The behavior of calpain-generated N- and C-terminal fragments of talin in integrin-mediated signaling pathways.
Our previous results showed that the binding of an adhesive ligand to integrin alphaIIbbeta(3) on the surface of platelets triggers the activation of calpain and the limited proteolysis of talin by calpain. To explore the physiological significance of the calpain-mediated cleavage of talin, we analyzed the behavior of the calpain-generated fragments of talin (N-terminal 47 kDa and C-terminal 190 kDa) during platelet activation by biochemical and immunoelectron microscopic studies. Intact talin and micro-calpain translocate from the Triton X-100-soluble fraction to the insoluble fraction upon platelet stimulation by thrombin, and the limited proteolysis of talin occurs in the Triton X-100-insoluble fraction, the cytoskeletal fraction. The fully autolyzed 76-kDa micro-calpain (active form) is found predominantly in the Triton X-100-insoluble fraction in stimulated platelets. While the N-terminal 47-kDa fragment remains in the Triton X-100-insoluble fraction, the C-terminal 190-kDa fragment is released into the Triton X-100-soluble fraction in a time-dependent manner. Immunoelectron microscopic observations revealed that the 47-kDa fragment locates on the submembrane zone just beneath the plasma membrane, including the open canalicular systems, while most of the 190-kDa fragment exists diffusely in the cytoplasm in thrombin-stimulated platelets. These findings suggest that calpain may contribute to the reorganization of the cytoskeleton in an integrin-mediated signaling pathway through the redistribution of the functional domain of talin.
Hayashi
M
M
Department of Protein Biochemistry, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakae-cho, Tokyo, Itabashi-ku, 173-0015, Japan.
Suzuki
H
H
Kawashima
S
S
Saido
T C
TC
Inomata
M
M
D000595:Amino Acid Sequence; D000918:Antibody Specificity; D001792:Blood Platelets / Q000648:ultrastructure; D002154:Calpain / Q000378:metabolism*; D002451:Cell Compartmentation; D002458:Cell Fractionation; D003593:Cytoplasm / Q000378:metabolism; D003599:Cytoskeleton / Q000378:metabolism; D006801:Humans; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D015539:Platelet Activation / Q000502:physiology*; D015398:Signal Transduction; D016608:Talin / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*
D010446:Peptide Fragments; D016608:Talin; D002154:Calpain