Abstract
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-Phe(6),Stat(13)]Bn(6-14)), and a pseudopeptide analogue, JMV641 (d-Phe-Gln-Trp-Ala-Val-Gly-His-Leupsi(CHOH-CH(2))-(CH(2))(2)-CH(3)), 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 Thr(297) in GRPR by Pro from the comparable position in NMBR, Phe(302) by Met, and Ser(305) by Thr decreased the affinity of each antagonist. Simultaneous replacement of Thr(297), Phe(302), and Ser(305) 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 A 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 Thr(297), Phe(302), and Ser(305) of the fourth EC domain of GRPR are the critical residues for determining GRPR selectivity and suggest that both receptor-ligand cation-pi interactions and hydrogen bonding are important for their high affinity interaction.
Highlights
The gastrin-releasing peptide (GRP)1 receptor, which mediates the diverse actions of the mammalian bombesin (Bn)related peptide [1, 2], GRP, has numerous high affinity peptide antagonists [3,4,5]
We have examined the molecular basis for the GRPR selectivity of two different classes of closely related high affinity Bn receptor peptide antagonists: a Bn statine analogue, JMV594 [36], and a Bn pseudopeptide analogue, JMV641 [37]
In this study we show that the selectivity of peptide antagonists JMV594 and JMV641 for the GRPR over the NMB receptor (NMBR) depends primarily on interactions with amino acids in the fourth extracellular region of the GRPR
Summary
Vol 276, No 39, Issue of September 28, pp. 36652–36663, 2001 Printed in U.S.A. Molecular Basis for Selectivity of High Affinity Peptide Antagonists for the Gastrin-releasing Peptide Receptor*. 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. We have examined the molecular basis for the GRPR selectivity of two different classes of closely related high affinity Bn receptor peptide antagonists: a Bn statine analogue, JMV594 [36], and a Bn pseudopeptide analogue, JMV641 [37]. Site-directed mutagenesis studies demonstrate that Thr297, Phe302, and Ser305 in this region of GRPR are the critical amino acids for high affinity binding and selectivity of these two antagonists. Computer modeling of this region of the GRPR demonstrates that these amino acids all face inward forward the proposed binding pocket and are all within 5 Å of it, suggesting that cation- and hydrogen bonding interactions between these antagonists and the above three amino acids are essential for receptor subtype selectivity and high affinity interaction
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