Glutamate Binding and Conformational Flexibility of Ligand-binding Domains Are Critical Early Determinants of Efficient Kainate Receptor Biogenesis

Martin B Gill,Pornpun Vivithanaporn,Geoffrey T Swanson

doi:10.1074/jbc.m900510200

Abstract

Intracellular glutamate binding within the endoplasmic reticulum (ER) is thought to be necessary for plasma membrane expression of ionotropic glutamate receptors. Here we determined the importance of glutamate binding to folding and assembly of soluble ligand-binding domains (LBDs), as well as full-length receptors, by comparing the secretion of a soluble GluR6-S1S2 protein versus the plasma membrane localization of GluR6 kainate receptors following mutagenesis of the LBD. The mutations were designed to either eliminate glutamate binding, thereby trapping the bilobate LBD in an "open" conformation, or "lock" the LBD in a closed conformation with an engineered interdomain disulfide bridge. Analysis of plasma membrane localization, medium secretion of soluble LBD proteins, and measures of folding efficiency suggested that loss of glutamate binding affinity significantly impacted subunit protein folding and assembly. In contrast, receptors with conformationally restricted LBDs also exhibited decreased PM expression and altered oligomeric receptor assembly but did not exhibit any deficits in subunit folding. Secretion of the closed LBD protein was enhanced compared with wild-type GluR6-S1S2. Our results suggest that glutamate acts as a chaperone molecule for appropriate folding of nascent receptors and that relaxation of LBDs from fully closed states during oligomerization represents a critical transition that necessarily engages other determinants within receptor dimers. Glutamate receptor LBDs therefore must access multiple conformations for efficient biogenesis.

Highlights

MAY 22, 2009 VOLUME 284 NUMBER 21 minants with accessory or chaperone proteins enhances forward transit from the endoplasmic reticulum (ER), insertion into the plasma membrane (PM), synaptic targeting, or a variety of other trafficking processes [3,4,5,6]
We compared the effect of an ligand-binding domains (LBDs) mutation that eliminated binding affinity for glutamate, T690A, on the efficiency of secretion of a soluble GluR6-S1S2 protein versus PM localization of the full-length receptor; this served as one way to test the autonomy of the LBD in quality control processes, because soluble LBDs fold properly, form binding sites similar to those in the fulllength receptor, and transit the secretory pathway to be released into culture media
We show here that glutamate binding within the ER is essential for the proper folding of the GluR6a KAR ligand-binding domain

Summary

To whom correspondence should be addressed

Dept. of Molecular Pharmacology and Biological Chemistry, Northwestern University, Feinberg School of Medicine, Searle 7-443, 303 E. Several recent studies have proposed that intracellular glutamate binding within the ER promotes proper folding and maturation of AMPA and kainate receptors and is required for forward trafficking of fully assembled receptors [10, 12,13,14,15]. This hypothesis is based in large part on the observation that mutagenesis of key glutamate-binding residues in the LBD greatly reduces or eliminates PM localization [7, 10, 12, 13]; the mechanistic basis of this putative quality control process is not understood. We propose that LBDs must be able to access multiple conformations for efficient KAR biogenesis

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION