Calcium modulates calmodulin/α-actinin 1 interaction with and agonist-dependent internalization of the adenosine A receptor.

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 receptor (AR), has an exceptionally long intracellular C terminus (AR-ct) that mediates interactions with a large array of proteins, including calmodulin and α-actinin. Here, we aimed to ascertain the α-actinin 1/calmodulin interplay whilst binding to AR and the role of Ca in this process. First, we studied the AR-α-actinin 1 interaction by means of native polyacrylamide gel electrophoresis, isothermal titration calorimetry, and surface plasmon resonance, using purified recombinant proteins. α-Actinin 1 binds the AR-ct through its distal calmodulin-like domain in a Ca-independent manner with a dissociation constant of 5-12μM, thus showing an ~100 times lower affinity compared to the AR-calmodulin/Ca complex. Importantly, calmodulin displaced α-actinin 1 from the AR-ct in a Ca-dependent fashion, disrupting the AR-α-actinin 1 complex. Finally, we assessed the impact of Ca on AR internalization in living cells, a function operated by the AR-α-actinin 1 complex. Interestingly, while Ca influx did not affect constitutive AR endocytosis, it abolished agonist-dependent internalization. In addition, we demonstrated that the AR/α-actinin interaction plays a pivotal role in receptor internalization and function. Overall, our results suggest that the interplay of AR with calmodulin and α-actinin 1 is fine-tuned by Ca, a fact that might power agonist-mediated receptor internalization and function.