Abstract
Many G protein-coupled receptors (GPCRs) signal through more than one subtype of heterotrimeric G proteins. For example, the C–C chemokine receptor type 5 (CCR5), which serves as a co-receptor to facilitate cellular entry of human immunodeficiency virus 1 (HIV-1), normally signals through the heterotrimeric G protein, Gi. However, CCR5 also exhibits G protein signaling bias and certain chemokine analogs can cause a switch to Gq pathways to induce Ca2+ signaling. We want to understand how much of the Ca2+ signaling from Gi-coupled receptors is due to G protein promiscuity and how much is due to transactivation and crosstalk with other receptors. We propose a possible mechanism underlying the apparent switching between different G protein signaling pathways. We show that chemokine-mediated Ca2+ flux in HEK293T cells expressing CCR5 can be primed and enhanced by ATP pretreatment. In addition, agonist-dependent lysosomal exocytosis results in the release of ATP to the extracellular milieu, which amplifies cellular signaling networks. ATP is quickly degraded via ADP and AMP to adenosine. ATP, ADP and adenosine activate different cell surface purinergic receptors. Endogenous Gq-coupled purinergic P2Y receptors amplify Ca2+ signaling and allow for Gi- and Gq-coupled receptor signaling pathways to converge. Associated secretory release of GPCR ligands, such as chemokines, opioids, and monoamines, should also lead to concomitant release of ATP with a synergistic effect on Ca2+ signaling. Our results suggest that crosstalk between ATP-activated purinergic receptors and other Gi-coupled GPCRs is an important cooperative mechanism to amplify the intracellular Ca2+ signaling response.
Highlights
G protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors that mediate many important physiological processes
HEK293T cells were transiently transfected with chemokine receptor type 5 (CCR5) and intracellular Ca2+ levels were detected by FLIPR Calcium 6 assay dye, a permeable dye sensitive to C a2+
As we have shown that the GCaMP6s system works well in our hands to reproduce the effects seen from the purinergic receptor inhibitors on CCR5-mediated C a2+ flux, we went on to investigate the large effect of CGS15943 further
Summary
G protein-coupled receptors (GPCRs) are the largest family of transmembrane receptors that mediate many important physiological processes. They are activated by ligand binding on the extracellular side and couple preferentially to a unique Gα protein sub-family on the intracellular side to activate specific downstream signaling pathways. Once activated by the GPCR, the G protein dissociates into the Gα and Gβγ subunits, and both of these subunits can go on to mediate downstream effector activity by activating or inhibiting enzymes or channels. Some GPCRs do not couple to a unique Gα protein and have the ability to signal through more than one Gα protein class (Asano et al 1984). GPCR signaling is further complicated by the ability for receptor crosstalk, which can modify
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