The >800 human G protein–coupled receptors (GPCRs) are responsible for transducing diverse chemical stimuli to alter cell state- and are the largest class of drug targets. Their myriad structural conformations and various modes of signaling make it challenging to understand their structure and function. Here, we developed a platform to characterize large libraries of GPCR variants in human cell lines with a barcoded transcriptional reporter of G protein signal transduction. We tested 7800 of 7828 possible single amino acid substitutions to the beta-2 adrenergic receptor (β₂AR) at four concentrations of the agonist isoproterenol. We identified residues specifically important for β₂AR signaling, mutations in the human population that are potentially loss of function, and residues that modulate basal activity. Using unsupervised learning, we identify residues critical for signaling, including all major structural motifs and molecular interfaces. We also find a previously uncharacterized structural latch spanning the first two extracellular loops that is highly conserved across Class A GPCRs and is conformationally rigid in both the inactive and active states of the receptor. More broadly, by linking deep mutational scanning with engineered transcriptional reporters, we establish a generalizable method for exploring pharmacogenomics, structure and function across broad classes of drug receptors.

The calcium release-activated calcium channel Orai regulates Ca²⁺ entry into non-excitable cells and is required for proper immune function. While the channel typically opens following Ca²⁺ release from the endoplasmic reticulum, certain pathologic mutations render the channel constitutively open. Previously, using one such mutation (H206A), we obtained low (6.7 Å) resolution X-ray structural information on Drosophila melanogaster Orai in an open conformation (Hou, Burstein, & Long, 2018). Here, we present a structure of this open conformation at 3.3 Å resolution using fiducial-assisted cryo-EM. The improved structure reveals the conformations of amino acids in the open pore, which dilates by outward movements of subunits. A ring of phenylalanine residues repositions to expose previously shielded glycine residues to the pore without significant rotational movement of the associated helices. Together with other hydrophobic amino acids, the phenylalanines act as the channel's gate. Structured M1-M2 turrets, not evident previously, form the channel's extracellular entrance.