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J. Biol. Chem., Vol. 279, Issue 15, 14639-14648, April 9, 2004

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Ryanodine Receptor Oligomeric Interaction

IDENTIFICATION OF A PUTATIVE BINDING REGION^*

Lynda M. Blayney, Spyros Zissimopoulos, Emma Ralph, Eleanor Abbot, Laura Matthews, and F. Anthony Lai

From the Wales Heart Research Institute, Department of Cardiology, University of Wales College of Medicine, Heath Park, Cardiff CF14 4XN, Wales, United Kingdom

Specific interactions between adjacent ryanodine receptor (RyR) molecules to form ordered two-dimensional arrays in the membrane have been demonstrated using electron microscopy both in situ, in tissues and cells, and in vitro, with the purified protein. RyR interoligomeric association has also been inferred from observations of simultaneous channel gating during multi-RyR channel recordings in lipid bilayers. In this study, we report experiments designed to identify the region(s) of the RyR molecule, participating in this reciprocal interaction. Using epitope-specific antibodies, we identified a RyR tryptic fragment that specifically bound the intact immobilized RyR. Three overlapping RyR fragments encompassing this epitope, expressed using an in vitro mammalian expression system, were immunoprecipitated by RyR. To refine the binding regions, smaller RyR fragments were expressed as glutathione S-transferase (GST) fusion proteins, and their binding to RyR was monitored using a "sandwich" enzyme-linked immunosorbent assay. Three GST-RyR fusion proteins demonstrated specific binding, dependent upon ionic strength. Binding was greatest at 50–150 mM NaCl for two GST-RyR constructs, and a third GST-RyR construct demonstrated maximum binding between 150 and 450 mM NaCl. The binding at high NaCl concentration suggested involvement of a hydrophobic interaction. In silico analysis of secondary structure showed evidence of coil regions in two of these RyR fragment sequences, which might explain these data. In GST pull-down assays, these same three fragments captured RyR2, and two of them retained RyR1. These results identify a region at the center of the linear RyR (residues 2540–3207 of human RyR2) which is able to bind to the RyR oligomer. This region may constitute a specific subdomain participating in RyR-RyR interaction.

Received for publication, July 23, 2003 , and in revised form, January 13, 2004.

^* This work was supported by British Heart Foundation Grants PG99087 and PG 0303915274. 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. Tel.: 44-29-2074-4256; Fax: 44-29-2074-3500; E-mail: blayney{at}cf.ac.uk.

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