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Understanding AMPA receptor function and assembly, one piece at a time: structural and biochemical analysis of GLuR4 ligand-binding and ecto-domains.

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Call No.	QP364.7 .G54 2008
Author	Gill, Avinash.
Title	Understanding AMPA receptor function and assembly, one piece at a time: structural and biochemical analysis of GLuR4 ligand-binding and ecto-domains.
Date	2008
Abstract	AMPA receptors are glutamate-gated ion channels that mediate most of the excitatory synaptic signals in the central nervous system. They form tetrameric assemblies that are combinations of the subunits GluR1-4, each of which contains a ligand-binding domain (LBD) that undergoes conformational changes upon binding to glutamate. Crystal structures of the GluR2 LBD have revealed an agonist-binding cleft, which is located between two lobes and which acts like a Venus flytrap. In general, agonist efficacy is correlated with the extent of cleft closure. Given the recent observation that cleft closure does not vary with relative efficacy for a homologous NMDA receptor subunit, we wished to test the applicability of the cleft closure:efficacy correlation for other AMPA-R subunits. The crystal structures of GluR4 LBD that are presented in this study show that full agonists are capable of inducing more cleft closure than partial agonist, in a manner similar to that observed for GluR2. However, a detailed analysis of GluR4-LBD:kainate interactions reveals the importance of subtle backbone conformational changes in the ligand-binding pocket in determining the magnitude of agonist-associated conformational changes. Furthermore, the GluR4 subunit is more sensitive to cleft-closure variation than GluR2. The structures also confirm the stereochemical compatibility of the GluR2 and GluR4 LBD dimerization interfaces, consistent with the assembly and activation of heteromeric AMPA receptors. In order to further investigate the mechanism of partial agonism, we mutated the "foot-in-the-door" residue Leu651 to a Val, thereby changing the steric environment but preserving chemistry at the location. Crystal structures of the mutant LBD revealed that a "twisting" domain movement in three-dimensional space could increase the efficacy of partial agonist kainate, without any further increase in two-dimensional cleft closure about the hinge axis. Finally, a proper understanding of AMPA receptor function can come only from high resolution studies of higher order multidomain assemblies of the receptor. Working towards this end, we have established an expression and purification system for carrying out crystallization studies of the GluR4 ectodomain, which comprises of the N-terminal domain and LBD, and exhibits similar ligand-binding properties as the intact full-length receptor.
Description	ix, 218 leaves : ill. (some col.) 28 cm.
Format	application/pdf
Advisor	Madden, Dean R., Trumpower, Bernald L., Kull, Jon F., Oswald, Robert E.
Subject	Glutamic acid -- Receptors. Ion channels -- Molecular aspects. Ligand binding (Biochemistry). Medical Term: Glutamates.
College	Dartmouth Medical School -- Department of Biochemistry.
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D.)--Dartmouth College.
Copyright	All rights reserved. No part of this book may be reproduced in any form by any electronic or mechanical means without permission in writing from the author.
DOI	http://dx.doi.org/10.1349/ddlp.243

Title	Page 1
Format	application/pdf
Issuing Body	Dartmouth Dissertations are made available through the Dartmouth Digital Library.
Degree	Thesis (Ph.D.)--Dartmouth College.
Full Text	UNDERSTANDING AMPA RECEPTOR FUNCTION AND ASSEMBLY, ONE PIECE AT A TIME: STRUCTURAL AND BIOCHEMICAL ANALYSIS OF GLUR4 LIGAND-BINDING AND ECTO-DOMAINS A Thesis Submitted to the Faculty in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry by Avinash Gill DARTMOUTH COLLEGE Hanover, New Hampshire October 2nd, 2008 Examining Committee: Dean R. Madden, Ph.D. Bernald L. Trumpower, Ph.D. F. Jon Kull, Ph.D. Robert E. Oswald, Ph.D. Brian W. Pogue, Ph.D. Dean of Graduate Studies

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Understanding AMPA receptor function and assembly, one piece at a time: structural and biochemical analysis of GLuR4 ligand-binding and ecto-domains.

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