A disintegrin and metalloprotease 12 (ADAM12) is implicated in the ectodomain shedding of heparin-binding epidermal growth factor-like growth factor (HB-EGF) in epidermal growth factor receptor (EGFR) transactivation. However, the activation mechanisms of ADAM12 remain elusive. To analyze the regulatory mechanisms of ADAM12 activation, we performed yeast two-hybrid screening using the cytoplasmic domain of ADAM12 as bait, and identified PACSIN3 and a novel protein that we designated Eve-1. The interaction analyses between ADAM12 and PACSIN3 or Eve-1 were performed by using Glutathione S-transferase (GST) fusion protein and revealed that the proline-rich region (amino acid residues 829-840) of ADAM12 was required for the binding to them. Knockdown of endogenous PACSIN3 and Eve-1 by small interfering RNA (siRNA) in HT1080 cells attenuated greatly the shedding of proHB-EGF induced by TPA and angiotensin II.
Based on these data, these identified proteins play a role in positively regulating the activity of ADAM12 in the signaling of angiotensin II- and TPA-induced HB-EGF shedding.