Molecular basis for selectivity of high affinity peptide antagonists for the gastrin-releasing peptide receptor

Few gastrointestinal hormones/neurotransmitters have high affinity peptide receptor antagonists, and little is known about the molecular basis of thei r selectivity or affinity. The receptor mediating the action of the mammali an bombesin (Bn) peptide, gastrin-releasing peptide receptor (GRPR), is an exception, because numerous classes of peptide antagonists are described. T o 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-Leu psi (CHOH-CH2)-(CH2)(2)-CH3), were studied. Each had high affi nity for the GRPR and >3,000-fold selectivity for GRPR over the closely rel ated neuromedin B receptor (NMBR). To investigate the basis for this, we us ed a chimeric receptor approach to make both GRPR loss of affinity and NMBR gain of affinity chimeras and a site-directed mutagenesis approach. Chimer ic or mutated receptors were transiently expressed in Balb/c 3T3. Only subs titution of the fourth extracellular (EC) domain of the GRPR by the compara ble NMBR domain markedly decreased the affinity for both antagonists. Subst ituting the fourth EC domain of NMBR into the GRPR resulted in a 300-fold g ain in affinity for JMV594 and an 11-fold gain for JMV641. Each of the 11 a mino acid differences between the GRPR and NMBR in this domain were exchang ed. The substitutions of Thr(297) in GRPR by Pro from the comparable positi on 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(30 5) in GRPR by the three comparable NMBR amino acids caused a 500-fold decre ase in affinity for both antagonists. Replacing the comparable three amino acids in NMBR by those from GRPR caused a gain in affinity for each antagon ist. Receptor modeling showed that each of these three amino acids faced in ward and was within 5 Angstrom of the putative binding pocket. These result s demonstrate that differences in the fourth EC domain of the mammalian Bn receptors are responsible for the selectivity of these two peptide antagoni sts. They demonstrate that Thr(297), Phe(302), and Ser(305) of the fourth E C 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.