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

Abstract

Traditional assays that monitor cAMP inhibition by opioid receptor ligands require second-messenger accumulation over periods of 10-20 minutes. Since receptor regulation occurs within a similar time frame, such assays do not discriminate the actual signal from its modulation. Here we used bioluminescence resonance energy transfer to monitor inhibition of cAMP production by d-opioid receptor (DOR) agonists in real time. cAMP inhibition elicited by different agonists over a period of 15 minutes was biphasic, with response buildup during the first 6 to 7 minutes, followed by a second phase of response decay or of no further increment. The rate at which the cAMP response disappeared was correlated with operational parameters describing ligand efficiency [log(t/K A )] to promote Ga i activation, as well as with ligand ability to promote internalization during the time course of the assay. Thus, ligands that displayed low signaling efficiency and poor sequestration (SB235863 ([8R-(4bS*,8aa,8ab,12bb)]7,10-dimethyl-1-methoxy- , which displayed high signaling efficiency and internalization. Moreover, inhibition of internalization by dynasore reduced or abolished response decay by internalizing ligands. Unlike acute responses, endocytic profiles were not predictive of whether an agonist would induce prolonged cAMP inhibition over sustained (30-120 minutes) DOR stimulation. Taken together, the data indicate that ligand ability to evoke Gprotein activation or promote endocytosis was predictive of response duration over short, but not over sustained periods of cAMP inhibition.

Molecular Pharmacology ofδ-Opioid Receptors

“…These quantitative differences do not allow us to unequivocally conclude whether they are attributable to ligand-specific conformations or are simply determined by ligand efficacy. Indeed, since TIPP, morphine, and TAN-67 behave as partial agonists in G protein activation (Quock et al, 1997;Tudashki et al, 2014), their reduced capacity to phosphorylate the receptor is most likely due to the fact that as partial agonists, these drugs induce a marginal increase in Ser363 phosphorylation. Unlike these quantitative differences, mutagenesis studies have clearly established that different agonists may phosphorylate DOPrs at different intracellular domains, implying that receptors occupied by different ligands expose different phosphorylation sites to regulatory kinases Liggett, 2011;Just et al, 2013).…”

“…The idea that some receptors may continue to signal after internalization must also be considered in the context of desensitization (Mullershausen et al, 2009;Irannejad and von Zastrow, 2014;Tudashki et al, 2014). According to the traditional view, GPCRs effectively signal to G proteins only when they are at the plasma membrane and, aside from noncanonical barrmediated signaling (Shukla et al, 2011), internalized receptors have been classically considered inactive, 670 simply trafficking toward degradation or recycling (Hanyaloglu and von Zastrow, 2008).…”

“…More recently, spectroscopic assays that allow real-time monitoring of GPCR signaling revealed that cAMP modulation by Gas and Gai/o-coupled receptors may take place after internalization has been completed (Calebiro et al, 2009;Ferrandon et al, 2009;Mullershausen et al, 2009;Irannejad et al, 2013), questioning sequestration as a mechanism of signal termination at least for enzymatic effectors. For example, by monitoring the kinetics of internalization and of cAMP inhibition by DOPr ligands, Tudashki et al (2014) showed that although sequestration may contribute to the initial rapid decay of acute cAMP responses, endocytosis does not control the magnitude or duration of cAMP inhibition over longer stimulation periods. For example, 60-minute exposure to agonists that induce either maximal internalization or no internalization at all produced similar cAMP inhibition, an observation that could not be attributed to differences in signaling efficiency (Tudashki et al, 2014).…”

“…For example, by monitoring the kinetics of internalization and of cAMP inhibition by DOPr ligands, Tudashki et al (2014) showed that although sequestration may contribute to the initial rapid decay of acute cAMP responses, endocytosis does not control the magnitude or duration of cAMP inhibition over longer stimulation periods. For example, 60-minute exposure to agonists that induce either maximal internalization or no internalization at all produced similar cAMP inhibition, an observation that could not be attributed to differences in signaling efficiency (Tudashki et al, 2014). The idea that intracellular signaling could make the duration of opiate-mediated cAMP inhibition independent of internalization may call into question the hypothesis that noninternalizing ligands such as morphine induce analgesic tolerance by producing sustained inhibition of AC function (Martini and Whistler, 2007) and raises the possibility that analgesic tolerance could rather be related to distinct substrates being modulated by membrane and intracellular AC signaling.…”

“…Since these early observations the repertoire of opioidmediated signals has continuously grown, first with the discovery that bg dimers derived from Gai/o proteins could modulate membrane-delimited enzymes (e.g., ACs, PLCb, and channels such as Cav2 and Kir3) and, more recently, with the finding that DOPrs also engage kinase signaling cascades. Concomitant with such multiplication of effectors, the availability of novel cell-based assays (Audet et al, 2008;Tudashki et al, 2014), Ga-specific antibodies (Garzón et al, 1997;Law and Reisine, 1997), antisense oligonucleotides (Standifer et al, 1996), and gene silencing/editing technologies enabled researchers to confirm that DOPrs pleiotropically couple to a multiplicity of G proteins beyond the classic Gai 1,2,3/o subtypes (reviewed in Piñeyro and Archer-Lahlou, 2007). This coupling diversity allows opioid receptors to trigger a great variety of signals (summarized in Table 4), whose contribution to the in vivo actions of DOPr ligands is the subject of active investigation.…”