Ligand- and cell-dependent determinants of internalization and cAMP modulation by delta opioid receptor (DOR) agonists

Abstract

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.

Endocytic Profiles ofδ-Opioid Receptor Ligands Determine the Duration of Rapid but Not Sustained cAMP Responses

“…This construct blocks internalization by interfering with binding of endogenous barr to clathrin (Krupnick et al, 1997) and, in the conditions used in this study, it inhibits internalization induced by 15-minute exposure to SNC-80 by ∼15% (Charfi et al, 2013). The basal BRET signal in cells that were transfected with mutant barr (0.272 6 0.012) or the corresponding vector pcDNA3 (0.266 6 0.008) were not different (P 5 0.6751; n 5 6), and neither were changes induced by 15-minute exposure to forskolin (controls: 24 6 4%; mutant barr: 27 6 4%; P 5 0.6154).…”

“…On the other hand, ligand signaling ability and endocytosis promoted over the time course of the experiment were predictive of the rate of decline in of the initial cAMP response. We previously showed that DOR ligands display different internalization profiles in HEK cells and neurons (Charfi et al, 2013). It would therefore be of interest to corroborate whether the time course of cAMP responses follow the distinct internalization profiles displayed by different ligands in HEK cells and neurons.…”

Molecular Pharmacology ofδ-Opioid Receptors

“…The structurally related dipeptide analog Dmt-Tic-NH-CH(CH 2 COOH)-Bid (UPF-512; Bid is 1H-benzimidazole-2-yl) is also a potent DOPr agonist (Balboni et al, 2002b). This compound displayed partial efficacy to inhibit cAMP production in human embryonic kidney (HEK) cells and to induce internalization in rat cortical neurons (Charfi et al, 2014) with anxiolytic-and antidepressant-like activities when administered peripherally to mice (Vergura et al, 2008). (Fig.…”

“…Several studies have shown that different ligands differ considerably in their ability to recruit barr to DOPr (Keith et al, 1996;Molinari et al, 2010;Audet et al, 2012;Charfi et al, 2014). The factors contributing to these differences are multiple.…”

“…It is also noteworthy that, in the case of GRK2/GRK3, poor phosphorylation of the receptor may be a direct consequence of low signaling efficacy since the ligand's ability to recruit (Li et al, 2003) and activate (Hong et al, 2009) this type of kinase is linked to G protein activation. Therefore, reduced capacity of partial agonists to activate the G protein may underlie low receptor phosphorylation (Kramer et al, 2000a;Audet et al, 2005;Navratilova et al, 2005;Hong et al, 2009) and poor barr recruitment, at least in certain immortalized cell lines (Charfi et al, 2014). It is also worth noting that prolonged ERK1/2 stimulation by partial but not by highly efficacious agonists (Eisinger et al, 2002;Eisinger and Schulz, 2004;Audet et al, 2005) may additionally interfere with barr recruitment.…”