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; Deborah L Segaloff

doi:10.1074/jbc.M502102200

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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

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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

The Journal of biological chemistry, Vol.280(28), pp.26169-26176

07/15/2005

DOI: 10.1074/jbc.M502102200

PMCID: PMC1237128

PMID: 15908694

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Abstract

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.

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Title: Subtitle: 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
Creators: Meilin Zhang - From the Department of Physiology and Biophysics, The University of Iowa, Iowa City, Iowa 52242 and
Dario Mizrachi - From the Department of Physiology and Biophysics, The University of Iowa, Iowa City, Iowa 52242 and
Francesca Fanelli - the Dulbecco Telethon Institute and Department of Chemistry, University of Modena e Reggio Emilia, Via Campi 183, 41100 Modena, Italy
Deborah L Segaloff - From the Department of Physiology and Biophysics, The University of Iowa, Iowa City, Iowa 52242 and
Resource Type: Journal article
Publication Details: The Journal of biological chemistry, Vol.280(28), pp.26169-26176
DOI: 10.1074/jbc.M502102200
PMID: 15908694
PMCID: PMC1237128
NLM abbreviation: J Biol Chem
ISSN: 0021-9258
eISSN: 1083-351X
Language: English
Date published: 07/15/2005
Academic Unit: Molecular Physiology and Biophysics; Obstetrics and Gynecology
Record Identifier: 9984083257602771

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