Demonstration by fluorescence resonance energy transfer of two sites of interaction between the low-density lipoprotein receptor-related protein and the amyloid precursor protein: Role of the intracellular adapter protein Fe65

Amyloid-beta, the major constituent of senile plaques in Alzheimer's diseas e, is derived from the amyloid precursor protein (APP) by proteolysis. Kuni tz protease inhibitor (KPI) containing forms of APP (APP751/770) interact w ith a multifunctional endocytic receptor, the low-density lipoprotein recep tor-related protein (LRP), which modulates its proteolytic processing affec ting production of amyloid-beta. We used fluorescence resonance energy tran sfer (FRET) using labeled LRP and APP in H4 cell line to examine the subcel lular localization and the molecular domains involved in the APP-LRP intera ction. KPI-containing forms of APP (APP770) demonstrated FRET with LRP that was sensitive to the LRP inhibitor receptor-associated protein (RAP), sugg esting an interaction between the extracellular domains of APP770 and LRP. APP695 also interacts with LRP to lesser degree (as measured by extracellul ar domain probes), and this ectodomain interaction is not altered by RAP. B y using C-terminally tagged LRP and APP, we demonstrate a second site of in teraction between the C termini of both APP695 and APP770 and the C terminu s of LRP, and that the interactions at these regions are not sensitive to R AP. We next examined the possibility that the C-termini APP-LRP interaction was mediated by Fe65, an adaptor protein that interacts with the cytoplasm ic tails of LRP and APP. FRET studies confirmed a close proximity between t he amino Fe65 phosphotyrosine binding (PTB) domain and LRP cytoplasmic doma in and between the carboxyl Fe65 PTB domain and the APP cytoplasmic domain. These findings demonstrate that LRP interaction with APP occurs via both e xtracellular and intracellular protein interaction domains.