The molecular basis for high affinity of a universal ligand for human bombesin receptor (BnR) family members
| Autor: | Nieves González, David H. Coy, Robert T. Jensen, Tomoo Nakagawa, Simon J. Hocart, Hirotsugu Uehara, Tatsuro Katsuno, Samuel A. Mantey |
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| Rok vydání: | 2012 |
| Předmět: |
Protein Conformation
Molecular Sequence Data Biology Ligands Biochemistry Article Cell Line Protein structure Cricetinae Animals Humans Amino Acid Sequence Binding site Tyrosine Peptide sequence G protein-coupled receptor Pharmacology Alanine chemistry.chemical_classification Binding Sites Cell Membrane Amino acid Bombesin receptor Receptors Bombesin chemistry Mutagenesis Site-Directed Peptides |
| Zdroj: | Biochemical Pharmacology. 84:936-948 |
| ISSN: | 0006-2952 |
| DOI: | 10.1016/j.bcp.2012.07.010 |
| Popis: | There is increased interest in the Bn-receptor family because they are frequently over/ectopically expressed by tumors and thus useful as targets for imaging or receptor-targeted-cytotoxicity. The synthetic Bn-analog, [D-Tyr(6), β-Ala(11), Phe(13), Nle(14)]Bn(6-14) [Univ.Lig] has the unique property of having high affinity for all three human BNRs (GRPR, NMBR, BRS-3), and thus could be especially useful for this approach. However, the molecular basis of this property is unclear and is the subject of this study. To accomplish this, site-directed mutagenesis was used after identifying potentially important amino acids using sequence homology analysis of all BnRs with high affinity for Univ.Lig compared to the Cholecystokinin-receptor (CCK(A)R), which has low affinity. Using various criteria 74 amino acids were identified and 101 mutations made in GRPR by changing each to those of CCK(A)R or to alanine. 22 GRPR mutations showed a significant decrease in affinity for Univ.Lig (>2-fold) with 2 in EC2[D97N, G112V], 1 in UTM6[Y284A], 2 in EC4[R287N, H300S] showing >10-fold decrease in Univ.Lig affinity. Additional mutations were made to explore the molecular basis for these changes. Our results show that high affinity for Univ.Lig by human Bn-receptors requires positively charged amino acids in extracellular (EC)-domain 4 and to a lesser extent EC2 and EC3 suggesting charge-charge interactions may be particularly important for determining the general high affinity of this ligand. Furthermore, transmembrane amino acids particularly in UTM6 are important contributing both charge-charge interactions as well as interaction with a tyrosine residue in close proximity suggesting possible receptor-peptide cation-π or H-bonding interactions are also important for determining its high affinity. |
| Databáze: | OpenAIRE |
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