Abstract
The mammalian bombesin (Bn) peptides, neuromedin B (NMB) and gastrin-releasing peptide (GRP), have widespread actions in many tissues, and their effects are mediated by two closely related G-protein-coupled receptors, the NMBR and GRPR. Little is known about the structural determinants of NMBR selectivity for NMB, in contrast to GRP selectivity for the GRPR, which has been extensively studied. To provide insight, chimeric NMBR-GRPR loss-of-affinity and gain-of-affinity mutants were made, as well as NH2-terminally truncated NMBR and point mutants using site-directed mutagenesis. Receptors were expressed in Balb-3T3-cells or CHOP cells, and affinities were determined. NMB had 115-fold greater affinity for NMBR than GRPR. Receptor-chimeric studies showed that NMBR selectivity for NMB was primarily determined by differences in the third extracellular (EC3) regions of GRPR-NMBR and adjacent upper-transmembrane-5 (TM5) region. In this region, 24 NMB gain-of-affinity GRPR mutants or NMBR loss-of-affinity point/combination mutants were made. Three gain-of-affinity mutant GRPRs [[A198I] (EC3), [H202Q] (EC3), [S215I] (upper TM5)] had increased NMB affinity (2.4–21-fold), and these results were confirmed with NMBR loss-of-affinity mutants [I199A,Q203H,I215S-NMBR]. The combination mutant [A198I,S215]GRPR had the greatest effect causing a complete NMB gain-of-affinity. The importance of differences at position 199NMBR or 203NMBR was studied by substituting amino acids with various properties. Our results show that NMBR selectivity for NMB is due to differences in the EC3 of NMBR-GRPR and the adjacent upper-TM5 region. Within these regions, isoleucines in NMBR [position 199 (EC3)] (instead of A198GRPR) and in 215NMBR (TM5) (instead of S214GRPR), as well as Q203NMBR (instead of H202GRPR) are responsible for high NMB-affinity/selectivity of NMBR. The effect at position 199 is primarily due to differences in hydrophobicity of the substitution, whereas steric factors and charge of the substitution at position 203 were important determinants of NMB selectivity.
- Bn, bombesin
- BRS-3, bombesin receptor subtype-3
- BSA, bovine serum albumin fraction V
- DMEM, Dulbecco's minimum essential medium
- EC, extracellular
- TM, transmembrane
- FBS, fetal bovine serum
- GRP, gastrin-releasing peptide
- GRPR, gastrin-releasing peptide receptor
- NMB, neuromedin B
- NMBR, neuromedin B receptor
- CRF, corticotropin-releasing factor
- CNS, central nervous system
- WT, wild type
- NKA, neurokinin A
- CCK, cholecystokinin
- CHOP, polyoma large T-antigen Chinese hamster ovary fibroblasts
- M10, cyclo(6β→10ϵ)(succinyl6)-d-(2′)Nal7-Arg8-Trp9-Lys10)-NH2
- PL017, H-Tyr-Pro-(N-Me))Phe-d-Pro-NH2
- PD168368, 3-(1H-indol-3-yl)-2-methyl-2-[3(4-nitrophenyl)-ureido]-N-(1-pyridin-2-yl-cyclohexylmethyl)-propionamide
- MDL103,392, (R,S)-1-[3-[3,4-dichlorophenyl]-1-(3,4,5-trimethoxybenzoyl)-pyrrolidin-3-yl]ethyl-4-phenylpiperidine-4-carboxamide
- CP96345, (2S,3S)-cis-2-(diphenylmethyl)-N-((2-methoxyphenyl)-methyl)-1-azabicyclo(2.2.2)-octan-3-amine
- JMV641, (d-Phe-Gln-Trp-Ala-Val-Gly-His-Leu psi(CHOH-CH2))-(CH2)(2)-CH3
- JMV594, [d-Phe6Stat13]Bn(6-14)
- NBI35965, (S)-6-cyclopropylmethyl-2-(2,4-dichlorophenyl)-7-ethyl-4-methyl-7,8-dihydro-6H-1,3,6a-tetraazaacenaphthylene mesylate.
Footnotes
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This research was supported by the Intramural Research Program of the National Institutes of Health National Institute of Diabetes and Digestive and Kidney Diseases.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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ABBREVIATIONS:
- Received March 27, 2009.
- Accepted July 22, 2009.
- © 2009 by The American Society for Pharmacology and Experimental Therapeutics
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