PT  - JOURNAL ARTICLE
AU  - Hanieh Bagheri Tudashki
AU  - Derek Robertson
AU  - Peter Schiller
AU  - Graciela Pineyro
TI  - Endocytic Profiles of δ-Opioid Receptor (DOR) Ligands Determine the Duration of Rapid but not Sustained cAMP Responses
AID  - 10.1124/mol.113.089003
DP  - 2013 Jan 01
TA  - Molecular Pharmacology
PG  - mol.113.089003
4099  - http://molpharm.aspetjournals.org/content/early/2013/10/30/mol.113.089003.short
4100  - http://molpharm.aspetjournals.org/content/early/2013/10/30/mol.113.089003.full
AB  - Traditional assays that monitor cAMP inhibition by opioid receptor ligands require second messenger accumulation over periods of 10-20 min. 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 (BRET) to monitor inhibition of cAMP production by delta opioid receptor (DOR) agonists in real time. cAMP inhibition elicited by different agonists over a period of 15 min was biphasic, with response build-up during the first 6-7 min, 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(Tau/KA)) to promote Gαi activation, as well as with ligand ability to promote internalization during the time course of the assay. Thus, ligands which displayed low signaling efficiency and poor sequestration (SB 235863, morphine) had minimal or no response decay. On the other hand, decay rate was pronounced for deltorphin II, DPDPE, Met-enkephalin and SNC-80, 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 sustained cAMP inhibition over sustained (30 -120 min) DOR stimulation. Taken together, the data indicate that ligand ability to evoke G protein activation or promote endocytosis were predictive of response duration over short but not sustained periods of cAMP inhibition.