A Hydrophobic Cluster between Transmembrane Helices 5 and 6 Constrains the Thyrotropin-Releasing Hormone Receptor in an Inactive Conformation
- Anny-Odile Colson1,
- Jeffrey H. Perlman2,
- Arti Jinsi-Parimoo2,
- Daniel R. Nussenzveig2,
- Roman Osman1 and
- Marvin C. Gershengorn2
- 1Department of Physiology and Biophysics, Mount Sinai School of Medicine of the City University of New York, New York, New York 10029 (A.-O.C., R.O.), and 2Division of Molecular Medicine, Department of Medicine, Cornell University Medical College and The New York Hospital, New York, New York 10021 (J.H.P., A.J.-P., D.R.N., M.C.G)
Abstract
We have studied the role of a highly conserved tryptophan and other aromatic residues of the thyrotropin-releasing hormone (TRH) receptor (TRH-R) that are predicted by computer modeling to form a hydrophobic cluster between transmembrane helix (TM)5 and TM6. The affinity of a mutant TRH-R, in which Trp279 was substituted by alanine (W279A TRH-R), for most tested agonists was higher than that of wild-type (WT) TRH-R, whereas its affinity for inverse agonists was diminished, suggesting that W279A TRH-R is constitutively active. We found that W279A TRH-R exhibited 3.9-fold more signaling activity than WT TRH-R in the absence of agonist. This increased basal activity was inhibited by the inverse agonist midazolam, confirming that the mutant receptor is constitutively active. Computer-simulated models of the unoccupied WT TRH-R, the TRH-occupied WT TRH-R, and various TRH-R mutants predict that a hydrophobic cluster of residues, including Trp279 (TM6), Tyr282, and Phe199 (TM5), constrains the receptor in an inactive conformation. In support of this model, we found that substitution of Phe199 by alanine or of Tyr282 by alanine or phenylalanine, but not of Tyr200 (by alanine or phenylalanine), resulted in a constitutively active receptor. We propose that a hydrophobic cluster including residues in TM5 and TM6 constrains the TRH-R in an inactive conformation via interhelical interactions. Disruption of these constraints by TRH binding or by mutation leads to changes in the relative positions of TM5 and TM6 and to the formation of an active form of TRH-R.
Footnotes
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Send reprint requests to: Dr. Marvin C. Gershengorn, Cornell University Medical College, 1300 York Avenue, Room A328, New York, NY 10021. E-mail:mcgersh{at}mail.med.cornell.edu
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This work was supported by National Research Service Award DK09647 (A.-O.C.), National Institutes of Health Physician Scientist Award DK02101 (J.H.P.), and National Institutes of Health Grant DK43036 (M.C.G., R.O.). A.-O.C. and J.H.P. contributed equally to this work.
- Abbreviations:
- WT
- wild-type
- GPCR
- G protein-coupled receptor
- TRH
- thyrotropin-releasing hormone
- TRH-R
- thyrotropin-releasing hormone receptor
- TM
- transmembrane helix
- IP
- inositol phosphate
- MeTRH
- [N-t-methylhistidine]thyrotropin-releasing hormone
- 1-desaza-TRH
- [desazapyro-Glu]thyrotropin-releasing hormone [Na-[(1R)-3-oxocyclopentanecarbonyl]-l-histidyl-l-prolineamide]
- bp
- base pair(s)
- PBS
- phosphate-buffered saline
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- Received May 8, 1998.
- Accepted August 26, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
