Aspartate 698 within a novel cation binding motif in alpha 4 integrin is required for cell adhesion.
The interactions of alpha 4 beta 1 integrin with vascular cell adhesion molecule (VCAM) and fibronectin play important roles in many physiological and pathological processes. To understand the mechanism of alpha 4 beta 1 integrin-mediated cell adhesion, we made mutant alpha 4 constructs. Three aspartic acid (Asp) residues in alpha 4, Asp-489, Asp-698, and Asp-811, were replaced with glutamic acids (Glu). The wild-type and mutant alpha 4 constructs were transfected into K562 cells, and stable transfectants with similar levels of alpha 4 surface expression were established. The Asp-->Glu substitutions did not affect alpha 4 beta 1 association or heterodimer formation as demonstrated by immunoprecipitation analysis. However, the glutamate substitutions at Asp-489 and Asp-698 severely impaired cell adhesion to VCAM and fibronectin, whereas the substitution at Asp-811 had no detectable effect on cell adhesion. In contrast to these results, isolated alpha 4 beta 1, containing the D489E or D698E substitution, was able to bind to VCAM, suggesting that these two residues are not critical for ligand recognition. In searching for a mechanism to explain inhibition of adhesion by Asp-489 and Asp-698 mutations, we found that the sequences flanking Asp-698 resemble the DxxxxxD-S-Sx divalent cation/ligand binding motif in beta integrins and the I-domains of alpha integrins. This suggests that Asp-698 in the alpha 4 integrin, which does not possess an I-domain, may also be involved in cation binding and may be part of a sequence functionally similar to that found in the I-domains of other alpha integrins.
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Institute for Bone and Joint Disorders and Cancer, Bayer Research Center, West Haven, Connecticut 06516, USA.
Conrad
P J
PJ
Webb
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Blue
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ML
D000595:Amino Acid Sequence; D015703:Antigens, CD / Q000235:genetics* / Q000378:metabolism; D001224:Aspartic Acid / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D002412:Cations / Q000378:metabolism; D002448:Cell Adhesion / Q000187:drug effects / Q000235:genetics*; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002460:Cell Line; D004252:DNA Mutational Analysis; D039441:Integrin alpha4; D039041:Integrin alpha4beta1; D016023:Integrins / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011233:Precipitin Tests; D011485:Protein Binding; D013329:Structure-Activity Relationship; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014162:Transfection
D015703:Antigens, CD; D002412:Cations; D015815:Cell Adhesion Molecules; D039041:Integrin alpha4beta1; D016023:Integrins; D039441:Integrin alpha4; D001224:Aspartic Acid; D013755:Tetradecanoylphorbol Acetate