G-protein-coupled receptors such as the proteinase-activated receptor 1 (PAR1) induce phosphorylation of mitogen-activated protein kinases through multiple pathways including transactivation of receptor tyrosine kinases. In vascular smooth muscle (VSM) cells, both a matrix-metalloproteinase (MMP)-dependent extracellular shedding of membrane-bound EGF receptor ligands and activation of the non-receptor tyrosine kinases Pyk2 and Src contributed to the thrombin-induced ERK1/2 phosphorylation. Surprisingly, disruption of the HB-EGF-mediated extracellular mode of EGF receptor transactivation also prevented the phosphorylation of the non-receptor tyrosine kinases Pyk2 and Src, locating these kinases downstream of the transactivated EGF receptor. The ionomycin-induced Pyk2 phosphorylation was partially sensitive to AG1478, heparin or the MMP-inhibitor BB2116, and the ionomycin-induced EGF receptor phosphorylation was almost completely blocked by these inhibitors of extracellular transactivation. Coimmuno-precipitation experiments revealed that, upon thrombin stimulation, a signaling complex consisting of Pyk2 and Src assembles at the EGF receptor. Reconstitution of the signaling molecules in HEK293 or VSM cells and subsequent determination of the EGF-induced Src kinase activity applying fluorescent sensor proteins demonstrated that a Ca(2+)-independent mode of Pyk2 activation is critical for the activation of Src downstream of the EGF receptor.