Mass-Dependent Cross-Signaling between TXA2 and PAF Receptors.
Authors: Huang Jin-Sheng , Dong Lanlan , Le Breton Guy C. 
Published: October 2019

We have previously demonstrated in human platelets that G-protein coupled receptors (GPCRs) which share a common G” subunit have the capacity to cross-signal when exposed to their respective ligands. This ligand-dependent cross-signaling was shown to involve a redistribution of GPCR-coupled G” subunits, a consequent shift in GPCR ligand affinity and a synergistic effector response (Djellas, et al. 1998; PNAS,95:10944 and Djellas, et al. 2000; Biochem.Pharmacol.59:1521). Based on these findings, we proposed that the phenomenon of ligand-dependent cross-signaling represents one manifestation of a more general process by which cells can dynamically regulate their different GPCR signaling priorities in response to various stimuli. In this model, the relative signaling capacity through different GPCRs is determined by the distribution of G proteins amongst the different GPCRs at a given point in time. The model further predicts that this G protein distribution is not only modulated by ligand-induced shifts in GPCR:G protein coupling affinities (ligand-dependent cross-signaling), but also by shifts in GPCR:G protein mass ratios (mass-dependent cross-signaling), such as those that may occur by up- or down-regulation of GPCRs or their respective G protein partners. The present study examined this mass-dependent GPCR cross-signaling process.

Specifically, we established a series of cell lines in which the expression levels of thromboxane A2 receptors (TPR) and platelet activating factor receptors (PAFR) can be progressively altered. These expression systems allowed an alteration of the GPCR:G protein mass ratio for specific GPCRs, i.e., TPR and PAFR. Using radioligand binding analysis and Western blot, we provide evidence that up-regulating TPR expression levels increased the TPR:G-protein mass ratio. This TPR up-regulation resulted in a reduced TPR ligand affinity for [3H] SQ29548. Thus increasing the mass of TPR relative to its G protein partners (Gq or G13) shifted TPRs to a lower ligand affinity state. A similar reduction in PAFR ligand affinity was observed when the PAFR:G-protein mass ratio was increased by elevating PAFR expression levels. Moreover, using an inducible TPR/PAFR co-expressing cell line, increasing the expression level of one of the receptors resulted in reduced ligand affinity for both receptors. Taken in combination with our previous studies, these findings suggest that increasing TPR: G protein mass ratio results in a shift in the TPR-G protein coupling status, such that a larger fraction of total TPR is in the uncoupled state. This reduction in TPR-G protein coupling in turn leads to lower TPR ligand affinity. The finding that expression of TPR also causes reduced ligand affinity for a separate GPCR, i.e. PAFR, suggests that there is a dynamic equilibrium between the distribution of G proteins amongst GPCRs, and that this equilibrium can be altered by changes in the GPCR: G protein mass ratio. In summary, both ligand-dependent and mass-dependent GPCR cross-signaling appear to result from a general mechanism by which cells modulate their GPCR-G protein coupling status, and hence their GPCR ligand affinities and signaling priorities.