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Originally published In Press as doi:10.1074/jbc.M009395200 on December 6, 2000
J. Biol. Chem., Vol. 276, Issue 11, 7741-7753, March 16, 2001
Evaluating the Signal Transduction Mechanism of the
Parathyroid Hormone 1 Receptor
EFFECT OF RECEPTOR-G-PROTEIN INTERACTION ON THE LIGAND BINDING
MECHANISM AND RECEPTOR CONFORMATION*
Sam R. J.
Hoare ,
Thomas J.
Gardella§, and
Ted B.
Usdin ¶
From the Unit on Cell Biology, Laboratory of
Genetics, National Institute of Mental Health,
Bethesda, Maryland 20892-4092 and the § Endocrine
Unit, Massachusetts General Hospital and Harvard Medical School,
Boston, Massachusetts 02114
Ligand binding to the PTH1
receptor is described by a "two-site" model, in which the
C-terminal portion of the ligand interacts with the N-terminal domain
of the receptor (N interaction), and the N-terminal region of the
ligand binds the juxtamembrane domain of the receptor (J interaction).
Previous studies have not considered the dynamic nature of receptor
conformation in ligand binding and receptor activation. In this study
the ligand binding mechanism was compared for the G-protein-coupled
(RG) and uncoupled (R) PTH1 receptor conformations. The two-site
model was confirmed by demonstration of spatially distinct binding
sites for PTH(3-34) and PTH(1-14): PTH(1-14), which binds
predominantly to the J domain, only partially inhibited binding of
125I-PTH(3-34); and PTH(3-34), shown to bind
predominantly to the N domain, only partially inhibited
PTH(1-14)-stimulated cAMP accumulation. To assess the effect of R-G
coupling, ligand binding to R was measured by displacement of
125I-PTH(3-34) with 30 µM guanosine
5'-3-O-(thio)triphosphate (GTP S) present, and binding to
RG was measured by displacement of 125I-[MAP]PTHrP(1-36)
(where MAP is model amphipathic peptide), a new radioligand that binds
selectively to RG. Agonists bound with higher affinity to RG than R,
whereas antagonists bound similarly to these states. The J interaction
was responsible for enhanced agonist binding to RG: residues 1 and 2 were required for increased PTH(1-34) affinity for RG; residue 5 of
MAP-PTHrP(1-36) was a determinant of R/RG binding selectivity, and
PTH(1-14) bound selectively to RG. The N interaction was insensitive
to R-G coupling; PTH(3-34) binding was GTP S-insensitive. Finally,
several observations suggest the receptor conformation is more
"closed" at RG than R. At the R state, an open conformation
is suggested by the simultaneous binding of PTH(1-14) and PTH(3-34).
At RG PTH(1-14) better occluded binding of 125I-PTH(3-34)
and agonist ligands bound pseudo-irreversibly, suggesting a more closed
conformation of this receptor state. The results extend the two-site
model to take into account R and RG conformations and suggest a model
for differences of receptor conformation between these states.
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
¶
To whom correspondence should be addressed: Laboratory of
Genetics, National Institute of Mental Health, Rm. 3D06, Bldg 36, 36 Convent Dr., Bethesda, MD 20892-4092. Tel.: 301- 402-6976; Fax:
301-435-5465; E-mail: usdin@codon.nih.gov.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

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Copyright © 2001 by the American Society for Biochemistry and Molecular Biology.
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