Abstract
G protein (heterotrimeric guanine nucleotide-binding protein)-coupled receptors belong to the largest family of membrane-embedded cell surface proteins and are involved in a diverse array of physiological processes. Despite progress in the mass spectrometry of membrane protein complexes, G protein-coupled receptors have remained intractable because of their low yield and instability after extraction from cell membranes. We established conditions in the mass spectrometer that preserve noncovalent ligand binding to the human purinergic receptor P2Y1. Results established differing affinities for nucleotides and the drug MRS2500 and link antagonist binding with the absence of receptor phosphorylation. Overall, therefore, our results are consistent with drug binding, preventing the conformational changes that facilitate downstream signaling. More generally, we highlight opportunities for mass spectrometry to probe effects of ligand binding on G protein-coupled receptors.