HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 284, Issue 1, 324-333, January 2, 2009

This Article Full Text Full Text (PDF) Supplemental Data All Versions of this Article: 284/1/324    most recent M804023200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Kenny, A. V. Articles by Stephenson, F. A. Search for Related Content PubMed PubMed Citation Articles by Kenny, A. V. Articles by Stephenson, F. A. Social Bookmarking                      What's this?

The Integrity of the Glycine Co-agonist Binding Site of N-Methyl-D-aspartate Receptors Is a Functional Quality Control Checkpoint for Cell Surface Delivery^*

From the School of Pharmacy, University of London, 29/39 Brunswick Square, London WC1N 1AX, United Kingdom

N-Methyl-D-aspartate receptors are a subclass of ligand-gated, heteromeric glutamatergic neurotransmitter receptors whose cell surface expression is regulated by quality control mechanisms. Functional quality control checkpoints are known to contribute to cell surface trafficking of non-N-methyl-D-aspartate glutamate receptors. Here we investigated if similar mechanisms operate for the surface delivery of NMDA receptors. Point mutations in the glycine binding domain of the NR1-1a subunit were generated: D732A, a mutation that results in an 3 x 10⁴ decrease in glycine binding affinity; D732E, a conservative change; and D723A, a residue in the same NR1-1a domain that has no effect on glycine binding affinity. Each NR1-1a subunit was co-expressed with NR2A in mammalian cells. Immunoblotting and immunoprecipitations showed that all mutants were expressed to similar levels as wild-type NR1-1a and associated with NR2A. Cell surface expression measured by an enzyme-linked immunosorbent assay found that whereas NR1-1a (D732E)/NR2A and NR1-1a (D723A)/NR2A trafficked as efficiently as NR1-1a/NR2A, there was a 90% decrease in surface expression for NR1-1a (D732A)/NR2A. This was confirmed by confocal microscopy imaging and cell surface biotinylation. Further imaging showed that NR1-1a (D732A) and co-transfected NR2A co-localized with an endoplasmic reticulum marker. Dichlorokynurenic acid, a competitive glycine site antagonist, partially rescued surface expression. Mutation of the NR1-1a ER retention motif showed that the ligand binding checkpoint is an early event preceding endoplasmic reticulum sorting mechanisms. These findings demonstrate that integrity of the glycine co-agonist binding site is a functional checkpoint requisite for efficient cell surface trafficking of assembled NMDA receptors.

Received for publication, May 27, 2008 , and in revised form, October 30, 2008.

^* This work was funded by the Biotechnology and Biological Sciences Research Council (BBSRC), United Kingdom. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table 1.

¹ To whom correspondence should be addressed. Tel.: 44-207-753-5877; Fax: 44-207-753-5964; E-mail: anne.stephenson{at}pharmacy.ac.uk.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				M. B. Gill, P. Vivithanaporn, and G. T. Swanson Glutamate Binding and Conformational Flexibility of Ligand-binding Domains Are Critical Early Determinants of Efficient Kainate Receptor Biogenesis J. Biol. Chem., May 22, 2009; 284(21): 14503 - 14512. [Abstract] [Full Text] [PDF]

				M. Horak and R. J. Wenthold Different Roles of C-terminal Cassettes in the Trafficking of Full-length NR1 Subunits to the Cell Surface J. Biol. Chem., April 10, 2009; 284(15): 9683 - 9691. [Abstract] [Full Text] [PDF]