The expression of contractile proteins in vascular smooth muscle (VSM) cells is controlled by still poorly defined mechanisms. A thrombin-inducible expression of smooth muscle-specific a-actin and myosin heavy chain requires transactivation of the epidermal growth factor (EGF) receptor and a biphasic activation of extracellular signal-regulated kinases (ERK1/2). Here we demonstrate that the sustained second phase of ERK1/2 phosphorylation requires de novo RNA and protein synthesis. Depolymerisation of the actin cytoskeleton by cytochalasin D or disruption of transit between the endoplasmic reticulum and the Golgi apparatus by brefeldin A prevented the second phase of ERK1/2 phosphorylation. We thus conclude that synthesis and trafficking of a plasma membrane-resident protein may be a critical intermediate. Analysis of the expression of protease-activated receptor 1 (PAR1), heparin-binding EGF (HB-EGF) and the EGF receptor revealed that pro-HB-EGF is significantly upregulated upon thrombin stimulation. The kinetic of HB-EGF expression closely matched that of the second phase of ERK1/2 phosphorylation. Since inhibition of matrix metalloproteases or of the EGF receptor strongly attenuated the late phase of ERK1/2 phosphorylation, the second phase of ERK1/2 activation is primarily relayed by shedding of EGF receptor ligands. The si-RNA-mediated knockdown of HB-EGF expression confirmed an important role of HB-EGF expression in triggering the second phase of ERK1/2 activation. Confocal imaging of a YFP-tagged HB-EGF construct demonstrates the rapid plasma membrane integration of the newly synthesized protein. These data imply that the hormonal control of contractile protein expression relies on an intermediate HB-EGF expression to sustain the signalling strength within the Ras/Raf/MEK/ERK cascade.