ADAM 12, a disintegrin metalloprotease, interacts with insulin-like growthfactor-binding protein-3

Insulin-like growth factor-binding protein (IGFBP)-3 binds the insulin-like growth factors with high affinity and modulates their actions. Proteolytic cleavage of IGFBP-3 may. regulate insulin-like growth factor bioavailabili ty IGFBP-3 is extensively degraded in serum during pregnancy; however, as y et the pregnancy-specific protease, or proteases, have not been identified. We utilized a yeast two-hybrid assay and a human placental cDNA library to investigate IGFBP-3-interacting proteins. A disintegrin and metalloproteas e-12 (ADAM 12), a member of a family of metalloprotease disintegrins that i s highly expressed in placental tissue, was identified as interacting with IGFBP-3, This interaction involved the cysteine-rich domain of ADAM 12. Unl ike other members of this family of disintegrin metalloproteases that are m embrane proteins, ADAM 12 exists as an alternatively spliced soluble secret ed protein. To verify the interaction between ADAM 12 and IGFBP-3, an expre ssion construct containing an ADAM 12-S c-DNA was transfected into COS-l ce lls. Go-precipitation was observed when conditioned medium was analyzed by immunoprecipitation with an antibody against either ADAM 12 or IGFBP-3 foll owed by Western blotting with anti-IGFBP-3 or anti-ADAM 12, Although minima l proteolysis of IGFBP-3 was observed in conditioned medium from control ce lls, this was increased similar to 4-fold in conditioned medium from ADAM 1 2-S-transfected cells. Recombinant ADAM 12-S partially purified from condit ioned medium on a heparin-Sepharose column also proteolyzed IGFBP-3. The de gradation pattern was similar to that seen with pregnancy serum, and the pr esence of ADAM 12-S in serum during pregnancy was confirmed. The data sugge st that ADAM 12-S has IGFBP-3 protease activity, and it may contribute to t he IGFBP-3 protease activity present in pregnancy serum.