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J. Biol. Chem., Vol. 283, Issue 26, 18048-18055, June 27, 2008

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Extended Hormone Binding Site of the Human Thyroid Stimulating Hormone Receptor

DISTINCTIVE ACIDIC RESIDUES IN THE HINGE REGION ARE INVOLVED IN BOVINE THYROID STIMULATING HORMONE BINDING AND RECEPTOR ACTIVATION^*

Sandra Mueller, Gunnar Kleinau, Holger Jaeschke¹, Ralf Paschke, and Gerd Krause¹

From the III. Medical Department, University of Leipzig, Ph.-Rosenthal-Strasse 27, D-04103 Leipzig, Germany and Leibniz-Institut für Molekulare Pharmakologie, Robert-Rössle-Strasse 10, D-13125 Berlin, Germany

The human thyroid stimulating hormone receptor (hTSHR) belongs to the glycoprotein hormone receptors that bind the hormones at their large extracellular domain. The extracellular hinge region of the TSHR connects the N-terminal leucine-rich repeat domain with the membrane-spanning serpentine domain. From previous studies we reasoned that apart from hormone binding at the leucine-rich repeat domain, additional multiple hormone contacts might exist at the hinge region of the TSHR by complementary charge-charge recognition. Here we investigated highly conserved charged residues in the hinge region of the TSHR by site-directed mutagenesis to identify amino acids interacting with bovine TSH (bTSH). Indeed, the residues Glu-297, Glu-303, and Asp-382 in the TSHR hinge region are essential for bTSH binding and partially for signal transduction. Side chain substitutions showed that the negative charge of Glu-297 and Asp-382 is necessary for recognition of bTSH by the hTSHR. Multiple combinations of alanine mutants of the identified positions revealed an increased negative effect on hormone binding. An assembled model suggests that the deciphered acidic residues form negatively charged patches at the hinge region resulting in an extended binding mode for bTSH on the hTSHR. Our data indicate that certain positively charged residues of bTSH might be involved in interaction with the identified negatively charged amino acids of the hTSHR hinge region. We demonstrate that the hinge region represents an extracellular intermediate connector for both hormone binding and signal transduction of the hTSHR.

Received for publication, January 17, 2008 , and in revised form, April 22, 2008.

^* This work was supported by Deutsche Forschungsgemeinschaft Grants KR 1223/1-2 and PA 423 12-2. 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 supplemental table.

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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