Human CCR5 Stable Cell Line - HEK293T

Many G protein-coupled receptors (GPCRs) transduce signals via more than one subtype of heterotrimeric G protein. For instance, CCR5-which serves as a co-receptor for HIV-1 entry-typically signals through the heterotrimeric G protein Gi. However, CCR5 also exhibits biased G protein signaling; certain chemokine analogs can induce a shift toward the Gq pathway, thereby triggering Ca2+ signaling. Here, researchers found that in CCR5-expressing HEK293T cells, chemokine-mediated Ca2+ flux can be primed and enhanced by ATP pretreatment. Furthermore, agonist-induced lysosomal exocytosis leads to the release of ATP into the extracellular environment, thereby amplifying cellular signaling networks. ATP is rapidly degraded, sequentially converting into ADP, AMP, and adenosine; these molecules can respectively activate distinct purinergic receptors on the cell surface. Endogenous Gq-coupled purinergic P2Y receptors amplify Ca2+ signals and facilitate the convergence of signaling pathways between Gi-coupled and Gq-coupled receptors. The secretion of GPCR ligands-such as chemokines, opioids, and monoamines-is often accompanied by the co-release of ATP, resulting in a synergistic effect on Ca2+ signaling. These findings indicate that signaling crosstalk between ATP-activated purinergic receptors and other Gi-coupled GPCRs constitutes a key synergistic mechanism for amplifying intracellular Ca2+ signal responses.

Here, researchers co-incubated CCR5-expressing cells with broad-spectrum G-protein and purinergic receptor inhibitors and stimulated them with the same ligands, but omitted the dual-injection step (Figure 1). The results showed that injection of buffer alone did not induce significant changes in Ca2+ flux (Figure 1a); however, in the buffer-incubated control group, ATP injection elicited the greatest change in Ca2+ flux, with a mean RFU value of 35,000 (Figure 1b). Both the non-selective adenosine receptor inhibitors CGS15943 and YM significantly attenuated this response. Regarding the Ca2+ flux induced by ATPγS stimulation, only YM exerted an inhibitory effect, whereas CGS15943 had no impact; this is because ATPγS does not degrade to produce adenosine capable of stimulating adenosine receptors (Figure 1e). CGS15943 also had no effect on the Ca2+ flux induced by carbachol stimulation (Figure 1f); this response was inhibited only by G-protein blockers (YM and PTX) and the non-selective P2 purinergic receptor antagonist suramin. This observation was unexpected, as there have been no prior reports of muscarinic M3 receptor activation being influenced by purinergic receptors; however, the inhibitory effect of suramin might be explained by its ability to non-specifically block the coupling of G proteins to GPCRs.

Figure 1. CCR5-expressing HEK293T cells were incubated with broadly acting inhibitors of G proteins and purinergic receptors. (Horioka M, et al., 2021)