A Two-state Model for the Diffusion of the A2A Adenosine Receptor in Hippocampal Neurons: AGONIST-INDUCED SWITCH TO SLOW MOBILITY IS MODIFIED BY SYNAPSE-ASSOCIATED PROTEIN 102 (SAP102)

Patrick Thurner,Ingrid Gsandtner,Oliver Kudlacek,Daniel Choquet,Christian Nanoff,Michael Freissmuth,Jürgen Zezula

doi:10.1074/jbc.m113.505685

Abstract

The A2A receptor is a class A/rhodopsin-like G protein-coupled receptor. Coupling to its cognate protein, Gs, occurs via restricted collision coupling and is contingent on the presence of cholesterol. Agonist activation slows diffusion of the A2A adenosine receptor in the lipid bilayer. We explored the contribution of the hydrophobic core and of the extended C terminus by examining diffusion of quantum dot-labeled receptor variants in dissociated hippocampal neurons. Single particle tracking of the A2A receptor(1-311), which lacks the last 101 residues, revealed that agonist-induced confinement was abolished and that the agonist-induced decrease in diffusivity was reduced substantially. A fragment comprising the SH3 domain and the guanylate kinase domain of synapse-associated protein 102 (SAP102) was identified as a candidate interactor that bound to the A2A receptor C terminus. Complex formation between the A2A receptor and SAP102 was verified by coimmunoprecipitation and by tracking its impact on receptor diffusion. An analysis of all trajectories by a hidden Markov model was consistent with two diffusion states where agonist activation reduced the transition between the two states and, thus, promoted the accumulation of the A2A receptor in the compartment with slow mobility. Overexpression of SAP102 precluded the access of the A2A receptor to a compartment with restricted mobility. In contrast, a mutated A2A receptor (with (383)DVELL(387) replaced by RVRAA) was insensitive to the action of SAP102. These observations show that the hydrophobic core per se does not fully account for the agonist-promoted change in mobility of the A2A receptor. The extended carboxyl terminus allows for regulatory input by scaffolding molecules such as SAP102.

Highlights

Agonist activation slows diffusion of the A2A receptor in the lipid bilayer
We explored the contribution of the hydrophobic core and of the extended C terminus by examining diffusion of quantum dot-labeled receptor variants in dissociated hippocampal neurons
Single Particle Tracking of the Wild-type A2A Receptor and of a C-terminally Truncated Version—Agonist-induced changes in the mobility of the A2A receptor may be mediated by binding of cholesterol to the hydrophobic core [14, 15] or by tethering via the extended C terminus. We addressed this directly by measuring the trajectories of the quantum dot-labeled wildtype receptor and of a C-terminally truncated version in neurite extensions of dissociated hippocampal neurons

Summary

Background

Results: In hippocampal neurons, the agonist-induced decrease in mobility was accounted for by both the hydrophobic receptor core and its extended C terminus, which recruited SAP102. A mutated A2A receptor (with 383DVELL387 replaced by RVRAA) was insensitive to the action of SAP102 These observations show that the hydrophobic core per se does not fully account for the agonist-promoted change in mobility of the A2A receptor. Impact of SAP102 on A2A Receptor Diffusion adenosine receptor can stimulate mitogen-activated protein kinase/ERK by a cAMP-independent pathway [11, 12] that is contingent on recruitment of ARNO (the exchange factor for ARF6) [13]. We examined previously the diffusion mode of the A2A receptor and an artificially palmitoylated version thereof by single particle tracking These experiments indicated that the absence of the palmitoylated cysteine was required for restricted collision coupling. The C terminus specifies access of the receptor to areas of low mobility by recruiting scaffolding molecules, one of which was identified as SAP102

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION