Diverse Pathways in GPCR-Mediated Activation of Ca2+ Mobilization in HEK293 Cells

Authors

Francesco De Pascali, Thomas Jefferson UniversityFollow
Asuka Inoue
Jeffrey L. Benovic, Thomas Jefferson UniversityFollow

Document Type

Article

Publication Date

11-1-2024

Comments

This article is the author's final published version in Journal of Biological Chemistry, Volume 300, Issue 11, November 2024, Article number 107882.

The published version is available at https://doi.org/10.1016/j.jbc.2024.107882.

Copyright © 2024 The Authors

Abstract

G protein-coupled receptors transduce extracellular stimuli into intracellular signaling. Ca²⁺ is a well-known second messenger that can be induced by G protein-coupled receptor activation through the primary canonical pathways involving Gα_q- and Gβγ-mediated activation of phospholipase C-β (PLCβ). While some G_s-coupled receptors are shown to trigger Ca²⁺ mobilization, underlying mechanisms remain elusive. Here, we evaluated whether G_s-coupled receptors including the β₂-adrenergic receptor (β₂AR) and the prostaglandin EP₂ and EP₄ receptors (EP₂R and EP₄R) that are endogenously expressed in human embryonic kidney 293 (HEK293) cells utilize common pathways for mediating Ca²⁺ mobilization. For the β₂AR, we found an essential role for G_q in agonist-promoted Ca²⁺ mobilization while genetic or pharmacological inhibition of G_s or G_i had minimal effect. β-agonist-promoted Ca²⁺ mobilization was effectively blocked by the G_q-selective inhibitor YM-254890 and was not observed in ΔGα_q/11 or ΔPLCβ cells. Bioluminescence resonance energy transfer analysis also suggests agonist-dependent association of the β₂AR with G_q. For the EP₂R, which couples to G_s, agonist treatment induced Ca²⁺ mobilization in a pertussis toxin-sensitive but YM-254890-insensitive manner. In contrast, EP₄R, which couples to G_s and G_i, exhibited Ca²⁺ mobilization that was sensitive to both pertussis toxin and YM-254890. Interestingly, both EP₂R and EP₄R were largely unable to induce Ca²⁺ mobilization in ΔGα_s or ΔPLCβ cells, supporting a strong dependency on G_s signaling in HEK293 cells. Taken together, we identify differences in the signaling pathways that are used to mediate Ca²⁺ mobilization in HEK293 cells where the β₂AR primarily uses G_q, EP₂R uses G_s and G_i, and EP₄R uses G_s, G_i, and G_q.

Recommended Citation

De Pascali, Francesco; Inoue, Asuka; and Benovic, Jeffrey L., "Diverse Pathways in GPCR-Mediated Activation of Ca2+ Mobilization in HEK293 Cells" (2024). Department of Biochemistry and Molecular Biology Faculty Papers. Paper 272.
https://jdc.jefferson.edu/bmpfp/272

Creative Commons License

This work is licensed under a Creative Commons Attribution 4.0 License.

PubMed ID

39395798

Language

English