Mass-Dependent Cross-Signaling between TXA₂ and PAF Receptors.

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 (

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 (G_q 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.