Interactions between Calmodulin, Adenosine A2A, and Dopamine D2 Receptors

Carmen Lluís,Vicent Casadó,Jose Luis Lanciego,Antoni Cortés,Marisol S. Aymerich,Josefa Mallol,Daniel Marcellino,Amina S. Woods,Sergi Ferré,Kjell Fuxe,Rafael Franco,Enric I. Canela,Gemma Navarro,Luigi Agnati

doi:10.1074/jbc.m109.034231

Abstract

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.

Highlights

G-protein-coupled receptors are able to form homo- and hetero-oligomers with unique biochemical and functional characteristics [1,2,3,4,5,6,7], and they are detected in vitro by using biophysical techniques (8 –10)
CaM binds to the N-terminal portion of the long third intracellular loop of the D2 receptor, within an Arg-rich epitope that is involved in the binding to Gi/o proteins and to the adenosine A2A receptor, with the formation of A2A-D2 receptor heteromers
By using bioluminescence resonance energy transfer (BRET) and sequential resonance energy transfer (SRET) techniques and analyzing MAPK signaling in transfected cells, evidence was obtained for CaM-A2A-D2 receptor oligomerization and a selective Ca2ϩmediated modulation of A2A and D2 receptor function in the A2A-D2 receptor heteromer

Summary

EXPERIMENTAL PROCEDURES

Animals—Male Sprague-Dawley rats (250 g) were obtained from Harlan (Barcelona, Spain). Immunoprecipitation—The pellet containing membrane proteins (P2Ј) was solubilized in immunoprecipitation buffer (phosphate-buffered saline (pH 7.4), complete protease inhibitor mixture, and 1% Igepal Ca-630 (Sigma)) for 1 h at 4 °C. BRET Assays—HEK-293T cells were transiently co-transfected with the indicated amounts of plasmid cDNAs corresponding to the indicated fusion proteins (see figure legends). For BRET measurements, the equivalent of 20 ␮g of cell protein was distributed in 96-well microplates (Corning 3600, white plates; Sigma), and 5 ␮M coelenterazine H (Molecular Probes, Inc., Eugene, OR) was added. By analogy with BRET, net SRET is defined as (long wavelength emission/short wavelength emission) Ϫ Cf, where Cf corresponds to long wavelength emission/short wavelength emission for cells expressing protein-Rluc, protein-GFP2, and the other protein partner not fused to a fluorescence protein (similar values were obtained measuring Cf in cells expressing protein-Rluc only and proteinGFP2). One-way analysis of variance and Student’s t test for unpaired samples were used for statistical comparisons

RESULTS

These findings correlate with the potential ability of CaM and

DISCUSSION

The results not only indicated that CaM may interact with