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Vol. 54, Issue 6, 968-978, December 1998
Department 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
Division 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)
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.
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