Cellular mechanisms that modulate kainate receptor trafficking and assembly



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Kainate receptors (KARs) in the mammalian brain play a variety of physiological roles that require selective assembly, intracellular trafficking, and synaptic targeting. Cytoplasmic and non-cytoplasmic determinants that modulate KAR expression at the plasma membrane have been recently characterized. The intracellular trafficking determinants are presumed to bind to chaperone proteins, but these proteins have not been identified for any KAR subunit. Here we identified two chaperone proteins that associated with the carboxy terminus but differently regulated the localization of KA2 receptors. We found that coatomer protein complex I (COPI) subunits interacted with KA2 subunits at the arginine-based ER retention/retrieval motif and these associations were decreased in heteromeric GluR6a/KA2 receptors. Disruptions of COPI and KA2 associations by alanine mutations at the arginine-rich domain and elimination of COPI vesicles were correlated with the increased expression at the plasma membrane of KA2 receptors, indicating that COPI proteins regulated the ER localization of receptors. KA2 receptors also co-precipitated with three isoforms of 14-3-3 proteins; only the KA2 and 14-3-3zeta association was correlated with higher plasma membrane expression of receptors. In addition to identifying cytoplasmic chaperone systems, we were interested in understanding the nature of a recently described trafficking checkpoint in non-cytoplasmic regions. Mutations in domains typically involved in glutamate binding and ion permeation disrupt expression of KARs at the plasma membrane. A conformational change of receptors after glutamate binding is proposed to permit egress of KARs from the ER. We mutated critical amino acid residues in the extracellular linker domain of GluR6a subunits and found that desensitization rates were only weakly correlated with plasma membrane expression levels. Alteration of these residues impaired other stages of receptor biosyntheis including assembly and degradation of mutated receptors. We found that mutations at the transduction linker collectively altered subunit assembly, degradation, desensitization and a post-assembly stage. Our characterizations of chaperone proteins and mechanisms that regulate intracellular trafficking provide a better understanding of cellular controls in the early stages of KAR biosynthesis.



plasma membrane expression, oligomerization, KA2, glutamate receptors, GluR6, biogenesis