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AT1 receptor mutant lacking heterotrimeric G protein coupling activates the Src-Ras-ERK pathway without nuclear
Koichi Seta1, Masakatsu Nanamori, J Gregory Modrall
1Cardiovascular Research Institute, Department of Cell Biology and Molecular Medicine, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark, New Jersey 07103, USA.
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 Galpha(i) or Galpha(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.
Insights
Angiotensin II (Ang II) activates Src and ERKs independently of G protein coupling, but the receptor
Area of Science:
- Molecular Cell Biology
- Receptor Signaling
- Signal Transduction Pathways
Background:
- Angiotensin II (Ang II) type 1 receptors (AT1Rs) are known to activate tyrosine kinases like Src.
- The role of heterotrimeric G protein coupling in AT1R-mediated tyrosine kinase activation and its functional consequences remain unclear.
- Understanding these pathways is crucial for deciphering Ang II's diverse cellular effects.
Purpose of the Study:
- To investigate whether AT1R-mediated tyrosine kinase activation occurs independently of G protein coupling.
- To elucidate the cellular functions associated with G protein-independent AT1R signaling.
- To determine the specific receptor domains involved in these distinct signaling pathways.
Main Methods:
- Utilized a mutant AT1a receptor (AT1a-i2m) lacking G protein coupling.
- Employed pharmacological inhibitors for protein kinase C, Ca(2+), Galpha(i), and Galpha(q).
- Investigated Src and ERK activation, phosphorylation of downstream targets (p90(RSK)), nuclear translocation, and cell proliferation.
- Manipulated nuclear exportin Crm-1 activity using leptomycin B.
Main Results:
- Ang II-induced Src activation was preserved in the G protein-uncoupled AT1a-i2m mutant.
- Src activation by Ang II required the carboxyl terminus of the AT1 receptor.
- ERKs were activated via a Src-Ras-dependent pathway in AT1a-i2m, leading to cytoplasmic target phosphorylation but not nuclear translocation or cell proliferation.
- G protein-independent ERK activation failed to inhibit Crm-1-mediated nuclear export, preventing nuclear signaling.
Conclusions:
- Src and ERKs can be activated by Ang II independently of heterotrimeric G protein coupling.
- The AT1 receptor's carboxyl terminus is essential for Ang II-mediated Src activation.
- Distinct roles exist for G protein-dependent and -independent signaling in Ang II's cellular responses, particularly concerning ERK nuclear signaling.
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