AT1 Receptor Mutant Lacking Heterotrimeric G Protein Coupling Activates the Src-Ras-ERK Pathway without Nuclear Translocation of ERKs

Abstract

Angiotensin II (Ang II) type 1 receptors (AT1Rs) activate tyrosine kinases, including Src. Whether or not tyrosine kinase activation by AT1R occurs independently of heterotrimeric G protein coupling and, if so, the cellular function of such a mechanism are unknown. To address these questions, we used an AT1aR intracellular second loop mutant, which lacks heterotrimeric G protein coupling (AT1a-i2m). Surprisingly, Ang II-induced Src activation was preserved in AT1a-i2m, which was not attenuated by inhibiting protein kinase C and Ca 2؉or by inhibiting G␣ i or G␣ q in CHO-K1 cells. By contrast, Ang II-induced Src activation was abolished in a C-terminally truncated AT1a-(1-309), where Ang II-induced inositol phosphate response was preserved. Ang II activates ERKs via a Src-Ras-dependent mechanism in AT1a-i2m. ERKs activated by AT1a-i2m phosphorylate their cytoplasmic targets, including p90 RSK , but fail to translocate into the nucleus or to cause cell proliferation. Ang II-induced nuclear translocation of ERKs by wild type AT1aR was inhibited by overexpression of nuclear exportin Crm-1, while that by AT1a-i2m was restored by leptomycin B, an inhibitor of Crm-1. In summary, while Src and ERKs are activated by Ang II even without heterotrimeric G protein coupling, the carboxyl terminus of the AT1 receptor is required for activation of Src. Interestingly, ERKs activated by heterotrimeric G protein-independent mechanisms fail to phosphorylate nuclear targets due to lack of inhibition of Crm-1-induced nuclear export of ERKs. These results suggest that heterotrimeric G protein-dependent and -independent signaling mechanisms play distinct roles in Ang II-mediated cellular responses.The signaling mechanism of the angiotensin II (Ang II) 1 type 1 (AT1) receptor has traditionally been portrayed as being dependent on heterotrimeric G proteins (1). The AT1 receptor activates phospholipase C␤ (PLC␤) via G␣ q proteins. This causes generation of inositol trisphosphates as well as diacylglycerol, which in turn causes release of Ca 2ϩ from the intracellular Ca 2ϩ store sites and activation of protein kinase C (PKC), respectively. The AT1 receptor also couples to G␣ i , thereby regulating adenylyl cyclase (2). Besides coupling with the heterotrimeric G proteins, activation of tyrosine kinases is also intimately involved in the AT1 receptor signaling (3, 4). Both nonreceptor type tyrosine kinases (Src, Fyn, Yes, Pyk2, focal adhesion kinase, and JAK2) and receptor type tyrosine kinases (EGF and platelet-derived growth factor receptors) are activated by the AT1 receptor (5-8). These tyrosine kinases regulate downstream signaling mechanisms, including PLC␥, Ras-Raf-MEK-ERK, and STAT (6, 9, 10), thereby playing a critical role in cell growth responses by Ang II.Several mechanisms have been shown to mediate tyrosine kinase activation by heterotrimeric G protein-coupled receptors (GPCRs; reviewed in Refs. 11-13). First, the downstream effectors of G␣ and G␤␥ mediate tyrosine kinase activation. For example, Ca 2ϩ and PKC activated through ...

Independent β-arrestin 2 and G protein-mediated pathways for angiotensin II activation of extracellular signal-regulated kinases 1 and 2

“…In contrast, ERK1͞2 activated by ␤-arrestin is excluded from the nucleus and is confined to a cytoplasmic compartment (8) where it presumably phosphorylates a distinct set of effectors. Consistent with our findings, ERK1͞2 activated by an AT 1A mutant receptor uncoupled from G proteins has been shown to localize to the cytoplasm and to be unable to induce activation of Elk1 (2). Similarly, stimulation of the DRY͞AAY mutant receptor with Ang II does not induce significant Elk1 activation (11).…”

“…1). In particular, several different angiotensin receptor mutants and angiotensin peptides, which are not able to activate G proteins, have been shown to activate the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinases 1 and 2 (ERK1͞2) (2)(3)(4). Nonetheless, the mechanisms mediating such G protein-independent signaling have remained unknown.…”

PEA-15 Is Inhibited by Adenovirus E1A and Plays a Role in ERK Nuclear Export and Ras-induced Senescence

“…Cytoplasmic accumulation of phosphorylated ERKs was observed in cells stimulated with angiotensin II and treated with protein kinase C inhibitors (32). Interestingly, upon treatment with leptomycin B, ERK1/2 nuclear localization was restored (32). These observations are consistent with a model in which ERK1/2 are translocated to the nucleus after MEK-dependent phosphorylation and exported to the cytoplasm via CRM1 by a factor normally inhibited by protein kinase C activity.…”

“…ated during Ras-induced senescence and, therefore, were unable to form stable complexes in these cells. Cytoplasmic accumulation of phosphorylated ERKs was observed in cells stimulated with angiotensin II and treated with protein kinase C inhibitors (32). Interestingly, upon treatment with leptomycin B, ERK1/2 nuclear localization was restored (32).…”

Cholecystokinin Stimulates Extracellular Signal-regulated Kinase through Activation of the Epidermal Growth Factor Receptor, Yes, and Protein Kinase C

“…Our finding that pertussis toxin had no effect on CCK-induced autophosphorylation of Src family tyrosine kinases argues against an involvement of G i proteins in CCKinduced Src activation despite the ability of CCK receptors to activate pertussis toxin-sensitive G proteins (48 -51). A recent study has shown that the G q -coupled angiotensin II receptor activates Src directly without the contribution of G proteins (75). However, whether CCK receptors utilize similar mechanisms to activate Src family kinases remains to be established.…”