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Media type:
E-Article
Title:
Amino acid motifs required for isolated β cytoplasmic domains to regulate ‘in trans’ β1 integrin conformation and function in cell attachment
Contributor:
Mastrangelo, Anthony M.;
Homan, Suzanne M.;
Humphries, Martin J.;
LaFlamme, Susan E.
Published:
The Company of Biologists, 1999
Published in:
Journal of Cell Science, 112 (1999) 2, Seite 217-229
Language:
English
DOI:
10.1242/jcs.112.2.217
ISSN:
0021-9533;
1477-9137
Origination:
Footnote:
Description:
ABSTRACT The role of β cytoplasmic domains in regulating β1 integrin conformation and function in cell attachment is not fully understood. In this study, we tested the ability of transiently expressed β cytoplasmic domains connected to an extracellular reporter domain to regulate ‘in trans’ the conformation of endogenous β1 integrins, and compared these effects on cell attachment. We found that chimeric receptors containing either the β1, β3 or β5 cytoplasmic domains inhibited the expression of the conformationally dependent 9EG7 and 12G10 epitopes on endogenous β1 integrins. In contrast, chimeric receptors containing the β4 or α5 cytoplasmic domain, or a control receptor lacking a cytoplasmic domain, had no effect. This inhibition occurred in a dose-dependent manner that required high levels of expression of the chimeric receptor. These results suggest that β1 integrin conformation can be regulated by conserved cytosolic interactions involving β cytoplasmic domains. This is further supported by our findings that mutations within amino acid motifs conserved among these β cytoplasmic domains, specifically the NXXY, NPXY and TST-like motifs, reduced the ability of these chimeric receptors to regulate β 1 integrin conformation. Interestingly, the chimeric receptors inhibited cell attachment in a similar dose-dependent manner and required intact NXXY, NPXY, and TST-like motifs. The β1 chimera also inhibited the binding of soluble fibronectin to endogenous β1 integrins. Thus, the concomitant inhibition in the expression of conformation-dependent integrin epitopes, cell attachment and ligand binding by the chimeras, suggests that the expression of the 9EG7 and 12G10 epitopes correlates with integrin function. However, Mn2+, which is an extracellular activator of integrin function, increased 9EG7 expression to basal levels in the presence of the β1 chimera, but did not rescue cell attachment to the same extent. Thus, although the β1 integrin conformation recognized by mAb 9EG7 may be required for cell attachment, it is not sufficient, suggesting that the β chimeras may be inhibiting both ligand binding and post-ligand binding events required for cell attachment. In addition, the inhibitory effects of the chimeric receptors on cell attachment were not reversed by the addition of the pharmacological agents that inhibit intracellular signals previously shown to inhibit integrin function. This finding, together with the requirement for high levels of the chimeric receptors and the fact that mutations in the same conserved motifs in heterodimeric β1 integrins have been reported to regulate β1 integrin conformation and function in cell attachment, suggest that β cytoplasmic domains regulate these processes by interacting with cytosolic factors and that the regulatory effect of the chimeras may be due to their ability to titrate proteins from endogenous integrins.