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Vol. 63, Issue 3, 751-765, March 2003
Departments of Pharmacology (S.R.J.H., S.K.S., P.D.C., D.E.G.) and
Peptide Chemistry (N.L.), Neurocrine Biosciences Inc., San Diego,
California
Mechanisms of nonpeptide ligand action at family B G protein-coupled
receptors are largely unexplored. Here, we evaluated corticotropin-releasing factor 1 (CRF1) receptor regulation
by nonpeptide antagonists. The antagonist mechanism was investigated at
the G protein-coupled (RG) and uncoupled (R) states of the receptor in
membranes from Ltk
cells expressing the cloned human
CRF1 receptor. R was detected with the antagonist
125I-astressin with 30 µM guanosine
5'-O-(3-thiotriphosphate present, and RG detected
using 125I-sauvagine. At the R state, nonpeptide
antagonists antalarmin, NBI 27914, NBI 35965, and DMP-696 only
partially inhibited 125I-astressin binding (22-32%
maximal inhibition). NBI 35965 accelerated 125I-astressin
dissociation and only partially increased the IC50 value of
unlabeled sauvagine, CRF, and urocortin for displacing 125I-astressin binding (by 4.0-7.1-fold). Reciprocal
effects at the R state were demonstrated using [3H]NBI
35965: agonist peptides only partially inhibited binding (by 13-40%)
and accelerated [3H]NBI 35965 dissociation. These data
are quantitatively consistent with nonpeptide antagonist and peptide
ligand binding spatially distinct sites, with mutual, weak negative
cooperativity (allosteric inhibition) between their binding. At the RG
state the compounds near fully inhibited 125I-sauvagine
binding at low radioligand concentrations (79-94 pM). NBI 35965 did
not completely inhibit 125I-sauvagine binding at high
radioligand concentrations (82 ± 1%, 1.3-2.1 nM) and slowed
dissociation of 125I-sauvagine and 125I-CRF.
The antagonist effect at RG is consistent with either strong allosteric
inhibition or competitive inhibition at one of the peptide agonist
binding sites. These findings demonstrate a novel effect of R-G
interaction on the inhibitory activity of nonpeptide antagonists:
Although the compounds are weak inhibitors of peptide binding to the R
state, they strongly inhibit peptide agonist binding to RG. Strong
inhibition at RG explains the antagonist properties of the compounds.
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