G-protein-coupled receptor-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 alpha-subunit.
Motion of integral membrane proteins to the plasma membrane in response to G-protein-coupled 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 alpha1-subunit is involved in the binding of adaptor protein 1. Mutation of Tyr-255 to a phenylalanine residue in the Na,K-ATPase alpha1-subunit greatly reduces the angiotensin II-dependent activation of Na,K-ATPase, recruitment of Na,K-ATPase molecules to the plasma membrane, and association of adaptor protein 1 with Na,K-ATPase alpha1-subunit molecules. To determine protein-protein interaction, we used fluorescence resonance energy transfer between fluorophores attached to the Na,K-ATPase alpha1-subunit and adaptor protein 1. Although angiotensin II activation of AT1 receptors induces a significant increase in the level of fluorescence resonance energy transfer between the two molecules, this effect was blunted in cells expressing the Tyr-255 mutant. Thus, results from different methods and techniques suggest that the Tyr-255-based sequence within the NKA alpha1-subunit 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.
Efendiev
Riad
R
College of Pharmacy, University of Houston, Houston, Texas 77204, USA. riad.efendi@uth.tmc.edu
Budu
Claudia E
CE
Bertorello
Alejandro M
AM
Pedemonte
Carlos H
CH
D033961:Adaptor Protein Complex 1 / Q000378:metabolism*; D000804:Angiotensin II / Q000737:chemistry; D000818:Animals; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism*; D004789:Enzyme Activation; D009154:Mutation; D009893:Opossums; D010649:Phenylalanine / Q000737:chemistry; D011485:Protein Binding; D011487:Protein Conformation; D051381:Rats; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014162:Transfection; D014443:Tyrosine / Q000737:chemistry*
D033961:Adaptor Protein Complex 1; D000804:Angiotensin II; D014443:Tyrosine; D010649:Phenylalanine; D000254:Sodium-Potassium-Exchanging ATPase