title: Calcium modulates calmodulin/α-actinin 1 interaction with and agonist-dependent internalization of the adenosine A2Areceptor.
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 A2Areceptor (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 Ca2+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 Ca2+-independent manner with a dissociation constant of 5-12μM, thus showing an ~100 times lower affinity compared to the A2AR-calmodulin/Ca2+complex. Importantly, calmodulin displaced α-actinin 1 from the A2AR-ct in a Ca2+-dependent fashion, disrupting the A2AR-α-actinin 1 complex. Finally, we assessed the impact of Ca2+on A2AR internalization in living cells, a function operated by the A2AR-α-actinin 1 complex. Interestingly, while Ca2+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 Ca2+, a fact that might power agonist-mediated receptor internalization and function.
authors.affiliation: Faculty of Biochemistry and Molecular Medicine, Biocenter Oulu, University of Oulu, Finland.
authors.forename: Henni
authors.initials: H
authors.lastname: Piirainen
authors.affiliation: Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona, Spain; Institut de Neurociències, Universitat de Barcelona, Spain.
authors.forename: Jaume
authors.initials: J
authors.lastname: Taura
authors.affiliation: Faculty of Biochemistry and Molecular Medicine, Biocenter Oulu, University of Oulu, Finland; Department of Biomedicine, University of Bergen, Norway.
authors.forename: Petri
authors.initials: P
authors.lastname: Kursula
authors.affiliation: Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona, Spain; Institut de Neurociències, Universitat de Barcelona, Spain. Electronic address: fciruela@ub.edu.
authors.forename: Francisco
authors.initials: F
authors.lastname: Ciruela
authors.affiliation: Faculty of Biochemistry and Molecular Medicine, 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: D000185:Actinin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000494:pharmacology; D058908:Adenosine A2 Receptor Agonists / Q000737:chemistry* / Q000494:pharmacology; D020816:Amino Acid Motifs; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D004705:Endocytosis / Q000187:drug effects; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D057809:HEK293 Cells; D006801:Humans; D007700:Kinetics; D010627:Phenethylamines / Q000737:chemistry* / Q000494:pharmacology; D011485:Protein Binding; D054730:Protein Interaction Domains and Motifs; D017433:Protein Structure, Secondary; D043705:Receptor, Adenosine A2A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: C526905:ACTN1 protein, human; D058908:Adenosine A2 Receptor Agonists; D002147:Calmodulin; D010627:Phenethylamines; D043705:Receptor, Adenosine A2A; D011994:Recombinant Proteins; D000185:Actinin; C061282:2-(4-(2-carboxyethyl)phenethylamino)-5'-N-ethylcarboxamidoadenosine; D000241:Adenosine; D002118:Calcium
keywords: Actin; Adenosine A(2A) receptor; Alpha-actinin; Ca(2+) binding; Carboxyl terminus; Crosslinking; Desensitization; GPCR; Internalization