Functional and structural analysis of VLA-4 integrin alpha 4 subunit cleavage.
The cell surface heterodimer VLA-4 (alpha 4 beta 1), a member of the integrin family of adhesion receptors, is involved in both cell-extracellular matrix and cell-cell adhesion. Unlike any other integrin alpha subunit, the intact (150 kDa) alpha 4 subunit of VLA-4 can sometimes be cleaved into two noncovalently associated fragments (80 and 70 kDa). Using biosynthetic and mixing experiments, we found that human alpha 4 cleavage is a regulated, compartmentalized event, occurring soon after maturation of the beta 1-associated alpha 4 subunit. Cleavage of alpha 4, which is increased following T cell activation, has been suggested to correlate with altered VLA-4 functions. To address directly the functional importance of alpha 4 cleavage, we have studied VLA-4-mediated adhesion functions in cells expressing intact alpha 4 in comparison with cells expressing cleaved alpha 4. For this purpose, we first sequenced the N terminus of the endogenously produced 70-kDa alpha 4 fragment and identified the alpha 4 cleavage site between Lys557-Arg558 and Ser559. To abolish cleavage, we converted Arg558 to Leu or Lys557 to Gln by site-directed mutagenesis of the alpha 4 cDNA and then transfected both mutant and wild type alpha 4 cDNAs into VLA-4-negative K562 cells. Whereas transfection with wild type alpha 4 cDNA yielded predominantly cleaved alpha 4 subunit, the Leu558-alpha 4 yielded only intact alpha 4 subunit, and Gln557-alpha 4 yielded mostly intact alpha 4 subunit. Transfectants with the intact or the cleaved alpha 4 were equally capable of engaging in VLA-4-dependent adhesion to vascular cell adhesion molecule-1 and to the Hep II fragment of fibronectin (40 kDa) and aggregated equally well in response to anti-alpha 4 antibodies. Thus, cleavage of the alpha 4 subunit in these transfectants did not alter any of the known VLA-4-mediated adhesion functions.
Teixidó
J
J
Division of Tumor Virology, Dana-Farber Cancer Institute, Boston, Massachusetts 02115.
Parker
C M
CM
Kassner
P D
PD
Hemler
M E
ME
D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal; D002448:Cell Adhesion; D002449:Cell Aggregation; D002460:Cell Line; D006801:Humans; D006868:Hydrolysis; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D016029:Receptors, Very Late Antigen / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D014162:Transfection
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D016029:Receptors, Very Late Antigen; D011994:Recombinant Proteins