Interactions between calmodulin, adenosine A2A, and dopamine D2 receptors.
The Ca(2+)-binding protein calmodulin (CaM) has been shown to bind directly to cytoplasmic domains of some G protein-coupled receptors, including the dopamine D(2) receptor. CaM binds to the N-terminal portion of the long third intracellular loop of the D(2) receptor, within an Arg-rich epitope that is also involved in the binding to G(i/o) proteins and to the adenosine A(2A) receptor, with the formation of A(2A)-D(2) receptor heteromers. In the present work, by using proteomics and bioluminescence resonance energy transfer (BRET) techniques, we provide evidence for the binding of CaM to the A(2A) receptor. By using BRET and sequential resonance energy transfer techniques, evidence was obtained for CaM-A(2A)-D(2) receptor oligomerization. BRET competition experiments indicated that, in the A(2A)-D(2) receptor heteromer, CaM binds preferentially to a proximal C terminus epitope of the A(2A) receptor. Furthermore, Ca(2+) was found to induce conformational changes in the CaM-A(2A)-D(2) receptor oligomer and to selectively modulate A(2A) and D(2) receptor-mediated MAPK signaling in the A(2A)-D(2) receptor heteromer. These results may have implications for basal ganglia disorders, since A(2A)-D(2) receptor heteromers are being considered as a target for anti-parkinsonian agents.
Navarro
Gemma
G
Institut d'Investigacions Biomèdiques August Pi i Sunyer, Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas, and Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain.
Aymerich
Marisol S
MS
Centro de Investigación Médica Aplicada Neurociencias (CIMA), Universidad de Navarra, Avda. Pio XII 55, 31008 Pamplona, Spain.
Marcellino
Daniel
D
Department of Neuroscience, Karolinska Institute, 17177 Stockholm, Sweden.
Cortés
Antoni
A
Institut d'Investigacions Biomèdiques August Pi i Sunyer, Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas, and Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain.
Casadó
Vicent
V
Institut d'Investigacions Biomèdiques August Pi i Sunyer, Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas, and Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain.
Mallol
Josefa
J
Institut d'Investigacions Biomèdiques August Pi i Sunyer, Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas, and Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain.
Canela
Enric I
EI
Institut d'Investigacions Biomèdiques August Pi i Sunyer, Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas, and Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain.
Agnati
Luigi
L
Department of Neuroscience, Karolinska Institute, 17177 Stockholm, Sweden.
Woods
Amina S
AS
National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Baltimore, Maryland 21224.
Fuxe
Kjell
K
Department of Neuroscience, Karolinska Institute, 17177 Stockholm, Sweden.
Lluís
Carmen
C
Institut d'Investigacions Biomèdiques August Pi i Sunyer, Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas, and Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain.
Lanciego
Jose Luis
JL
Centro de Investigación Médica Aplicada Neurociencias (CIMA), Universidad de Navarra, Avda. Pio XII 55, 31008 Pamplona, Spain.
Ferré
Sergi
S
National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Baltimore, Maryland 21224.
Franco
Rafael
R
Institut d'Investigacions Biomèdiques August Pi i Sunyer, Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas, and Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain; Centro de Investigación Médica Aplicada Neurociencias (CIMA), Universidad de Navarra, Avda. Pio XII 55, 31008 Pamplona, Spain. Electronic address: rfranco@unav.es.
D000241:Adenosine / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D002147:Calmodulin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D004298:Dopamine / Q000378:metabolism; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology; D008297:Male; D008969:Molecular Sequence Data; D046912:Multiprotein Complexes; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods; D051381:Rats; D017207:Rats, Sprague-Dawley; D043705:Receptor, Adenosine A2A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017448:Receptors, Dopamine D2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
D002147:Calmodulin; D046912:Multiprotein Complexes; D043705:Receptor, Adenosine A2A; D017448:Receptors, Dopamine D2; D011993:Recombinant Fusion Proteins; D000241:Adenosine; D004298:Dopamine