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J. Biol. Chem., Vol. 282, Issue 1, 518-525, January 5, 2007

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Contacts between Extracellular Loop Two and Transmembrane Helix Six Determine Basal Activity of the Thyroid-stimulating Hormone Receptor^*

Gunnar Kleinau, Maren Claus, Holger Jaeschke, Sandra Mueller, Susanne Neumann^¶, Ralf Paschke, and Gerd Krause¹

From the Leibniz-Institut für molekulare Pharmakologie (FMP), Robert-Rössle-Str. 10, D-13125 Berlin, Germany, the III. Medical Department, University of Leipzig, Ph.-Rosenthal-Str. 27, D-04103 Leipzig, Germany, and the ^¶NIDDK, National Institutes of Health, Bethesda, Maryland, 20814

A number of alanine mutations in extracellular loop two (ECL2) of the thyroid-stimulating hormone receptor (TSHR) were found to increase or decrease basal activity when compared with the wild type receptor. K565A was identified as a mutant with decreased basal activity, and strongly impaired hormone induced signaling activity. To gain insights into how ECL2 mutants affect basal activity, we focused on constitutively activating pathogenic mutant I568V in ECL2, which exhibits elevated basal activity. Because our molecular model suggests that Ile-568 is embedded in an environment of hydrophobic residues provided by transmembrane helix bundle, we tested mutants in this region to identify potential interaction partner(s) for Ile-568. Indeed, the double mutant I568V/I640L (ECL2/TMH6) suppresses the increased basal activity exhibited by I568V alone. We suggest a spatial and functional relationship between ECL2 and TMH6 in which side chain interaction between Ile-568 and Ile-640 constrains the receptor in a conformation with low basal activity. Although the single mutant I640L exhibits basal activity lower than wild type, its differently branched and bulkier side chain complements the reduced side chain bulk in I568V, restoring wild type basal activity to the double mutant. This scenario is confirmed by the reciprocal double mutant I640V/I568L. The combination of basally increased activity of I640V and basally decreased activity of mutant I568L also restores basal activity of wild type TSHR. These and other mutant phenotypes reported here support a dynamic interface between TMH6 and ECL2. Disruption of this critical interface for signaling by introduction of mutations in TSHR can either increase or decrease basal activity.

Received for publication, June 28, 2006 , and in revised form, October 30, 2006.

^* This work was supported by grants from the Deutsche Forschungsgemein-schaft (Grants KR 1273/1-1 and PA 423 /12-1) and the Intramural Research Program of the NIDDK, National Institutes of Health. 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 a table and three figures.

¹ To whom correspondence should be addressed. Tel.: 49-30-94793228; Fax: 49-30-94793230; E-mail: gkrause{at}fmp-berlin.de.

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