Phosphorylation of Tyrosine 319 of the Angiotensin II Type 1 Receptor Mediates Angiotensin II-induced Trans-activation of the Epidermal Growth Factor Receptor

Abstract

Although tyrosine kinases are critically involved in the angiotensin II (Ang II) type 1 (AT1) receptor signaling, how AT1 receptors activate tyrosine kinases is not fully understood. We examined the structural requirements of the AT1 receptor for transactivation of the epidermal growth factor (EGF) receptor (EGFR). Studies using carboxyl terminal-truncated AT1 receptors indicated that the amino acid sequence between 312 and 337 is required for activation of EGFR. The role of the conserved YIPP motif in this sequence in transactivation of EGFR was investigated by mutating tyrosine 319. Ang II failed to activate EGFR in cells expressing AT1-Y319F, whereas EGFR was activated even without Ang II in cells expressing AT1-Y319E, which mimics the AT1 receptor phosphorylated at Tyr-319. Immunoblot analyses using anti-phospho Tyr-319-specific antibody showed that Ang II increased phosphorylation of Tyr-319. EGFR interacted with the AT1 receptor but not with AT1-Y319F in response to Ang II stimulation, whereas the EGFR-AT1 receptor interaction was inhibited in the presence of dominant negative SHP-2. The requirement of Tyr-319 seems specific for EGFR because Ang II-induced activation of other tyrosine kinases, including Src and JAK2, was preserved in cells expressing AT1-Y319F. Extracellular signal-regulated kinase activation was also maintained in AT1-Y319F through activation of Src. Overexpression of wild type AT1 receptor in cardiac fibroblasts enhanced Ang II-induced proliferation. By contrast, overexpression of AT1-Y319F failed to enhance cell proliferation. In summary, Tyr-319 of the AT1 receptor is phosphorylated in response to Ang II and plays a key role in mediating Ang II-induced transactivation of EGFR and cell proliferation, possibly through its interaction with SHP-2 and EGFR.The signaling mechanism of the angiotensin II (Ang II) 1 type 1 (AT1) receptor has traditionally been portrayed to be dependent on heterotrimeric G proteins, including G␣ q and G␣ i proteins and their downstream targets, primarily phospholipase C (1). This results in inositol triphosphate generation, which in turn causes an increase in intracellular calcium concentrations and diacylglycerol formation, leading to activation of protein kinase C. However, recent investigations revealed that tyrosine phosphorylation is also intimately involved in AT1 receptor signaling (2-6). Ang II-induced ERK1/2 activation, for example, requires tyrosine kinase activation, including Src family tyrosine kinases (7, 8) and epidermal growth factor receptor (EGFR) (9, 10). It is unclear, however, how AT1 receptors, which lack intrinsic tyrosine kinase activities, are able to stimulate tyrosine kinases.We have recently shown that an AT1 receptor second intracellular loop mutant, lacking heterotrimeric G protein coupling, is able to activate Src tyrosine kinase (11). This suggests that heterotrimeric G protein-independent mechanisms are able to activate Src. Furthermore, increasing lines of evidence suggest that the carboxyl terminus (C-tail) of the AT1 receptor ...

G Protein Coupling and Second Messenger Generation Are Indispensable for Metalloprotease-dependent, Heparin-binding Epidermal Growth Factor Shedding through Angiotensin II Type-1 Receptor

“…In conflict with our findings, several recent reports have argued against the requirement of G protein-derived second messengers for tyrosine kinase activation thorough the AT 1 receptor (36, 37, 52). Seta and Sadoshima showed that phosphorylation of the AT 1 receptor at Tyr 319 is a prerequisite for EGFR transactivation because it can provide a docking site for protein-protein interaction, a proposed mechanism for the transactivation (37). However, this mutant is able to stimulate HB-EGF shedding and EGFR transactivation in our study.…”

“…However, recent studies using distinct mutants of AT 1 demonstrated G proteinindependent activation of tyrosine kinases (36,37,52). In particular, it has been reported that in a mutant of a conserved YIPP motif in the C terminus of AT 1 , AT 1 Y319F, EGFR transactivation was attenuated, whereas G q coupling remained intact (37). By contrast, we observed comparable HB-EGF shedding by AT 1 Y319F in COS7 cells (Fig.…”

“…The mutant also stimulated ERK in COS7 cells that was blocked by an EGFR kinase inhibitor or a metalloprotease inhibitor (66). The discrepancy may be due to the different nature of the cells utilized together with distinct experimental strategies (Seta and Sadoshima used cells expressing an exogenous EGFR gene) (37).…”

“…We also confirmed that AngII-induced EGFR transactivation remained intact in COS7 cells expressing AT 1 -(1-317) (data not shown). However, recent studies using distinct mutants of AT 1 demonstrated G proteinindependent activation of tyrosine kinases (36,37,52). In particular, it has been reported that in a mutant of a conserved YIPP motif in the C terminus of AT 1 , AT 1 Y319F, EGFR transactivation was attenuated, whereas G q coupling remained intact (37).…”

“…Adenoviral Infection-Adenovirus constructs encoding wild type rat AT 1 receptor and a carboxyl-terminal mutant, AT 1 Y319F, in which carboxyl-terminal Tyr 319 was replaced with Phe 319 were generated as described previously (29,36,37). The adenoviral vector containing the inhibitor of G q signaling (GqI), comprised of the amino acids 305-359 of murine G␣ q , was constructed as described previously (38).…”