Signaling bias refers to G protein-coupled receptor ligand ability to preferentially activate one type of signal over another. Bias to evoke signaling as opposed to sequestration has been proposed as a predictor of opioid ligand potential for generating tolerance. Here we measured whether delta opioid receptor agonists preferentially inhibited cyclase activity over internalization in HEK cells. Efficacy (τ) and affinity (KA) values were estimated from functional data and bias was calculated from efficiency coefficients (log τ/KA). This approach better represented the data as compared to alternative methods that estimate bias exclusively from τ values. Log (τ/KA) coefficients indicated that SNC-80 and UFP-512 promoted cyclase inhibition more efficiently than DOR internalization as compared to DPDPE (bias factor for SNC-80: 50 and for UFP-512: 132). Molecular determinants of internalization were different in HEK293 cells and neurons with βarrs contributing to internalization in both cell types, while PKC and GRK2 activities were only involved in neurons. Rank orders of ligand ability to engage different internalization mechanisms in neurons were compared to rank order of Emax values for cyclase assays in HEK cells. Comparison revealed a significant reversal in rank order for cyclase Emax values and βarr-dependent internalization in neurons, indicating that these responses were ligand-specific. Despite this evidence, and because kinases involved in internalization were not the same across cellular backgrounds, it is not possible to assert if the magnitude and nature of bias revealed by rank orders of maximal responses is the same as the one measured in HEK cells.

This study assessed how conformational information encoded by ligand binding to d-opioid receptors (DORs) is transmitted to Kir3.1/Kir3.2 channels. Human embryonic kidney 293 cells were transfected with bioluminescence resonance energy transfer (BRET) donor/acceptor pairs that allowed us to evaluate independently reciprocal interactions among signaling partners. These and coimmunoprecipitation studies indicated that DORs, Gbg, and Kir3 subunits constitutively interacted with one another. GaoA associated with DORs and Gbg, but despite being part of the complex, no evidence of its direct association with the channel was obtained. DOR activation by different ligands left DOR-Kir3 interactions unmodified but modulated BRET between DOR-GaoA, DOR-Gbg, GaoA-Gbg, and GbgKir3 interfaces. Ligand-induced BRET changes assessing GbgKir3.1 subunit interaction 1) followed similar kinetics to those monitoring the GaoA-Gbg interface, 2) displayed the same order of efficacy as those observed at the DOR-Gbg interface, 3) were sensitive to pertussis toxin, and 4) were predictive of whether a ligand could evoke channel currents. Conformational changes at the Gbg/Kir3 interface were lost when Kir3.1 subunits were replaced by a mutant lacking essential sites for Gbg-mediated activation. Thus, conformational information encoded by agonist binding to the receptor is relayed to the channel via structural rearrangements that involve repositioning of Gbg with respect to DORs, GaoA, and channel subunits. Further, the fact that BRET changes at the Gbg-Kir3 interface are predictive of a ligand's ability to induce channel currents points to these conformational biosensors as screening tools for identifying GPCR ligands that induce Kir3 channel activation.