Activity-based, bioorthogonal imaging of phospholipase D reveals spatiotemporal dynamics of GPCR-Gq signaling.

Canonically, G-protein-coupled receptor (GPCR) signaling is transient and confined to the plasma membrane (PM). Deviating from this paradigm, the parathyroid hormone receptor (PTHR1) stimulates sustained G(s) signaling at endosomes. In addition to G(s), PTHR1 activates G(q) signaling; yet, in contrast to the PTHR1-G(s) pathway, the spatiotemporal dynamics of the G(q) branch of PTHR1 signaling and its relationship to G(s) signaling remain largely ill defined. Recognizing that a downstream consequence of G(q) signaling is the activation of phospholipase D (PLD) enzymes, we leverage activity-based, bioorthogonal imaging tools for PLD signaling to visualize and quantify the G(q) branch of PTHR1 signaling. We establish that PTHR1-G(q) signaling is short lived, exclusively at the PM, and antagonized by PTHR1 endocytosis. Our data support a model wherein G(q) and G(s) compete for ligand-bound receptors at the PM and more broadly highlight the utility of bioorthogonal tools for imaging PLDs as probes to visualize GPCR-G(q) signaling.