title: Human adenosine A2A receptor binds calmodulin with high affinity in a calcium-dependent manner.
abstract: Understanding how ligands bind to G-protein-coupled receptors and how binding changes receptor structure to affect signaling is critical for developing a complete picture of the signal transduction process. The adenosine A2A receptor (A2AR) is a particularly interesting example, as it has an exceptionally long intracellular carboxyl terminus, which is predicted to be mainly disordered. Experimental data on the structure of the A2AR C-terminus is lacking, because published structures of A2AR do not include the C-terminus. Calmodulin has been reported to bind to the A2AR C-terminus, with a possible binding site on helix 8, next to the membrane. The biological meaning of the interaction as well as its calcium dependence, thermodynamic parameters, and organization of the proteins in the complex are unclear. Here, we characterized the structure of the A2AR C-terminus and the A2AR C-terminus-calmodulin complex using different biophysical methods, including native gel and analytical gel filtration, isothermal titration calorimetry, NMR spectroscopy, and small-angle X-ray scattering. We found that the C-terminus is disordered and flexible, and it binds with high affinity (Kd = 98 nM) to calmodulin without major conformational changes in the domain. Calmodulin binds to helix 8 of the A2AR in a calcium-dependent manner that can displace binding of A2AR to lipid vesicles. We also predicted and classified putative calmodulin-binding sites in a larger group of G-protein-coupled receptors.
authors.affiliation: Faculty of Biochemistry and Molecular Medicine, University of Oulu, Finland; Biocenter Oulu, University of Oulu, Finland.
authors.forename: Henni
authors.initials: H
authors.lastname: Piirainen
authors.affiliation: Institute of Biotechnology, Program in Structural Biology & Biophysics, University of Helsinki, Finland.
authors.forename: Maarit
authors.initials: M
authors.lastname: Hellman
authors.affiliation: Institute of Biotechnology, Program in Structural Biology & Biophysics, University of Helsinki, Finland.
authors.forename: Helena
authors.initials: H
authors.lastname: Tossavainen
authors.affiliation: Institute of Biotechnology, Program in Structural Biology & Biophysics, University of Helsinki, Finland.
authors.forename: Perttu
authors.initials: P
authors.lastname: Permi
authors.affiliation: Faculty of Biochemistry and Molecular Medicine, University of Oulu, Finland; Biocenter Oulu, University of Oulu, Finland; Department of Biomedicine, University of Bergen, Norway.
authors.forename: Petri
authors.initials: P
authors.lastname: Kursula
authors.affiliation: Faculty of Biochemistry and Molecular Medicine, University of Oulu, Finland; Biocenter Oulu, University of Oulu, Finland. Electronic address: veli-pekka.jaakola@oulu.fi.
authors.forename: Veli-Pekka
authors.initials: VP
authors.lastname: Jaakola
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D011485:Protein Binding; D043705:Receptor, Adenosine A2A / Q000737:chemistry* / Q000378:metabolism
chemical_list: D002147:Calmodulin; D043705:Receptor, Adenosine A2A; D002118:Calcium