Cornichon-2 modulates AMPA receptor-transmembrane AMPA receptor regulatory protein assembly to dictate gating and pharmacology.

Neuronal AMPA receptor complexes comprise a tetramer of GluA pore-forming subunits as well as accessory components, including transmembrane AMPA receptor regulatory proteins (TARPs) and cornichon-2/3 (CNIH-2/3). The mechanisms that control AMPA receptor complex assembly remain unclear. AMPA receptor responses in neurons differ from those in cell lines transfected with GluA plus TARPs γ-8 or γ-7, which show unusual resensitization kinetics and non-native AMPA receptor pharmacologies. Using tandem GluA/TARP constructs to constrain stoichiometry, we show here that these peculiar kinetic and pharmacological signatures occur in channels with four TARP subunits per complex. Reducing the number of TARPs per complex produces AMPA receptors with neuron-like kinetics and pharmacologies, suggesting a neuronal mechanism controls GluA/TARP assembly. Importantly, we find that coexpression of CNIH-2 with GluA/TARP complexes reduces TARP stoichiometry within AMPA receptors. In both rat and mouse hippocampal neurons, CNIH-2 also associates with AMPA receptors on the neuronal surface in a γ-8-dependent manner to dictate receptor pharmacology. In the cerebellum, however, CNIH-2 expressed in Purkinje neurons does not reach the neuronal surface. In concordance, stargazer Purkinje neurons, which express CNIH-2 and γ-7, display AMPA receptor kinetics/pharmacologies that can only be recapitulated recombinantly by a low γ-7/GluA stoichiometry. Together, these data suggest that CNIH-2 modulates neuronal AMPA receptor auxiliary subunit assembly by regulating the number of TARPs within an AMPA receptor complex to modulate receptor gating and pharmacology.

Two modes of interaction between the membrane-embedded TARP stargazin's C-terminal domain and the bilayer visualized by electron crystallography.

Glutamate-mediated neurotransmission through ligand-gated, ionotropic glutamate receptors is the main form of excitatory neurotransmission in the vertebrate central nervous system where it plays central roles in learning, memory and a variety of disorders. Acting as auxiliary subunits, transmembrane α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR) regulatory proteins (TARPs) are essential regulators for glutamate-mediated neurotransmission in the central nervous system. Here, we report the first electron crystallographic reconstructions of full-length mouse stargazin (γ-2) at ∼20Å resolution in a membrane bilayer environment. Formation of ordered arrays required anionic lipids and was modulated by cholesterol and monovalent cations. Projection structures revealed that the C-termini of stargazin monomers closely interacted with the bilayer surface in an extended conformation that placed the C-terminal PDZ-binding motif ∼100Å away from the transmembrane domain and in close proximity to a membrane re-entrant region. The C-termini interaction with the bilayer was modulated by the ionic strength of the solution and overall protein secondary structure increased when membrane-bound. Our data suggest that stargazin interactions with and within the membrane play significant roles in TARP structure and directly visualize TARP functional mechanisms essential for AMPAR trafficking and clustering.

Identifying interactions between transmembrane helices from the adenosine A2A receptor.

The human adenosine A(2A) receptor (A(2A)R) is an integral membrane protein and a member of the G-protein-coupled receptor (GPCR) superfamily, characterized by seven transmembrane (TM) helices. Although helix-helix association in the lipid bilayer is known to be an essential step in the folding of GPCRs, the determinants of their structures, folding, and assembly in the cell membrane are poorly understood. Previous studies in our group showed that while peptides corresponding to all seven TM domains of A(2A)R form stable helical structures in detergent micelles and lipid vesicles, they display significant variability in their helical propensity. This finding suggested to us that some TM domains might need to interact with other domains to properly insert and fold in hydrophobic environments. In this study, we assessed the ability of TM peptides to interact in pairwise combinations. We analyzed peptide interactions in hydrophobic milieus using circular dichroism spectroscopy and Förster resonance energy transfer. We find that specific interactions between TM helices occur, leading to additional helical content, especially in weakly helical TM domains, suggesting that some TM domains need a partner for proper folding in the membrane. The approach developed in this study will enable complete analysis of the TM domain interactions and the modeling of a folding pathway for A(2A)R.

Oligomerization of the fifth transmembrane domain from the adenosine A2A receptor.

The human adenosine A2A receptor (A(2A)R) belongs to one of the largest family of membrane proteins, the G-protein coupled receptors (GPCRs), characterized by seven transmembrane (TM) helices. Little is known about the determinants of their structures, folding, assembly, activation mechanisms, and oligomeric states. Previous studies in our group showed that peptides corresponding to all seven TM domains form stable helical structures in detergent micelles and lipid vesicles. However, the peptides behave differently; TM5 is the only peptide to have a ratio [theta]222/[theta]208 obtained by circular dichroism (CD) spectroscopy>1. This finding suggested to us that TM5 might self-associate. In the present study, we investigate the unique properties of the TM5 domain. We performed detailed analyses of TM5 peptide behavior in membrane-mimetic environments using CD spectroscopy, fluorescence spectroscopy and Förster resonance energy transfer, and gel electrophoresis. We find that TM5 peptide has the ability to self-associate to form oligomeric structures in various hydrophobic milieus and that these oligomers are highly resistant to temperature and chemical denaturation. We also find that mutation of the full-length A(2A)R at position M193, which is located in the fifth TM domain, noticeably alters A(2A)R monomer: dimer ratio as observed on SDS-PAGE. Our results suggest that parallel association of TM5 dimers may play a role in the known adenosine A2A receptor dimerization. This study represents the first evidence of an individual GPCR transmembrane domain self-association.