Mapping the sequence determinants of constitutive activity in GPCRs

G protein-coupled receptors (GPCRs) signal not only in response to ligands but also in their absence. This basal, or constitutive, activity has distinct physiological consequences and is the only functional measurement available for orphan receptors. It is also hard to compare receptors because each is reported relative to its own baseline. Here, we measure basal activity on a single scale across the human GPCR family. Pairing a barcoded library of all human GPCRs with a transcriptional cAMP reporter, we scored 901 receptor constructs in one pool experiment. Basal activity forms a continuous distribution, and many of the highest-scoring recipes have prior reports of ligand-independent activity, with orphan being strongly represented. We then exhaustively mutated one of them, GPR161, and measured its effects on protein abundance, surface expression, and cAMP signaling. Of the 528 positions, only 10 specifically alter signaling independent of expression. Constraints on basal signaling are dominated by reepotro biogenesis, which requires multiphenotype scanning to resolve. Together, these datasets place basal activity on a common scale across the GPCR family and provide a template for dissecting it at residue resolution.