Interactions between Intracellular Domains as Key Determinants of the Quaternary Structure and Function of Receptor Heteromers

Gemma Navarro,Rafael Franco,Josefa Mallol,Arnau Cordomi,Jordi Ortiz,Hanne Hoffmann,Enric I Canela,Leonardo Pardo,Antoni Cortés,Amina S Woods,Vicent Casadó,Estefania Moreno,Carme Lluís,Sergi Ferré

doi:10.1074/jbc.m110.115634

Abstract

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.

Highlights

It was inferred that different molecular mechanisms were involved in G protein-coupled receptor (GPCR) homo- and heteromerization
By using mutated A2A, CB1, and D2 receptors, we investigated the relevance of electrostatic interactions [16] between intracellular domains in the determination of the quaternary structure of GPCR heteromers between A2A, CB1, and D2 receptors
RRRKRVN epitope is most probably involved in A2A-D2 receptor heteromerization [28, 29], we explored the possibility that IL3 of the D2 receptor could interact with IL3 of the CB1 receptor, The D2 short isoform (D2S), an alternative splicing that lacks 29 amino acid residues of IL3 [30], including 266NRRRVEAARR275, was used

Summary

Interactions between Intracellular Domains as Key

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 A2A, cannabinoid CB1, and dopamine D2 receptors. In these interactions, arginine-rich epitopes form salt bridges with phosphorylated serine or threonine residues from CK1/2 consensus sites. Mutation experiments indicated that the interactions of the intracellular domains of the CB1 receptor with A2A and D2 receptors are fundamental for the correct formation of the quaternary structure needed for the function (MAPK signaling) of the A2A-CB1-D2 receptor heteromers. The same intracellular domains involved in A2ACB1-D2 receptor heteromultimerization were involved in

Quaternary Structure of Receptor Heteromers

EXPERIMENTAL PROCEDURES

RESULTS

Computational Model of the Quaternary Structure of the

DISCUSSION