Related Experiment Videos
Calpain cleavage of integrin beta cytoplasmic domains
1Department of Vascular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. martin.pfaff@ens-lyon.fr
Abstract:
We showed previously that the calcium-dependent protease, calpain, cleaves the cytoplasmic domain of the integrin beta3 subunit. To investigate whether susceptibility to calpain is a common feature of all integrin beta subunits, and to map calpain cleavage sites in different integrin beta tails, we treated recombinant cytoplasmic domains of integrin beta1A, beta1D, beta2, beta3 and beta7 subunits with purified calpain in vitro. We found that the cytoplasmic domains of all these integrin chains were cleaved by calpain. HPLC followed by mass spectrometry was used to identify calpain cleavage sites. These sites were clustered in the C-terminal half of the integrin beta cytoplasmic domains in regions flanking the two NXXY motifs, suggesting the possibility that the structural framework provided by these motifs is recognized by calpain. We used the knowledge of these cleavage sites to develop cleavage site-specific antibodies and to demonstrate cleavage of the beta1A cytoplasmic domain in intact platelets stimulated with calcium ionophore or thrombin. Thus susceptibility to calpain cleavage is common to integrin beta subunits, can be induced in intact cells, and appears to favor regions surrounding two conserved NXXY motifs.
Insights
Calpain, a calcium-dependent protease, cleaves integrin beta subunits, common to all tested. Cleavage sites, identified using HPLC and mass spectrometry, are near conserved NXXY motifs.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Calpain is a calcium-dependent protease.
- Integrins are cell surface receptors involved in cell adhesion.
- Previous work showed calpain cleaves integrin beta3.
Purpose of the Study:
- To determine if all integrin beta subunits are susceptible to calpain cleavage.
- To map calpain cleavage sites within integrin beta tails.
- To investigate the role of NXXY motifs in calpain recognition.
Main Methods:
- In vitro cleavage of recombinant integrin beta subunits (beta1A, beta1D, beta2, beta3, beta7) by purified calpain.
- High-Performance Liquid Chromatography (HPLC) and mass spectrometry for cleavage site identification.
- Development of cleavage site-specific antibodies for detection in intact platelets.
Main Results:
- All tested integrin beta subunit cytoplasmic domains were cleaved by calpain.
- Cleavage sites were mapped to the C-terminal half of the domains, flanking conserved NXXY motifs.
- Calpain cleavage of the beta1A subunit was demonstrated in intact platelets stimulated with calcium ionophore or thrombin.
Conclusions:
- Susceptibility to calpain cleavage is a common feature of integrin beta subunits.
- Calpain cleavage of integrins can be induced in intact cells.
- Cleavage sites favor regions around conserved NXXY motifs, suggesting a structural recognition mechanism.