Intracellular Trafficking of KA2 Kainate Receptors Mediated by Interactions with Coatomer Protein Complex I (COPI) and 14-3-3 Chaperone Systems

Abstract

Assembly and trafficking of neurotransmitter receptors are processes contingent upon interactions between intracellular chaperone systems and discrete determinants in the receptor proteins. Kainate receptor subunits, which form ionotropic glutamate receptors with diverse roles in the central nervous system, contain a variety of trafficking determinants that promote either membrane expression or intracellular sequestration. In this report, we identify the coatomer protein complex I (COPI) vesicle coat as a critical mechanism for retention of the kainate receptor subunit KA2 in the endoplasmic reticulum. COPI subunits immunoprecipitated with KA2 subunits from both cerebellum and COS-7 cells, and ␤-COP protein interacted directly with immobilized KA2 peptides containing the arginine-rich retention/retrieval determinant. Association between COPI proteins and KA2 subunits was significantly reduced upon alanine substitution of this signal in the cytoplasmic tail of KA2. Temperature-sensitive degradation of COPI complex proteins was correlated with an increase in plasma membrane localization of the homologous KA2 receptor. Assembly of heteromeric GluR6a/KA2 receptors markedly reduced association of KA2 and COPI. Finally, the reduction in COPI binding was correlated with an increased association with 14-3-3 proteins, which mediate forward trafficking of other integral signaling proteins. These interactions therefore represent a critical early checkpoint for biosynthesis of functional KARs. Kainate receptors (KARs)2 play a variety of roles in the mammalian central nervous system that include contributions to postsynaptic neurotransmission at a subset of excitatory synapses and presynaptic modulation of both excitatory and inhibitory transmission (1, 2). These diverse physiological roles of KARs require selective assembly, subcellular trafficking, and targeting of these proteins to their functional sites. Elucidating the cellular mechanisms that control these processes is important for understanding the full spectrum of KAR-mediated signaling in the brain.Oligomerization and intracellular trafficking of KARs are controlled in part through interactions with chaperone proteins that bind to discrete cytoplasmic domains on the receptor proteins themselves. For example, GluR6a KAR subunits contain a forward trafficking determinant in their cytoplasmic tail and therefore are highly expressed as homomeric receptors on the plasma membrane of heterologous cell lines (3), whereas the KA2 subunit has an endoplasmic reticulum (ER) retention/retrieval signal and consequently does not reach the plasma membrane in the absence of other KAR subunits (4). The KA2 ER retention/retrieval signal consists primarily of an arginine-rich domain similar to that characterized on several other ionotropic glutamate receptor subunits, including NR1 (5) and GluR5-2c (6), as well as other signaling proteins such as ATP-sensitive potassium channels (K ATP ) (7) and GABA B receptors (8). Although a number of trafficking motifs in KAR subunits have been id...

Exciting Times: New Advances Towards Understanding the Regulation and Roles of Kainate Receptors

“…ER retention sequences in the C-terminus of GluK5 facilitate its interaction with the COPI coat complex, driving retrograde Golgi to ER trafficking of KARs, and acting as an ER retrieval mechanism [74]. This interaction is disrupted by the heteromerization of GluK2 with GluK5 and binding to 14-3-3ζ, which promotes forward trafficking to the cell surface, both driving ER exit and favouring assembly of heteromeric KARs [74].…”

Endocytosis of the Glutamate Receptor Subunit GluK3 Controls Polarized Trafficking

“…The differential membrane targeting of KAR subunit splice variants, as for many channels and receptors (Ellgaard and Helenius, 2003), relies on retention in the ER based on the presence of lysine (KKXX) or arginine (RXR) amino acid sequences (Ma and Jan, 2002). The arginine-based motifs have been shown to mediate COPI complex binding, which is involved in retrieval transport of ER resident proteins (Vivithanaporn et al, 2006). The RXR site present in GluK1c and GluK5 clearly prevents export of these subunits to the plasma membrane (Ren et al, 2003a,b).…”

Kainate receptor trafficking

“…14-3-3 proteins are involved in a range of cellular processes including the regulation of enzyme activity, stimulation of protein-protein interactions, and the localization and function of ion channels. 93,94 ERretained GluK5 appears to be bound to COPI, whereas subunit assembly with GluK2 releases GluK5 from the ER and increases its interaction with 14-3-3ζ and subsequent surface expression 81 suggesting that the ER retention signals are masked following oligomerization and highlighting how correct receptor assembly can lead to the switching of interacting partners to regulate a trafficking checkpoint within the biosynthetic pathway.…”

“…[77][78][79] These subunits contain an RXR motif in the intracellular region between the second and third membrane domains 80 and an intracellular C-terminal arginine-rich (RRRRR) ER retention/retrieval motif. This motif is a binding site for the coatomer protein I (COPI) 81 a component of the retrograde trafficking protein complex that mediates the transport of defective proteins back from the Golgi to the ER. 82 In addition, GluK5 contains a di-leucine endocytic motif that favors internalization of any subunits that do get trafficked to the plasma membrane.…”

“…COPI has been shown to bind GluK5 to mediate its ER retention. 81 Interestingly, disruption of COPI or the arginine-based ER retention motifs enhance binding of GluK5 to 14-3-3ζ . 14-3-3 proteins are involved in a range of cellular processes including the regulation of enzyme activity, stimulation of protein-protein interactions, and the localization and function of ion channels.…”