A missense mutation in the seven-transmembrane domain of the human Ca ²⁺ receptor converts a negative allosteric modulator into a positive allosteric modulator

G protein-coupled receptors (GPCRs) are the most common targets of drug action. Allosteric modulators bind to the seven-transmembrane domain of family 3 GPCRs and offer enhanced selectivity over orthosteric ligands that bind to the large extracellular N terminus. We characterize a novel negative allosteric modulator of the human Ca²⁺ receptor, Compound 1, that retains activity against the E837A mutant that lacks a response to previously described positive and negative modulators. A related compound, JKJ05, acts as a negative allosteric modulator on the wild type receptor but as a positive modulator on the E837A mutant receptor. This positive modulation critically depends on the primary amine in JKJ05, which appears to interact with acidic residue Glu ⁷⁶⁷ in our model of the seven-transmembrane domain of the receptor. Our results suggest the need for identification of possible genetic variation in the allosteric site of therapeutically targeted GPCRs.