Eylem Seç
Analysis of integrin activation and divalent cation binding by site-directed mutagenesis
Başlık:
Analysis of integrin activation and divalent cation binding by site-directed mutagenesis
Yazar:
Kline, Adam D., author.
ISBN:
9780438043749
Yazar Ek Girişi:
Fiziksel Tanımlama:
1 electronic resource (284 pages)
Genel Not:
Source: Dissertation Abstracts International, Volume: 76-08C.
Özet:
Integins are alpha/beta heterodimers. They are the principal receptors involved in cell-extracellular matrix interactions and play roles in differentiation, proliferation, survival, and gene expression. A system for the functional analysis of mutated integrin beta1 subunit was established. Mouse GD25, beta1 knockout cells were transfected with human integrin beta1 subunit using a series of expression vectors, including pIRES1hyg, pZEOSV2+ and pECE. Stable transfectants of the pECE construct were obtained and found to be fully functional. Alanine scanning mutations were subsequently made between asparagine 207 and serine 222 of beta1. This region contains the epitopes of several stimulatory and inhibitory monoclonal antibodies and was identified as a potential integrin site involved in intramolecular activation/effector binding. Mutant beta1 integrins were expressed in GD25 cells and were subsequently analysed for their ability to attach and spread on ligand. The mutations made did not appear to affect gross integrin function, but had compromised signalling in response to antibody binding. Divalent cations are critical for integrin ligand binding but to date the locations of their binding sites are unclear. By analysing cation regulation of ligand and antibody binding to truncated alpha5beta1-Fc tagged proteins, its has been shown that all functionally relevant sites lie N-terminal to amino acid 613 in the alpha5 subunit, and between amino acids 121 and 455 in the beta1 subunit. Site directed mutagenesis of oxygenated residues in the alpha5 subunit corresponding to the predicted Mg2+ binding site was carried out. The cation regulation of ligand and antibody binding remained unchanged in the mutants. Site directed mutagenesis of predicted alpha5 Ca2+ binding, EF hand-like sites in the beta-propeller region was also carried out. Mutants of 2 out of 3 predicted Ca2+ binding sites were successfully expressed with Ca2+ induced inhibition of ligand and antibody binding being decreased in both mutants. Finally, the potential cation binding site in the beta1 subunit A-domain-like region was mutated. Mn2+ and Mg2+ stimulation of antibody binding was ablated in this mutant. A model for integrin divalent cation binding was derived. Extrapolation from the co-crystal of the integrin alpha2 A-domain with a triple helical collagen peptide indicates a ligand bridging role for divalent cation at the binding site in the pi subunit A-domain-like region.
Notlar:
School code: 1543
Konu Başlığı:
Tüzel Kişi Ek Girişi:
Mevcut:*
Yer Numarası | Demirbaş Numarası | Shelf Location | Lokasyon / Statüsü / İade Tarihi |
---|---|---|---|
XX(684311.1) | 684311-1001 | Proquest E-Tez Koleksiyonu | Arıyor... |
On Order
Liste seç
Bunu varsayılan liste yap.
Öğeler başarıyla eklendi
Öğeler eklenirken hata oldu. Lütfen tekrar deneyiniz.
:
Select An Item
Data usage warning: You will receive one text message for each title you selected.
Standard text messaging rates apply.