Discovery of high affinity bombesin receptor subtype 3 agonists.
Human bombesin receptor subtype 3 (BRS-3) was cloned based on its homology to the human gastrin-releasing peptide (GRP) receptor and neuromedin B (NMB) receptor. Some bombesin-like peptides were shown to activate BRS-3 expressed in Xenopus laevis oocytes, but only at relatively high concentrations, which suggests that BRS-3 is an orphan receptor. To study the pharmacology of BRS-3 in the context of a mammalian cell, we used BR2 cells, which are Balb/3T3 fibroblasts transfected with BRS-3 cDNA. A number of bombesin-like peptides found in mammals and amphibians stimulated calcium mobilization in BR2 cells but exhibited no effect on nontransfected parental Balb/3T3 cells. Of these peptides, NMB (EC50 approximately 1-10 microM) was the most active for stimulation of calcium mobilization. Testing of a series of NMB analogs truncated at the amino terminus and carboxyl terminus indicated that the minimal size of NMB required for retention of full activity was Ac-NMB(3-10). Systematically replacing each residue with alanine, or changing its chirality, demonstrated that the carboxyl-terminal residues His8, Phe9, and Met10 of NMB are important for optimal activity. We also tested whether a number of bombesin (BN) analogs that are potent pure or partial antagonists of the GRP receptor can activate BRS-3 in BR2 cells. One such analog, D-Phe6-BN(6-13) propyl amide, activated BRS-3-mediated calcium mobilization with an EC50 level of 84 nM. Through additional synthesis, we generated a significantly more potent analog, D-Phe6-Phe13-BN(6-13) propyl amide, which displayed an EC50 level of 5 nM for activation of BRS-3. Taken together, our data show that the core portions of bombesin-like peptides required for activation of BRS-3 are similar to those necessary for activation of the GRP and NMB receptors and thus provide pharmacological evidence that BRS-3 is in the BN receptor family. Furthermore, we have identified an agonist of BRS-3, namely D-Phe6-Phe13-BN(6-13) propyl amide, which is roughly 1000-fold more potent than BRS-3 agonists described previously.
Wu
J M
JM
Department of Protein Biochemistry and Biophysics, Berlex Biosciences, Richmond, California 94804-0099, USA.
Nitecki
D E
DE
Biancalana
S
S
Feldman
R I
RI
D016475:3T3 Cells / Q000187:drug effects / Q000648:ultrastructure; D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D019886:Gastrin-Releasing Peptide; D006801:Humans; D051379:Mice; D008807:Mice, Inbred BALB C; D008969:Molecular Sequence Data; D015287:Neurokinin B / Q000031:analogs & derivatives / Q000494:pharmacology; D010455:Peptides / Q000494:pharmacology; D018004:Receptors, Bombesin / Q000819:agonists* / Q000145:classification / Q000378:metabolism; D013329:Structure-Activity Relationship; D014162:Transfection; D014982:Xenopus laevis
D018076:DNA, Complementary; D010455:Peptides; D018004:Receptors, Bombesin; D019886:Gastrin-Releasing Peptide; D015287:Neurokinin B; C038393:neuromedin B; D002118:Calcium