Calcium modulates calmodulin/alpha-actinin 1 interaction with and agonist-dependent internalization of the adenosine A(2A) receptor

摘要

Adenosine receptors are G protein-coupled receptors that sense extracellular adenosine to transmit intracellular signals. One of the four adenosine receptor subtypes, the adenosine A(2A) receptor (A(2A)R), has an exceptionally long intracellular C terminus (A(2A)R-ct) that mediates interactions with a large array of proteins, including calmodulin and a-actinin. Here, we aimed to ascertain the a-actinin 1/calmodulin interplay whilst binding to A(2A)R and the role of Ca2+ in this process. First, we studied the A(2A)R-a-actinin 1 interaction by means of native polyacrylamide gel electrophoresis, isothermal titration calorimetry, and surface plasmon resonance, using purified recombinant proteins. a-Actinin 1 binds the A2AR-ct through its distal calmodulin-like domain in a Ca2+-independent manner with a dissociation constant of 5-1211M, thus showing an 100 times lower affinity compared to the A(2A)R-calmodulin/Ca2+ complex. Importantly, calmodulin displaced a-actinin 1 from the A(2A)R-ct in a Ca2+-dependent fashion, disrupting the A(2A)R-a-actinin 1 complex. Finally, we assessed the impact of Ca2+ on A(2A)R internalization in living cells, a function operated by the A(2A)R-alpha-actinin 1 complex. Interestingly, while Ca2+ influx did not affect constitutive A(2A)R endocytosis, it abolished agonist-dependent internalization. In addition, we demonstrated that the A(2A)R/alpha-actinin interaction plays a pivotal role in receptor internalization and function. Overall, our results suggest that the interplay of A(2A)R with calmodulin and alpha-actinin 1 is fine-tuned by Ca2+, a fact that might power agonist-mediated receptor internalization and function. (C) 2017 Elsevier B.V. All rights reserved.