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Identification of four amino acids in the gastrin-releasing peptide receptor that are required for high affinity agonist binding.

Authors :
Akeson, M
Sainz, E
Mantey, S A
Jensen, R T
Battey, J F
Source :
Journal of Biological Chemistry; July 1997, Vol. 272 Issue: 28 p17405-9, 5p
Publication Year :
1997

Abstract

The bombesin family of G-protein-coupled receptors includes the gastrin-releasing peptide receptor (GRP-R), the neuromedin B receptor (NMB-R), bombesin receptor subtype 3 (BRS-3), and bombesin receptor subtype 4 (bb4). All species homologues of GRP-R, NMB-R, and bb4 bind bombesin with dissociation constants in the nanomolar range; by comparison, human BRS-3 binds bombesin at much lower affinity (Kd >> 1 microM). We used this difference to help identify candidate residues that were potentially critical for forming the bombesin binding pocket. We reasoned that amino acids essential for bombesin binding would be conserved among all homologues of bb4, NMB-R, and GRP-R; conversely, at least one of these amino acids would not be conserved among homologues of BRS-3. Amino acid sequence alignment revealed nine residues that fit this model. We replaced each of these amino acids in mouse GRP-R with the homologous amino acid in human BRS-3. Four substitutions resulted in a significant decrease in bombesin affinity (R288H, Q121R, P199S, and A308S). The analogous mutations in BRS-3 (R127Q, H294R, S205P, and S315A) together resulted in a receptor with a 100-fold increase in bombesin and GRP affinities relative to wild-type BRS-3. From this, we propose a preliminary map of some of the amino acids comprising the agonist binding pocket.

Details

Language :
English
ISSN :
00219258 and 1083351X
Volume :
272
Issue :
28
Database :
Supplemental Index
Journal :
Journal of Biological Chemistry
Publication Type :
Periodical
Accession number :
ejs7208122