G protein-coupled receptors mobilize intracellular calcium via the Gs-βγ-PLCβ module

Abstract

Accurate regulation of calcium is essential for many physiological processes. To fine-tune these processes, the cell can draw on a large portfolio of diverse mechanisms to govern calcium mobilization, in which GPCRs present an integral component. A key element of how the cell mobilizes calcium using GPCRs involves the large group of phospholipases Cß. These enzymes are canonically activated by Gq-coupled receptors, but it has been found that Gi coupled receptors can also activate PLCß2 and PLCß3 via their Gßγ subunit. Notably, this Gßγ-specific effect is fully dependent on active Gq. Upon activation, PLCß enzymes hydrolyze the membrane component PIP₂ to DAG and IP₃. While DAG triggers various cellular effectors, IP₃ mediates a cytosolic increase in calcium via activation of endoplasmically located InsP₃R. <br /> By studying the Gi-ßγ-PLCß-Ca²⁺ pathway and identifying active Gq as the driver of Gi-ßγ-mediated Ca²⁺release, we came up with a new hypothesis. Could Gq be the missing link that would allow even other members of the G protein family to release calcium via their Gßγ subunit? Therefore, we hypothesized that activation of Gq may permit Gs-ßγ to activate PLCß enzymes, resulting in the release of intracellularly stored calcium. To test this hypothesis, we used a variety of CRISPR/Cas9-edited cells, engineered molecules, and inhibitors alongside illuminating techniques such as fluorescence-based calcium measurement, HTRF-based cAMP accumulation, label-free whole-cell biosensing, real-time BRET and Nanobit measurements. <br /> However, examination of Gq-dependent Gs-calcium revealed that Gs-GPCRs trigger more than one Gq-dependent calcium release pathway in non-excitable HEK293 cells. This exploration led us to discriminate two distinct pathways utilized by Gs-GPCRs: i) a cAMP-dependent and ii) a cAMP-independent, previously unrecognized pathway. Both pathways are tightly governed by active Gaq, yet they diverge in terms of the underlying action. Because Gas mainly orchestrates cAMP-mediated effects, we focused on the unknown cAMP-independent Gs-mediated calcium release and were able to identify Gs-ßγ as the driving force. It appears that the Gs-ßγ module activates Gßγ-sensitive PLCß isoforms that mediate a calcium release from intracellular stores. Given the cell- and tissue-specific nature of calcium regulation, we extended our investigation to cells of greater physiological relevance. Thereby, we revealed a Gq-dependent Gs-mediated calcium response even in primary cell lines, including mouse brown adipose tissue and mouse embryonic fibroblasts, expanding the physiological significance of Gs-ßγ-mediated calcium release. Therefore, our results illustrate the remarkable adaptability of GPCR calcium signaling and highlight its capacity to rapidly adapt to ever-changing conditions (externally & internally). The Gs-GPCR system appears to be a competent regulator of calcium dynamics, whether through Gas-mediated, cAMP-dependent mechanisms or through a Gs-ßγ-mediated and cAMP-independent mechanism.