Calpain cleavage of integrin beta cytoplasmic domains.
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.
Pfaff
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Department of Vascular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA. martin.pfaff@ens-lyon.fr
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D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000378:metabolism; D018821:CD18 Antigens / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002154:Calpain / Q000378:metabolism*; D017124:Conserved Sequence; D003593:Cytoplasm / Q000737:chemistry; D006801:Humans; D039641:Integrin beta Chains; D019012:Integrin beta1 / Q000378:metabolism*; D039661:Integrin beta3; D016023:Integrins / Q000378:metabolism; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D015539:Platelet Activation; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013379:Substrate Specificity; D013917:Thrombin / Q000378:metabolism
D015703:Antigens, CD; D018821:CD18 Antigens; D039641:Integrin beta Chains; D019012:Integrin beta1; D039661:Integrin beta3; D016023:Integrins; D010446:Peptide Fragments; D010980:Platelet Membrane Glycoproteins; D011994:Recombinant Proteins; C091368:integrin beta7; D000001:Calcimycin; D013917:Thrombin; D002154:Calpain