is mediated by interaction with the NPXY motif. Results of experiments designed to test this hypothesis will be presented. The NPXY motif has previously been shown to modulate the internalization and trafficking of βPP. Thus it is possible that the interaction of hFE65L with the cytoplasmic domain of βPP plays a role in these cellular events.