Minimum alpha chain cytoplasmic tail sequence needed to support integrin-mediated adhesion.
Previously, we found that deletion of the integrin alpha 4 and alpha 2 subunit cytoplasmic domains, just after the conserved GFFKR motif, causes a loss of adhesive activity mediated by VLA-4 or VLA-2, respectively (Kassner, P.D., and Hemler, M. E. (1993) J. Exp. Med. 178, 649-60; Kawaguchi, S., and Hemler, M. E. (1993) J. Biol. Chem. 268, 16279-12685). Here, we show for alpha 4 and alpha 2 chains (expressed in MIP101 and Chinese hamster ovary cells) that adding only 3-4 amino acids after the GFFKR motif restores substantial adhesive activity and that 5-7 amino acids confers maximal adhesive activity (to VCAM-1, CS1 peptide, or collagen, respectively). Point mutations within the most critical 5 alpha 4 residues had no effect on alpha 4 adhesive activity, nor did exchange of the alpha 4 tail with that of alpha 2. Thus, only a short and relatively nonspecific stretch of alpha chain cytoplasmic domain amino acids may be required to achieve maximal integrin adhesive activity. Also, comprehensive divalent cation titration assays revealed (i) that deletion of alpha chain cytoplasmic domains caused a marked decrease in the efficiency of divalent cation utilization during cell adhesion assays and (ii) that cytoplasmic domain deletion effects could be either suppressed or accentuated depending on the type and amount of divalent cation and the cellular environment utilized. Notably, integrin alpha chain tail deletions did not appear to alter the intrinsic ability to interact with ligand because deletion effects were minimal in the presence of metabolic energy inhibitors and were absent during cell-free ligand binding assays.
Kassner
P D
PD
Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115.
Kawaguchi
S
S
Hemler
M E
ME
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D016466:CHO Cells; D002413:Cations, Divalent; D002448:Cell Adhesion / Q000235:genetics; D002478:Cells, Cultured; D003094:Collagen / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D003593:Cytoplasm / Q000378:metabolism; D004734:Energy Metabolism / Q000187:drug effects; D016023:Integrins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009838:Oligodeoxyribonucleotides; D017384:Sequence Deletion
D002413:Cations, Divalent; D016023:Integrins; D009838:Oligodeoxyribonucleotides; D003094:Collagen