APP and α2AAR interact through their intracellular domains. A, B) Deletion of the α2AAR 3iloop significantly reduced its ability to interact with APP. HEK293 cells coexpressing APP with HA-α2AAR or HA-α2AAR-Δ3iloop were treated with 10 µM NE (plus 1 µM prazosin and 1 µM propranolol) or vehicle for 5 min. Cell lysates were subjected to IP assays with an HA Ab. A) Representative Western blots. B) Quantification of the amount of APP in the IP complex. Data (means ± sem) are expressed as fold change vs. vehicle control (defined as 1.0) (n = 3 in each group). *P < 0.05; **P < 0.01 (Student’s t test). C, D) Removal of the APP-C abolished its interaction with the α2AAR. HEK293 cells coexpressing HA-α2AAR with APP or APPΔC were treated with 10 µM NE (plus 1 µM prazosin and 1 µM propranolol) or vehicle for 5 min. Cell lysates were subjected to IP assays. C) Representative Western blots. D) Quantification of the amount of APP and APPΔC in the IP complex. Data (means ± sem) are expressed as fold change vs. vehicle control (defined as 1.0) (n = 3 in each group). **P < 0.01 (Student’s t test). E) The C99 fragment of APP formed a complex with HA-α2AAR. HEK293 cells coexpressing C99, with or without HA-α2AAR were stimulated with vehicle or 10 µM NE (plus 1 µM prazosin and 1 µM propranolol), and cell lysates were subjected to IP assays. Representative Western blots are shown. F) In vitro GST pull-down assay showing direct interaction between the α2AAR 3iloop and APP C-terminal domain. Purified GST or GST-fused APP-C was incubated with [35S]-labeled, in vitro translated α2AAR-3iloop. Input probe represents one-tenth of the total input in each reaction.