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J. Biol. Chem., Vol. 280, Issue 28, 26169-26176, July 15, 2005

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The Formation of a Salt Bridge Between Helices 3 and 6 Is Responsible for the Constitutive Activity and Lack of Hormone Responsiveness of the Naturally Occurring L457R Mutation of the Human Lutropin Receptor^*

Meilin Zhang, Dario Mizrachi, Francesca Fanelli¶||, and Deborah L. Segaloff**

From the Department of Physiology and Biophysics, The University of Iowa, Iowa City, Iowa 52242 and the ^¶Dulbecco Telethon Institute and Department of Chemistry, University of Modena e Reggio Emilia, Via Campi 183, 41100 Modena, Italy

The human lutropin receptor (hLHR) plays a pivotal role in reproductive endocrinology. A number of naturally occurring mutations of the hLHR have been identified that cause the receptor to become constitutively active. To gain further insights into the structural basis for the activation of the hLHR by activating mutations, we chose to examine a particularly strong constitutively activating mutation of this receptor, L457R, in which a leucine that is highly conserved among rhodopsin-like G protein-coupled receptors in helix 3 has been substituted with arginine. Using both disruptive as well as reciprocal mutagenesis strategies, our studies demonstrate that the ability of L457R to stabilize an active form of the hLHR is because of the formation of a salt bridge between the replacing amino acid and Asp-578 in helix 6. Such a lock between the transmembrane portions of helices 3 and 6 is concurrent with weakening the connections between the cytosolic ends of the same helices, including the interaction found in the wild-type receptor between Arg-464, of the (E/D)R(Y/W) motif, and Asp-564. This structural effect is properly marked by the increase in the solvent accessibility of selected amino acids at the cytosolic interfaces between helices 3 and 6. The integrity of the conserved amino acids Asn-615 and Asn-619 in helix 7 is required for the transfer of the structural change from the activating mutation site to the cytosolic interface between helices 3 and 6. The results of in vitro and computational experiments further suggest that the structural trigger of the constitutive activity of the L457R mutant may also be responsible for its lack of hormone responsiveness.

Received for publication, February 24, 2005 , and in revised form, April 27, 2005.

^* This work was supported in part by National Institutes of Health Grant HD22196 (to D. L. S.), Telethon-Italy Grant TCP00068and Compagnia S. Paolo Grant TCP00068CSP01 (to F. F.), and University of Iowa Diabetes and Endocrinology Research Center Grant DK25295. 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.

Supported by National Institutes of Health Training Grant DK07759.

^|| Assistant Telethon Scientist.

^** To whom correspondence should be addressed: Dept. of Physiology and Biophysics, 5–470 Bowen Science Bldg., The University of Iowa, IA City, IA 52242. Tel.: 319-335-7850; Fax: 319-335-7330; E-mail: deborah-segaloff{at}uiowa.edu.

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