GPCR-mediated traffic of Na,K-ATPase to the plasma membrane requires the binding of adaptor protein 1 to a Tyr-255 based sequence in the α-subunit*

Motion of integral membrane proteins to the plasma membrane in response to G-proteincoupled receptor signals requires selective cargo recognition motifs that bind adaptor protein 1 and clathrin. Angiotensin II, through the activation of AT1 receptors, promotes the recruitment to the plasma membrane of Na,K-ATPase molecules from intracellular compartments. We present evidence to demonstrate that a tyrosine-based sequence (IVVY-255) present within the Na,K-ATPase α1-subunit is involved in binding of adaptor protein 1. Mutation of Tyr-255 to a phenylalanine residue in the Na,K-ATPase α1-subunit greatly reduces the angiotensin II-dependent activation of Na,KATPase, recruitment of Na,K-ATPase molecules to the plasma membrane and association of adaptor protein 1 with Na,KATPase α1-subunit molecules. To determine protein-protein interaction, we used fluorescence resonance energy transfer between fluorophores attached to the Na,KATPase α1-subunit and adaptor protein 1. While angiotensin II activation of AT1 receptors induces a significant increase in the level of FRET between the two molecules, this effect was blunted in cells expressing the Tyr255 mutant. Thus, results from different methods and techniques suggest that the Tyr255 based sequence within the NKA α1subunit is the site of adaptor protein 1 binding in response to the G-protein coupled receptor signals produced by angiotensin II binding to AT1 receptors.