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

J. Biol. Chem., Vol. 279, Issue 3, 1627-1636, January 16, 2004

This Article Full Text Full Text (PDF) All Versions of this Article: 279/3/1627    most recent M303811200v1 Alert me when this article is cited 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 Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Breitinger, U. Articles by Becker, C.-M. Search for Related Content PubMed PubMed Citation Articles by Breitinger, U. Articles by Becker, C.-M. Social Bookmarking                      What's this?

Conserved High Affinity Ligand Binding and Membrane Association in the Native and Refolded Extracellular Domain of the Human Glycine Receptor 1-Subunit^*

Ulrike Breitinger, Hans-Georg Breitinger, Finn Bauer, Karim Fahmy¶, Daniela Glockenhammer, and Cord-Michael Becker||

From the Institut für Biochemie, Emil-Fischer-Zentrum, Friedrich-Alexander-Universität Erlangen-Nürnberg, D-91054 Erlangen, Germany, the Lehrstuhl Biopolymere, Universität Bayreuth, D-95440 Bayreuth, Germany, and the ^¶Institut für Kern- und Hadronenphysik, Forschungszentrum Rossendorf, D-01314 Dresden, Germany

The strychnine-sensitive glycine receptor (GlyR) is a ligand-gated chloride channel composed of ligand binding - and gephyrin anchoring -subunits. To identify the secondary and quaternary structures of extramembraneous receptor domains, the N-terminal extracellular domain (1-(1–219)) and the large intracellular TM3–4 loop (1-(309–392)) of the human GlyR 1-subunit were individually expressed in HEK293 cells and in Escherichia coli. The extracellular domain obtained from E. coli expression was purified in its denatured form and refolding conditions were established. Circular dichroism and Fourier-transform-infrared spectroscopy suggested 25% -helix and 48% -sheet for the extracellular domain, while no -helices were detectable for the TM3–4 loop. Size exclusion chromatography and sucrose density centrifugation indicated that isolated glycine receptor domains assembled into multimers of distinct molecular weight. For the extracellular domain from E. coli, we found an apparent molecular weight compatible with a 15mer by gel filtration. The N-terminal domain from HEK293 cells, analyzed by sucrose gradient centrifugation, showed a bimodal distribution, suggesting oligomerization of 5 and 15 subunits. Likewise, for the intracellular domain from E. coli, a single molecular mass peak of 49 kDa indicated oligomerization in a defined native structure. As shown by [³H]strychnine binding, expression in HEK293 cells and refolding of the isolated extracellular domain reconstituted high affinity antagonist binding. Cell fractionation, alkaline extraction experiments, and immunocytochemistry showed a tight plasma membrane association of the isolated GlyR N-terminal protein. These findings indicate that distinct functional characteristics of the full-length GlyR are retained in the isolated N-terminal domain.

Received for publication, April 11, 2003 , and in revised form, October 16, 2003.

^* This work was supported by the Deutsche Forschungsgemeinschaft (SFB 353) and Fonds der Chemischen Industrie. 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.

^|| To whom correspondence should be addressed: Institut für Biochemie, Emil-Fischer-Zentrum, Universität Erlangen-Nürnberg, Fahrstrasse 17, D-91054 Erlangen, Germany. Tel.: 49-9131-85-24190; Fax: 49-9131-85-22485; E-mail: cmb{at}biochem.uni-erlangen.de.

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

This article has been cited by other articles:

				A. M. Person, K. L. Bills, H. Liu, S. K. Botting, J. Lindstrom, and G. B. Wells Extracellular Domain Nicotinic Acetylcholine Receptors Formed by {alpha}4 and {beta}2 Subunits J. Biol. Chem., December 2, 2005; 280(48): 39990 - 40002. [Abstract] [Full Text] [PDF]