Effects of mutations in the highly conserved DRY motif on binding affinity, expression, and G-protein recruitment of the human angiotensin II type-2 receptor
| Autor: | Tom N. Grammatopoulos, James A. Weyhenmeyer, Susan A. Moore, Bridget C Lavin, Anjali S Patel, Nancy Huang, Robert D. Andres |
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| Rok vydání: | 2002 |
| Předmět: |
Agonist
Angiotensin receptor medicine.drug_class Amino Acid Motifs Mutant CHO Cells Biology Receptor Angiotensin Type 2 Second Messenger Systems Radioligand Assay Cellular and Molecular Neuroscience GTP-Binding Proteins Cricetinae medicine Animals Humans Receptor Molecular Biology Receptors Angiotensin Angiotensin II Wild type Ligand (biochemistry) Molecular biology Biochemistry Guanosine 5'-O-(3-Thiotriphosphate) Mutagenesis Site-Directed Signal transduction Protein Binding |
| Zdroj: | Molecular Brain Research. 109:161-167 |
| ISSN: | 0169-328X |
| DOI: | 10.1016/s0169-328x(02)00552-1 |
| Popis: | The signaling pathways for the seven transmembrane G-protein coupled angiotensin II receptors (AT 1 and AT 2 ) are just beginning to be understood. While these receptors play an important role in the development and differentiation of many tissues, including the cardiovascular and central nervous systems, information about amino acid motifs involved in angiotensin II-mediated signaling is only available for the AT 1 receptor subtype. In the present study, we mutated the conserved DRY 141–143 motif in the AT 2 receptor, which is thought to be involved in G-protein recruitment. Expression of wild type and mutant receptors in CHO-K1 cell plasma membranes was confirmed using radioligand binding analyses. Our findings indicate a significant change in the binding affinities (kD) and capacities ( B max ) of the mutant receptors relative to wild type. Alanine substitutions of D 141 and DRY 141–143 resulted in a significant decrease of binding affinity for both Sar 1 Ile 8 –angiotensin II (SarIle–Ang II) (mixed agonist/antagonist) and angiotensin II (agonist). The binding affinities following alanine substitutions of R 142 and Y 143 were not significantly different from wild type receptor. Interestingly, the R 142 –A and Y 143 –A mutants revealed a significant decrease in binding levels from wild type with SarIle–Ang II, but not angiotensin II. The effect of GTPγS on angiotensin II binding affinity between wild type and mutant receptors was similarly significant. The D 141 –A, Y 143 –A, and DRY 141–143 –AAA mutant receptors showed a marked decrease in GTPγS-induced angiotensin II affinity shift. The R 142 –A GTPγS binding affinity shift was not different from the wild type receptor. Our results support the hypothesis that the DRY motif plays a significant role in the binding affinity, structural stability and G-protein recruiting of the AT 2 receptor. |
| Databáze: | OpenAIRE |
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