Preliminary mutational analysis of the human kinin B2receptor for nonpeptide antagonist ligands recognition
| Autor: | Alessandro Giolitti, Laura Quartara, Stefania Meini, Carlo Alberto Maggi, Paola Cucchi, Sabrina Zappitelli, Luigi Rotondaro |
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| Rok vydání: | 2002 |
| Předmět: |
Models
Molecular Receptor Bradykinin B2 Physiology G protein Stereochemistry DNA Mutational Analysis CHO Cells Pharmacology Bradykinin Ligands Binding Competitive Cricetinae Physiology (medical) Animals Humans Point Mutation Bradykinin Receptor Antagonists Chemistry Ligand Receptors Bradykinin Mutagenesis Antagonist General Medicine Kinin In vitro Cell culture Quinolines B2 Bradykinin Receptor Protein Binding |
| Zdroj: | Canadian Journal of Physiology and Pharmacology. 80:303-309 |
| ISSN: | 1205-7541 0008-4212 |
| DOI: | 10.1139/y02-027 |
| Popis: | FR173657, LF16,0335, and LF16,0687 are nonpeptide antagonists, endowed with high affinity and selectivity for the human kinin B2receptor. The kinin B2receptor belongs to the family of G-protein-coupled receptors with seven transmembrane (TM) helices. In the present study, we aimed, through computer-assisted modeling and mutagenesis, to identify residues in the human B2receptor (hB2R) amino acid sequence that are involved in nonpeptide antagonist binding in order to build up experimental data as a first step towards a molecular model of nonpeptide ligands binding site. Fourteen amino acid residues within the TM segments were mutated to alanine. The wild type and mutant receptors were stably expressed in Chinese hamster ovary (dhfr) cells and tested for their ability to bind agonist ([3H]bradykinin) and peptide antagonist ([3H]MEN11270) radioligands. The affinity of nonpeptide ligands was determined by heterologous competition experiments using the above radioligands. We found that some mutations in TM2 (W86A) and TM7 (Y295A, N297A) impair the binding affinity of the three nonpeptide antagonists. On the other hand, some mutated residues in TM3 (S117A) and TM6 (W256A) reduce the affinity of LF16,0335 and LF16,0687 only. Results are discussed in order to build up a hypothesis for the likely different interactions of various nonpeptide ligands with the B2receptor.Key words: binding, bradykinin B2receptor, G-protein-coupled receptor, mutagenesis, nonpeptide. |
| Databáze: | OpenAIRE |
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