Smad signaling pathway bation with cinacalcet or NPS R568 increased the potency of Cao 2 for mediating Cai 2 mobilization, whereas NPS 2143 decreased Cao 2 potency, in a concentration dependent manner. Surprisingly, the Emax was also reduced in the presence of high concentrations of all three modulators. This effect was subsequently determined to be a function of modulator preincubation time, reduction in the preincubation period abolished the modulator effects onmaximal agonist responsiveness but also attenuated the degree of positive or negative modulation of agonist potency. The latter finding likely reflects a lack of equilibrium of the modulator due to the short exposure time, and thus, all subsequent analyses were performed on data derived from experiments using the longer preincubation time of 20 min. Another consistent feature of the effects of the positive allosteric modulators was noted on the Cao 2 C/R curveshape. Specifically, the C/R curve Hill slopes became progressively shallower with increasing calcimimetic concentrations. In the case of Cai 2 mobilization, they exhibited Hill mGluR slope values of nH3.85 0.42 in the presence of 30 nM cinacalcet and nH 3.65 0.18 in the presence of 3 M NPS R568.
This finding may reflect complex changes in the cooperative binding of Cao 2 to the CaSR in the presence of modulator or modulator induced changes in the stimulus response bcr-abl coupling of the CaSR. An attractive alternative explanation is allosteric potentiation of CaSR agonists, e.g. Cao 2 and/or Mgo 2 present at basal concentrations in the medium. Theoretical allosteric receptor model simulations incorporating ambient orthosteric agonist recapitulated these findings, indicating that this is the most parsimonious explanation of the data. Additionalexperiments in which the Cao 2 in the buffer was increased from 0.1 to 1 mM also demonstrated a greater relative shallowing of the Hill slope in the presence of cinacalcet. When these experiments were repeated using pERK1/2 or PM ruffling as functional measures of CaSR activation, similar observations were made with regard to the direction of modulator effects on orthosteric agonist potency and the effects of calcimimeticsonthe Hill slope. However, itwasalso noted that thePMruffling assay was substantially more sensitive to lower concentrations of allosteric modulator than either theCai 2mobilization Marbofloxacin orpERK1/2assays and that the modulator had no significant effect on the agonist maximal response.
Quantification of stimulus bias engendered by cinacalcet, NPS R568, and NPS 2143 In operational terms, allosteric modulator effects are described by the following: 1 the avidity with which the modulator binds to the allosteric site on its receptor, 2 the magnitude and direction of the mortality allosteric effect on the binding affinity of the orthosteric ligand, and 3 the magnitude and direction of the allosteric effect on the signaling efficacy of the orthosteric ligand. It is often not possible to determine each of these parameters separately for many GPCRs, especially in the absence of commercially available radioli gand binding assays for the CaSR, but the composite cooperativity parameter, can be derived from functional datasets as a global measure of allosteric modulation, together with the modulator KB value. In terms of these ligand.