Disintegrin-like/cysteine-rich region of ADAM 12 is an active cell adhesion domain.
ADAM (a disintegrin and metalloprotease) proteins contain structural homology to the P-III class of snake venom metalloproteases (SVMPs) and are postulated to function, by analogy to these SMVPs, as cell adhesion molecules. ADAM 12 has been implicated in fusion of myoblasts, but its mechanism of action is not known. Instead of the RGD-like cell-binding motif present in SVMP disintegrins, the disintegrin domain of ADAM 12 contains a unique SNS sequence and therefore its adhesive potential has been controversial. In this report we demonstrate that the disintegrin-like/cysteine-rich (DC) domain of ADAM 12 constitutes a functional cell adhesion domain. We have expressed the DC domain of mouse ADAM 12 in insect cells and shown that the recombinant protein supported adhesion of C2C12 myoblasts and NIH 3T3 fibroblasts in a divalent cation-dependent manner. A sulfhydryl-specific biotinylation reagent revealed, however, that the overall conformation and flexibility of the cell-binding region of ADAM 12 DC domain may be significantly different from those of the SVMP disintegrins. Moreover, the disulfide bond structure of the DC domain was critical for its function, as incubation of the recombinant protein with reducing agents abolished subsequent cell adhesion. Recombinant DC bound to C2C12 cells with high affinity (K(D) approximately 0.10 microM, total number of binding sites n approximately 4.6 x 10(5)/cell). Adhesive properties of the DC domain of ADAM 12 produced in insect cells were further confirmed by cell surface binding of the DC domain expressed in C2C12 cells and secreted to the medium, consistent with the role of ADAM 12 in cell-cell interactions and myoblast fusion.
Zolkiewska
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Department of Biochemistry, Kansas State University, Manhattan, Kansas, 66506, USA. zolkiea@ksu.edu
D051722:ADAM Proteins; D000072199:ADAM12 Protein; D000595:Amino Acid Sequence; D000818:Animals; D002448:Cell Adhesion; D002460:Cell Line; D003545:Cysteine; D019483:Disintegrins / Q000235:genetics* / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics* / Q000378:metabolism*; D008666:Metalloendopeptidases / Q000235:genetics* / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism
D019483:Disintegrins; D008565:Membrane Proteins; D011994:Recombinant Proteins; D051722:ADAM Proteins; D000072199:ADAM12 Protein; D008666:Metalloendopeptidases; D003545:Cysteine