An Anionic Residue at Position 564 Is Important for Maintaining the Inactive Conformation of the Human Lutropin/Choriogonadotropin Receptor
- 1Department of Laboratory Medicine, Kyoto University School of Medicine, Kyoto 606-8507, Japan (S.K., T.M.), and 2Division of Endocrinology, Department of Pediatrics, Northwestern University Medical School and Children’s Memorial Hospital, Chicago, Illinois 60614 (A.S.)
Abstract
Gonadotropin-independent, male-limited precocious puberty is caused by a variety of mutations in the lutropin/choriogonadotropin receptor (LHR) that produce constitutive receptor activation. Two of these mutations encode replacement of conserved aspartate residues at positions 564 and 578 with glycine. We previously used site-directed mutagenesis to study the functional role of the Asp578 side chain in transmembrane helix 6, and concluded that it is its ability to serve as a properly positioned interhelical hydrogen bond acceptor, rather than its negative charge, that is important for stabilizing the inactive state of the LHR. We now report the effects of substituting seven different amino acids for the Asp564 residue located at the carboxyl terminus of the third intracellular loop. Glycine, alanine, valine, leucine, phenylalanine, and asparagine produced constitutive activation in a COS-7 cell expression system (3–5-fold increase in basal cAMP), but glutamate did not, indicating that a negative charge at position 564 may be important for maintaining the inactive LHR conformation. Characterization of double-mutant receptors showed that certain substitutions at Asp564 and Asp578 have a cumulative effect on basal receptor activity, perhaps because they mimic different aspects of the activation process normally triggered by hormone binding.
Footnotes
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Send reprint requests to: Dr. S. Kosugi, Department of Laboratory Medicine, Kyoto University School of Medicine, Room 223, First Clinical Research Building, Kyoto University Hospital, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. E-mail:kosugi{at}kuhp.kyoto-u.ac.jp or Dr. A. Shenker, Children’s Memorial Hospital, Box 225, 2300 Children’s Plaza, Chicago, IL 60614. E-mail: ashenker{at}nwu.edu
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This work was in part supported by grants-in-aid from the Japanese Ministry of Education (Nos. 0644128, 06671024, 07671129, 07557353, and 08671152), Mochida Foundation for Medical and Pharmaceutical Research, Kowa Foundation for Life Science, Shimizu Foundation for Immunology Research, Kyoto University Foundation, Kurozumi Foundation, Inamori Foundation, Fujiwara Foundation, Clinical Pathology Research Foundation of Japan, and SRF for Biomedical Research (all to S.K.)
- Abbreviations:
- LHR
- lutropin/choriogonadotropin receptor
- GPCR
- G protein-coupled receptor
- TM
- transmembrane helix
- i3
- third intracellular loop
- WT
- wild-type
- hCG
- human chorionic gonadotropin
- AR
- adrenergic receptor
- IP
- inositol phosphate
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
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- Received August 28, 1997.
- Accepted February 2, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
