Interactions between intracellular domains as key determinants of the quaternary structure and function of receptor heteromers.
G protein-coupled receptor (GPCR) heteromers are macromolecular complexes with unique functional properties different from those of its individual protomers. Little is known about what determines the quaternary structure of GPCR heteromers resulting in their unique functional properties. In this study, using resonance energy transfer techniques in experiments with mutated receptors, we provide for the first time clear evidence for a key role of intracellular domains in the determination of the quaternary structure of GPCR heteromers between adenosine A(2A), cannabinoid CB(1), and dopamine D(2) receptors. In these interactions, arginine-rich epitopes form salt bridges with phosphorylated serine or threonine residues from CK1/2 consensus sites. Each receptor (A(2A), CB(1), and D(2)) was found to include two evolutionarily conserved intracellular domains to establish selective electrostatic interactions with intracellular domains of the other two receptors, indicating that these particular electrostatic interactions constitute a general mechanism for receptor heteromerization. Mutation experiments indicated that the interactions of the intracellular domains of the CB(1) receptor with A(2A) and D(2) receptors are fundamental for the correct formation of the quaternary structure needed for the function (MAPK signaling) of the A(2A)-CB(1)-D(2) receptor heteromers. Analysis of MAPK signaling in striatal slices of CB(1) receptor KO mice and wild-type littermates supported the existence of A(1)-CB(1)-D(2) receptor heteromer in the brain. These findings allowed us to propose the first molecular model of the quaternary structure of a receptor heteromultimer.
Navarro
Gemma
G
Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas, Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain.
Ferré
Sergi
S
Intramural Research Program, National Institute on Drug Abuse, Department of Health and Human Services, Baltimore, Maryland 21224.
Cordomi
Arnau
A
Laboratori de Medicina Computacional, Unitat de Bioestadística, Universitat Autónoma de Barcelona, 08193 Bellaterra, Spain.
Moreno
Estefania
E
Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas, Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain.
Mallol
Josefa
J
Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas, Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain.
Casadó
Vicent
V
Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas, Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain.
Cortés
Antoni
A
Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas, Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain.
Hoffmann
Hanne
H
Neuroscience Institute and Department of Biochemistry and Molecular Biology, Facultat de Medicina, Universitat Autónoma de Barcelona, 08193 Bellaterra, Spain.
Ortiz
Jordi
J
Neuroscience Institute and Department of Biochemistry and Molecular Biology, Facultat de Medicina, Universitat Autónoma de Barcelona, 08193 Bellaterra, Spain.
Canela
Enric I
EI
Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas, Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain.
Lluís
Carme
C
Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas, Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain.
Pardo
Leonardo
L
Laboratori de Medicina Computacional, Unitat de Bioestadística, Universitat Autónoma de Barcelona, 08193 Bellaterra, Spain.
Franco
Rafael
R
Centro de Investigación Biomédica en Red Sobre Enfermedades Neurodegenerativas, Department of Biochemistry and Molecular Biology, Faculty of Biology, University of Barcelona, 08028 Barcelona, Spain; Centro de Investigación Médica Aplicada, Universidad de Navarra, 31008 Pamplona, Spain.
Woods
Amina S
AS
Intramural Research Program, National Institute on Drug Abuse, Department of Health and Human Services, Baltimore, Maryland 21224. Electronic address: awoods@intra.nida.nih.gov.
D000596:Amino Acids / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D002460:Cell Line; D000939:Epitopes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D020935:MAP Kinase Signaling System / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D008958:Models, Molecular; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013329:Structure-Activity Relationship
D000596:Amino Acids; D000939:Epitopes; D043562:Receptors, G-Protein-Coupled