Recombinant soluble human alpha 3 beta 1 integrin: purification, processing, regulation, and specific binding to laminin-5 and invasin in a mutually exclusive manner.
Using insect cells, we expressed large quantities of soluble human integrin alpha 3 beta 1 ectodomain heterodimers, in which cytoplasmic and transmembrane domains were replaced by Fos and Jun dimerization motifs. In direct ligand binding assays, soluble alpha 3 beta 1 specifically bound to laminin-5 and laminin-10, but not to laminin-1, laminin-2, fibronectin, various collagens, nidogen, thrombospondin, or complement factors C3 and C3b. Soluble alpha 3 beta1 integrin also bound to invasin, a bacterial surface protein, that mediates entry of Yersinia species into the eukaryotic host cell. Invasin completely displaced laminin-5 from the alpha 3 beta 1 integrin, suggesting sterically overlapping or identical binding sites. In the presence of 2 mM Mg2+, alpha 3 beta 1's binding affinity for invasin (Kd = 3.1 nM) was substantially greater than its affinity for laminin-5 (Kd > 600 nM). Upon addition of 1 mM Mn2+, or activating antibody 9EG7, binding affinity for both laminin-5 and invasin increased by about 10-fold, whereas the affinity decreased upon addition of 2 mM Ca2+. Thus, functional regulation of the purified soluble integrin alpha 3 beta 1 ectodomain heterodimer resembles that of wild-type membrane-anchored beta 1 integrins. The integrin alpha 3 subunit was entirely cleaved into disulfide-linked heavy and light chains, at a newly defined cleavage site located C-terminal of a tetrabasic RRRR motif. Within the alpha 3 light chain, all potential N-glycosylation sites bear N-linked mannose-rich carbohydrate chains, suggesting an important structural role of these sugar residues in the stalk-like region of the integrin heterodimer. In conclusion, studies of our recombinant alpha 3 beta 1 integrin have provided new insights into alpha 3 beta1 structure, ligand binding function, specificity, and regulation.
Eble
J A
JA
Dana Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115, USA. eble@uni-muenster.de
Wucherpfennig
K W
KW
Gauthier
L
L
Dersch
P
P
Krukonis
E
E
Isberg
R R
RR
Hemler
M E
ME
D018829:Adhesins, Bacterial; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D002460:Cell Line; D004330:Drosophila; D005822:Genetic Vectors / Q000138:chemical synthesis / Q000378:metabolism; D006801:Humans; D039201:Integrin alpha3beta1; D016023:Integrins / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D004915:Leukemia, Erythroblastic, Acute; D011485:Protein Binding; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D012995:Solubility; D017186:Titrimetry; D014407:Tumor Cells, Cultured; D015007:Yersinia / Q000502:physiology; D000097794:Kalinin
D018829:Adhesins, Bacterial; D001426:Bacterial Proteins; D015815:Cell Adhesion Molecules; D039201:Integrin alpha3beta1; D016023:Integrins; D011993:Recombinant Fusion Proteins; D000097794:Kalinin; C053476:invasin, Yersinia