Precipitated EGFR and SHC were immunoblotted with anti-phosphotyrosine antibody followed by reprobing of the same filters with anti-EGFR and anti-SHC antibody, respectively

Precipitated EGFR and SHC were immunoblotted with anti-phosphotyrosine antibody followed by reprobing of the same filters with anti-EGFR and anti-SHC antibody, respectively. can function as an effector of GPCR-mediated signalling and represents a key element of the cellular receptor cross-talk network. Keywords: amphiregulin/EGFR/HNSCC/TACE/transactivation Introduction Interreceptor communication between G protein-coupled receptors (GPCRs) and the epidermal growth factor receptor (EGFR) occurs in diverse cell types including fibroblasts, keratinocytes, astrocytes, PC-12 cells and easy muscle cells (Daub (Peschon et al., 1998). In addition, the absence of functional TACE results in impaired basal solubilization of a variety of other EGF-like ligands and cell surface molecules such as AR and HB-EGF (Merlos-Suarez et al., 2001; Sunnarborg et al., 2002). ADAM10-deficient mice have been reported to die very early in embryogenesis with multiple defects of the developing central nervous system, somites and cardiovascular system (Hartmann et al., 2002). It is not known, however, whether these Rabbit Polyclonal to CHFR developmental Dagrocorat defects are due to impaired growth factor precursor shedding. On the other hand, mice lacking MDC9/ADAM9 have no evident major abnormalities during development or adult Dagrocorat life (Weskamp et al., 2002). Moreover, proHB-EGF processing is comparable in embryonic fibroblasts isolated from ADAM9(C/C) and wild-type mice, arguing against an essential role for ADAM9 in proHB-EGF shedding in these cells. The HB-EGF-dependent mechanism of EGFR signal transactivation has gained further experimental support by studies on GPCR mitogenic signalling in vascular easy muscle cells (Eguchi < 0.03 for the difference between agonists versus BB94?+?agonists. In addition to the decrease of cell surface proAR, GPCR stimulation resulted in the accumulation of mature AR in cell culture medium as determined by sandwich enzyme-linked immunosorbent assay (ELISA) (Physique?2C). The finding that AR release in response to carbachol was substantially lower compared with LPA stimulation suggested a direct correlation between the amount of released AR and EGFR tyrosine phosphorylation content in response to GPCR ligands (Physique?1). Moreover, pre-incubation with batimastat completely prevented GPCR- and TPA-induced accumulation of AR in cell culture medium (Physique?2C), confirming the metalloprotease dependency of AR release. Ectodomain shedding of proAR is usually a prerequisite to GPCR-induced EGFR activation and EGFR-characteristic cellular responses We used three approaches to determine if AR function is required for GPCR-induced EGFR tyrosine phosphorylation and downstream Dagrocorat cellular responses. First, the endogenous expression of proAR, proHB-EGF and proTGF- was silenced by small interfering RNA (siRNA) in SCC-9 cells. Efficient and specific knockdown of target gene expression was monitored by RTCPCR (Physique?3A), confirming that gene silencing occurred by mRNA degradation. Concomitantly, the effect of siRNAs around the EGFR transactivation signal was examined. As shown in Physique?3B, siRNA to proAR completely blocked GPCR-induced EGFR tyrosine phosphorylation. SiRNAs to proHB-EGF and proTGF-, however, did not significantly alter the transactivation signal, demonstrating a specific requirement for proAR. In addition, we examined whether inhibition of proAR expression affects the GPCR-induced chemotactic migration of head and neck cancer cells towards fibronectin < 0.001 for control siRNA?+?LPA versus proAR siRNA?+?LPA. Secondly, we examined the effect of AR neutralizing antibodies on EGFR tyrosine phosphorylation by LPA in the squamous cell carcinoma cell lines Dagrocorat SCC-4, SCC-9, SCC-15 and SCC-25. The results show that pre-treatment with either a polyclonal goat or a monoclonal mouse antibody raised against the ectodomain of human AR inhibited the EGFR transactivation signal (Physique?4A, upper panel; representative data shown for the polyclonal anti-AR antibody in SCC-9 cells). Comparable results were obtained upon stimulation of head and neck cancer cells with carbachol (data not shown). In contrast, specific inhibition of HB-EGF by.