Abstract

Calcium sensing receptor (CaSR) positive allosteric modulators (PAMs) are therapeutically important. However, few are approved for clinical use, in part due to complexities in assessing allostery at a receptor where the endogenous agonist (extracellular calcium, Ca2+o) is present in all biological fluids. Such complexity impedes efforts to quantify and optimize allosteric drug parameters (affinity, cooperativity, efficacy) that dictate PAM structure-activity relationships (SAR). Furthermore, an under-appreciation of the structural mechanisms underlying CaSR activation hinders predictions of how PAM SAR relates to in vitro and in vivo activity. Herein we combined site-directed mutagenesis and calcium mobilization assays with analytical pharmacology to compare modes of PAM binding, positive modulation, and agonism. We demonstrate that 3-(2-chlorophenyl)-N-((1R)-1-(3-methoxyphenyl)ethyl)-1-propanamine (NPS R568) binds to a 7 transmembrane domain (7TM) cavity common to Class C GPCRs and utilised by (αR)-(−)-α-methyl-N-[3-[3-[trifluoromethylphenyl]propyl]-1-napthalenemethanamine (cinacalcet) and 1-benzothiazol-2-yl-1-(2,4-dimethylphenyl)-ethanol (AC265347), but there are subtle distinctions in the contribution of select residues to the binding and transmission of cooperativity by PAMs. Further, we reveal some common activation mechanisms utilized by different CaSR activators, but also demonstrate some differential contributions of residues within the 7TM bundle and extracellular loops (ECLs) to the efficacy of the PAM-agonist, AC265347, versus cooperativity. Finally, we show that PAMS potentiate the affinity of divalent cations. Our results support the existence of both global and ligand-specific CaSR activation mechanisms and reveal that allosteric agonism is mediated in part via distinct mechanisms to positive modulation.