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 α-Subunit

Abstract

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 tyrosinebased sequence (IVVY-255) present within the Na,K-ATPase ␣1-subunit is involved in the binding of adaptor protein 1. Mutation of Tyr-255 to a phenylalanine residue in the Na,KATPase ␣1-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 ␣1-subunit molecules. To determine protein-protein interaction, we used fluorescence resonance energy transfer between fluorophores attached to the Na,K-ATPase ␣1-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 ␣1-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.

Mutations in ap1b1 Cause Mistargeting of the Na+/K+-ATPase Pump in Sensory Hair Cells

“…Sorting motifs are usually present within the cytoplasmic C-terminal tails of cargo membrane proteins. Despite the absence of a canonical sorting signal, previous evidence from cell culture experiments suggests that the α-subunit of the basolateral pump NKA is sorted by AP-1 [40]. We hypothesized that if NKA is an AP-1 dependent cargo, then the pump would be mislocalized in ap1b1 mutant HCs.…”

“…However, in the case of NKA, a canonical AP-1 sorting motif has not been identified in NKA. Instead, novel motifs within the α-subunit of NKA appear to be necessary for basolateral targeting in cell lines [40], [49], [50]. Additionally, unlike other well-known AP-1 dependent cargoes, the post-Golgi transport pathway taken by NKA to the plasma membrane does not involve recycling endosomes, but rather goes directly from the TGN to the plasma membrane [12].…”

The structure of the Na+,K+-ATPase and mapping of isoform differences and disease-related mutations

“…By contrast, large clusters of isoform differences map at surfaceexposed regions of the A-and N-domains, possibly reflecting isoform specific interactions with other cellular components. A recent example of an isoform specific interaction is in trafficking, where adaptor protein 1 was shown to recruit alpha1 to the membrane via Tyr 255 (Efendiev et al 2008), a residue not conserved in alpha2 and alpha3.…”

A hypomorphic mutation of the gamma-1 adaptin gene (Ap1g1) causes inner ear, retina, thyroid, and testes abnormalities in mice

“…3, 4). Na + /K + ATPase, a plasma membrane enzyme that is essential for maintaining sodium and potassium concentration gradients and polarized membrane potentials, requires AP-1 for proper targeting to the plasma membrane (Efendiev et al 2008). Na + /K + ATPase was mislocalized to the apical rather than basolateral surface of sensory hair cells in the ap1b1 mutant zebrafish and was thought to underlie their auditory and balance dysfunction (Clemens Grisham et al 2013).…”