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 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.
Tokita
K
K
Digestive Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1804, USA.
Katsuno
T
T
Hocart
S J
SJ
Coy
D H
DH
Llinares
M
M
Martinez
J
J
Jensen
R T
RT
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D000818:Animals; D001665:Binding Sites; D018076:DNA, Complementary / Q000378:metabolism; D020128:Inhibitory Concentration 50; D007700:Kinetics; D008715:Methionine / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry*; D010649:Phenylalanine / Q000737:chemistry; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D051381:Rats; D018004:Receptors, Bombesin / Q000037:antagonists & inhibitors* / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D014162:Transfection
D000596:Amino Acids; D018076:DNA, Complementary; D010455:Peptides; D018004:Receptors, Bombesin; D012694:Serine; D010649:Phenylalanine; D008715:Methionine