Kinetics of binding with HEK293A-mGlu₅-low cell membranes. Competition association binding with [³H]methoxy-PEPy and indicated concentrations of each PAM: A) MPPA; B) CDPPB; C) compound 2c. Data are represented as means + S.D. (duplicate measurements) from six (MPPA), four (CDPPB competition experiments), eight (CDPPB vehicle experiments), or three (compound 2c) independent experiments.

Intrinsic PAM agonist activity in HEK293A-mGlu₅-low cells. (A) Peak Ca²⁺ mobilization measurement measured 90 seconds after PAM or DHPG addition at 37°C expressed as percent maximal DHPG response. (B) IP₁ accumulation measured 1 hour after PAM or DHPG addition at 37°C expressed as percent maximal (max) DHPG response. (C) ERK1/2 phosphorylation measurement after a 5-minute incubation of PAM or DHPG addition at 37°C. In the presence of 300 µM LY341495, the response to PAMs or DHPG is diminished for intracellular Ca²⁺ (iCa²⁺) mobilization (D), IP₁ accumulation (E), or ERK1/2 phosphorylation (F). Data in (D–F) are expressed as percent maximal DHPG response in the absence of LY341495, in which 0% is defined by vehicle (veh) treated in the presence of LY341495. The effect of LY341495 on basal responses in each assay is shown in Supplemental Fig 1. The dashed line in (D–F) shows the response to DHPG concentration-response relationship (from A–C) in the absence of LY341495 for reference. Data are means + S.D. (duplicate measurements) from 3–11 independent experiments (refer to Table 2 for exact numbers).

Potentiation of orthosteric agonist responses in HEK293A-mGlu₅-low cells. Ca²⁺ mobilization (intracellular: iCa²⁺) after stimulation with orthosteric agonist alone or simultaneous addition of PAM and 100 nM l-glutamate (Glu) (A) or DHPG (B). IP₁ accumulation in response to incubation with orthosteric agonist alone or coincubation with PAM and 1 µM l-glutamate(C) or DHPG (D). Phosphorylated ERK1/2 levels after stimulation with orthosteric agonist alone or simultaneous addition of PAMs and 500 nM l-glutamate (E) or DHPG (F). Data points are means + S.D. (triplicate measurements) from three to six independent experiments (refer to Table 3 and Supplemental Table 1 for exact numbers). Data were normalized to buffer or vehicle (veh.) treated as 0% and to the maximal (max) l-glutamate (A and C), maximal DHPG (B and D), or 10% FBS (E and F) responses as 100%.

Real-time measurement of mGlu₅ internalization. HEK293A cells were transiently transfected with HA-SNAP-mGlu₅ and EAAT3 (HEK293A-SNAP-mGlu₅), and internalization was measured as a change in fluorescence over time. (A–C) Indicated concentrations of each PAM were added at t = 0 minutes, and surface mGlu₅ levels were tracked for 66 minutes. (D–F) PAM-induced mGlu₅ internalization in the presence of 300 µM LY341495. (G–I) Potentiation of l-glutamate–induced mGlu₅ internalization by indicated PAMs. The l-glutamate concentration was increased by partially blocking the EAAT3 glutamate transporter with 30 µM DL-TBOA (DL-threo-β-Benzyloxyaspartic acid). (J–L) Potentiation of 1 μM DHPG-induced mGlu₅ internalization by indicated PAMs. Data points are means + S.D. (triplicate measurements) from three independent experiments, and solid lines are nonlinear regression fit to an exponential model of one-phase association.

Concentration-response relationships for agonism and potentiation of mGlu₅ internalization. From the kinetic measurements in Fig. 5, the area under the curve was calculated for each ligand concentration and normalized to the maximal (max) orthosteric agonist response measured in parallel. Each PAM was tested alone (A) and in the presence of 300 µM LY341495 (B). Each PAM was assessed for potentiation of l-glutamate (by partially blocking l-glutamate (Glu) transport with 30 µM DL-TBOA) (C) or 1 µM DHPG (D) induced mGlu₅ internalization. Data are means + S.D. (triplicate measurement) from three or four (l-glutamate) independent experiments. For reference, the control curve for DHPG (without LY341495) is shown by the dashed line in (B). Error bars not shown lie within the dimensions of the symbol, veh. denotes vehicle.

Assessment of biased agonism or modulation in HEK293A-mGlu₅-low or HEK293A-SNAP-mGlu₅ cells. (A) For CDPPB intrinsic agonism, ∆log(τ/K_A) values (relative to DHPG) were derived from concentration-response curves in the presence of LY341495. (B) Cooperativity factors for each functional response are presented relative to the value calculated from Ca²⁺ mobilization. Cooperativity with DHPG is depicted in squares. Cooperativity with l-glutamate is depicted in triangles. For select PAMs and functional outputs, cooperativity could not be determined (not applicable: n.a.) or was indistinguishable from neutral because of intrinsic PAM agonist activity. Data are means and 95% CI from three to five independent experiments (refer to Tables 2 and 4 for exact numbers). *P < 0.05, one-way ANOVA with Tukey’s multiple comparisons test.