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

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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 E max values for cyclase assays in HEK cells. Comparison revealed a significant reversal in rank order for cyclase E max 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.

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Abbreviations

PBS:

Phosphate-buffered saline

BSA:

Bovine serum albumin

DOR:

D-opioid receptor

DPDPE:

[d-Pen(2), d-Pen(5)]-enkephalin

SNC-80:

(1)-4-[(αR)-α-((2S,5R)-4-Allyl-2,5-dimethyl-1-piperazinyl)-3- methoxybenzyl]-N,N-diethylbenzamide

TIPP:

H-Tyr-Tic-Phe-Phe-OH

UFP-512:

H-Dmt-Tic-NH-CH(CH2-COOH)-Bid

Emax:

Maximal effect

GPCR:

G protein-coupled receptor

HEK:

Human embryonic kidney

PKC:

Protein kinase C

GRK:

G protein-coupled receptor kinase

βarr:

βarrestin

ARM390:

AR-M100390

ADL5859:

N,N-diethyl-4-(5-hydroxyspiro[chromene-2,4′-piperidine]-4-yl) benzamide

ADL5747:

N,N-diethyl-3-hydroxy-4-(spiro[chromene-2,4′-piperidine]-4-yl) benzamide

cAMP:

Cyclic adenosine monophosphate

EC50:

Term half maximal effective concentration

BRET:

Bioluminescence resonance energy transfer

5-HT4R:

5-Hydroxytryptamine receptor 4

PMA:

Phorbol 12-myristate 13-acetate

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Acknowledgments

This work was supported by grants from the Natural Sciences and Engineering Research Council of Canada (NSERC) to GP [311997]; from the Canadian Institutes of Health Research (CIHR) to GP [MOP 79432], PWS [MOP 89716] and LET [MOP-106556]. PWS was also supported by the National Institutes of Health (NIH) [DA004443]. IC and KN hold a studentship from Ste-Justine Hospital Research Center and DT held a CIHR salary award. The authors would also like to thank Dr. Arthur Christopoulos for his insightful and helpful comments on curve fitting using the operational model.

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Correspondence to Graciela Pineyro.

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18_2013_1461_MOESM1_ESM.pdf

Supplementary material 1 (PDF 53 kb) Supplementary Fig. 1. Time course of DOR internalization in HEK cells. HEK293 cells stably expressing Flag-DORs were exposed to different agonists (10 μM) for the indicated time periods. Internalization was expressed as  % of receptors present at the membrane before exposure to agonist and represent mean ± SEM of 10 independent experiments carried out in triplicate. Statistical analysis by two way ANOVA revealed an effect for drug (p < 0.0001), for time (p < 0.0001) and an interaction (p < 0.0001). Post-hoc Bonferroni comparisons indicated a significant effect of time for all drugs (p < 0.01), except morphine

18_2013_1461_MOESM1_ESM.pdf

Supplementary material 1 (PDF 53 kb) Supplementary Fig. 2. A single affinity state could not account for cyclase and internalization responses of DOR agonists. (A) cAMP accumulation and (B) DOR internalization data were fit with the operational model as described in the methodology section, except that KA values for the agonists were fixed from published references as follows: KASNC-80: 5,75 nM; KAUFP512: 100 pM; KAMOR: 213.80 nM; KATIPP: 1.29 nM and KADPDPE: 12.02 nM

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Charfi, I., Nagi, K., Mnie-Filali, O. et al. Ligand- and cell-dependent determinants of internalization and cAMP modulation by delta opioid receptor (DOR) agonists. Cell. Mol. Life Sci. 71, 1529–1546 (2014). https://doi.org/10.1007/s00018-013-1461-7

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