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J. Biol. Chem., Vol. 276, Issue 39, 36652-36663, September 28, 2001
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From the Few gastrointestinal
hormones/neurotransmitters have high affinity peptide receptor
antagonists, and little is known about the molecular basis of their
selectivity or affinity. The receptor mediating the action of the
mammalian bombesin (Bn) peptide, gastrin-releasing peptide receptor
(GRPR), is an exception, because numerous classes of peptide
antagonists are described. To investigate the molecular basis for their
high affinity for the GRPR, two classes of peptide antagonists, a
statine analogue, JMV594
([D-Phe6,Stat13]Bn(6-14)),
and a pseudopeptide analogue, JMV641
(D-Phe-Gln-Trp-Ala-Val-Gly-His-Leu
Molecular Basis for Selectivity of High Affinity Peptide
Antagonists for the Gastrin-releasing Peptide Receptor*
,,
Digestive Diseases Branch, NIDDK, National
Institutes of Health, Bethesda, Maryland 20892-1804, the
§ Department of Medicine, Peptide Research Laboratories,
Tulane University Health Sciences Center, New Orleans, Louisiana 70112, and ¶ Faculté de Pharmacie, Universités de
Montpellier, Montpellier 34060, France
(CHOH-CH2)-(CH2)2-CH3), were studied. Each had high affinity for the GRPR and >3,000-fold selectivity for GRPR over the closely related neuromedin B receptor (NMBR). To investigate the basis for this, we used a chimeric receptor
approach to make both GRPR loss of affinity and NMBR gain of affinity
chimeras and a site-directed mutagenesis approach. Chimeric or mutated
receptors were transiently expressed in Balb/c 3T3. Only
substitution of the fourth extracellular (EC) domain of the GRPR by the
comparable NMBR domain markedly decreased the affinity for both
antagonists. Substituting the fourth EC domain of NMBR into the GRPR
resulted in a 300-fold gain in affinity for JMV594 and an 11-fold gain
for JMV641. Each of the 11 amino acid differences between the GRPR and
NMBR in this domain were exchanged. The substitutions of
Thr297 in GRPR by Pro from the comparable position in NMBR,
Phe302 by Met, and Ser305 by Thr decreased the
affinity of each antagonist. Simultaneous replacement of
Thr297, Phe302, and Ser305 in GRPR
by the three comparable NMBR amino acids caused a 500-fold decrease in
affinity for both antagonists. Replacing the comparable three amino
acids in NMBR by those from GRPR caused a gain in affinity for each
antagonist. Receptor modeling showed that each of these three amino
acids faced inward and was within 5 Å of the putative binding pocket.
These results demonstrate that differences in the fourth EC domain of
the mammalian Bn receptors are responsible for the selectivity of these
two peptide antagonists. They demonstrate that Thr297,
Phe302, and Ser305 of the fourth EC domain of
GRPR are the critical residues for determining GRPR selectivity and
suggest that both receptor-ligand cation-
interactions and hydrogen
bonding are important for their high affinity interaction.
*
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 and reprint requests should be
addressed: Dr. Robert T. Jensen, NIH/NIDDK/DDB, Bldg. 10, Rm. 9C-103, 10 Center Dr., MSC 1804, Bethesda MD 20892-1804. Tel.: 301-496-4201; Fax: 301-402-0600; E-mail: robertj@bdg10.niddk.nih.gov.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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