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 signaling at endosomes. In addition to G, PTHR1 activates G signaling; yet, in contrast to the PTHR1-G pathway, the spatiotemporal dynamics of the G branch of PTHR1 signaling and its relationship to G signaling remain largely ill defined. Recognizing that a downstream consequence of G 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 branch of PTHR1 signaling. We establish that PTHR1-G signaling is short lived, exclusively at the PM, and antagonized by PTHR1 endocytosis. Our data support a model wherein G and G 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 signaling.