Piirainen, Henni, Taura, Jaume, Kursula, Petri, Ciruela, Francisco and Jaakola, Veli-Pekka (2017) Calcium modulates calmodulin/α-actinin 1 interaction with and agonist-dependent internalization of the adenosine A2A receptor. Biochimica et biophysica acta. ISSN 0006-3002

Abstract

Adenosine receptors are G protein-coupled receptors that sense extracellular adenosine to transmit intracellular signals. One of the four adenosine receptor subtypes, the adenosine A2A receptor (A2AR), has an exceptionally long intracellular C terminus (A2AR-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 A2AR and the role of Ca(2+) in this process. First, we studied the A2AR-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 A2AR-ct through its distal calmodulin-like domain in a Ca(2+)-independent manner with a dissociation constant of 5-12μM, thus showing a~100 times lower affinity compared to the A2AR-calmodulin/Ca(2+) complex. Importantly, calmodulin displaced α-actinin 1 from the A2AR-ct in a Ca(2+)-dependent fashion, disrupting the A2AR-α-actinin 1 complex. Finally, we assessed the impact of Ca(2+) on A2AR internalization in living cells, a function operated by the A2AR-α-actinin 1 complex. Interestingly, while Ca(2+) influx did not affect constitutive A2AR endocytosis, it abolished agonist-dependent internalization. In addition, we demonstrated that the A2AR/α-actinin interaction plays a pivotal role in receptor internalization and function. Overall, our results suggest that the interplay of A2AR with calmodulin and α-actinin 1 is fine-tuned by Ca(2+), a fact that might power agonist-mediated receptor internalization and function.

Item Type:	Article
Keywords:	Adenosine A(2A) receptor; Ca(2+) binding; GPCR; actin; alpha-actinin; carboxyl terminus; crosslinking; desensitization; internalization
Date Deposited:	31 Jan 2017 00:45
Last Modified:	31 Jan 2017 00:45
URI:	https://oak.novartis.com/id/eprint/32070