Using Laser tweezers-based technologies to assess single-molecular pair interactions between integrin alpha(IIb)beta(3) and the disintegrin rhodostomin in living cell
| Autor: | Chia-Fen Hsieh, 謝嘉芬 |
|---|---|
| Rok vydání: | 2003 |
| Druh dokumentu: | 學位論文 ; thesis |
| Popis: | 97 Recent evidence demonstrated that the integrin conformational changes during receptor activation modulate its binding to extracellular matrix (ECM); however, experimental assessment of ligand-receptor binding following the initial molecular interaction has rarely been carried out at single-molecule resolution. In this study, laser tweezers were used to assess the dynamic molecular binding behaviors by a live Chinese hamster ovary cell that expressed integrin alpha(IIb)beta(3) (CHO-alpha(IIb)beta(3) cell), to the bead carrier coated with the snake venom rhodostomin that served as an integrin alpha(IIb)beta(3)-activating ligand. The investigation of dynamics between ligand and receptor were divided into two parts: In the first part, we investigated the role of integrin 脉alpha(IIb)beta(3) highly conserved cytoplasmic region when integrin initially binds to the ligand. There were two conserved regions of integrin beta(3)-subunit cytoplasmic domain. Both of them were amino acid sequence of asparagine-X-X-tyrosine (NXXY). One was located at 744-747 and another one was located at 756-759 amino acids of integrin beta(3)-subunit cytoplasmic domain. We found that phosphorylations at tyrosines 747 and 759 play a major role in regulating the dynamic interactions between integrin alpha(IIb)beta(3) and rhodostomin. Moreover, the mutation at tyrosine 759 decreased the ligand-binding ability more than mutation at tyrosine 747. In the second part, we investigated the dynamic changes of rhodostomin-integrin alpha(IIb)beta(3) binding force during integrin clustering process. A progressive increase of total binding force over time was noticed when the bead bound to a CHO-alpha(IIb)beta(3) cell; such an increase was due mainly to the recruitment of more interacting molecules to the bead-cell interface. When the binding strength exerted by a single ligand-receptor pair was derived from the “polyvalent” measurements, surprisingly, a stepped decrease of “monovalent binding force” was noted (from 4.15 to 2.54 pN), that appeared to occur during the ligand-induced integrin clustering process. On the other hand, the mutant rhodostomin defective in clustering integrins exhibited only one (1.81 pN) unit binding strength. |
| Databáze: | Networked Digital Library of Theses & Dissertations |
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