Targeting opioid receptors with pharmacological chaperones.

G Model YPHRS-2648; No. of Pages 11

ARTICLE IN PRESS Pharmacological Research xxx (2013) xxx–xxx

Contents lists available at ScienceDirect

Pharmacological Research journal homepage: www.elsevier.com/locate/yphrs

Review

Targeting opioid receptors with pharmacological chaperones Ulla E. Petäjä-Repo ∗ , Jarkko J. Lackman Department of Anatomy and Cell Biology and Medical Research Center Oulu, Institute of Biomedicine, University of Oulu, FI-90014 Oulu, Finland

a r t i c l e

i n f o

Article history: Received 5 November 2013 Received in revised form 5 December 2013 Accepted 5 December 2013 Keywords: Endoplasmic reticulum Opioid receptor Pharmacological chaperone Protein folding Quality control Up-regulation

a b s t r a c t G protein-coupled receptors (GPCRs) are polytopic membrane proteins that have a pivotal role in cellular signaling. Like other membrane proteins, they fold in the endoplasmic reticulum (ER) before they are transported to the plasma membrane. The ER quality control monitors the folding process and misfolded proteins and slowly folding intermediates are targeted to degradation in the cytosol via the ubiquitin–proteasome pathway. The high efficiency of the quality control machinery may lead to the disposal of potentially functional receptors. This is the major underlying course for loss-of-function conformational diseases, such as retinitis pigmentosa, nephrogenic diabetes insipidus and early onset obesity, which involve mutant GPCRs. During the past decade, it has become increasingly evident that small-molecular lipophilic and pharmacologically selective receptor ligands, called pharmacological chaperones (PCs), can rescue these mutant receptors from degradation by stabilizing newly synthesized receptors in the ER and enhancing their transport to the cell surface. This has raised the interesting prospect that PCs might have therapeutic value for the treatment of conformational diseases. At the same time, accumulating evidence has indicated that wild-type receptors might also be targeted by PCs, widening their therapeutic potential. This review focuses on one GPCR subfamily, opioid receptors that have been useful models to unravel the mechanism of action of PCs. In contrast to most other GPCRs, compounds that act as PCs for opioid receptors, including widely used opioid drugs, target wild-type receptors and their common natural variants. © 2013 Elsevier Ltd. All rights reserved.

Contents 1. 2. 3. 4.

5. 6. 7.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The opioid receptor family . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Opioid receptor pharmacological chaperones (ORPCs) enhance receptor maturation and cause up-regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The mechanism of action of ORPCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. ORPCs are membrane-permeable ligands that can bind to intracellular receptor precursors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. ORPCs induce structural stabilization of receptor precursors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. ORPCs enhance the dissociation of receptor precursors from the ER quality control machinery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.4. ORPCs rescue receptor precursors from premature ER-associated degradation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Wild-type opioid receptors respond to ORPCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Opioid receptor up-regulation in endogenous tissues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions and future perspectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: ER, endoplasmic reticulum; ERAD, ER-associated degradation; GPCR, G protein-coupled receptor; OR, opioid receptor; PC, pharmacological chaperone; QC, quality control; SNP, single nucleotide polymorphism. ∗ Corresponding author at: Department of Anatomy and Cell Biology, Institute of Biomedicine, University of Oulu, P.O. Box 5000, FI-90014 Oulu, Finland. Tel.: +358 294 485193; fax: +358 8 537 5172. E-mail address: ulla.petaja-repo@oulu.fi (U.E. Petäjä-Repo). 1043-6618/$ – see front matter © 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.phrs.2013.12.001

Please cite this article in press as: Petäjä-Repo UE, Lackman JJ. Targeting opioid receptors with pharmacological chaperones. Pharmacol Res (2013), http://dx.doi.org/10.1016/j.phrs.2013.12.001


ARTICLE IN PRESS U.E. Petäjä-Repo, J.J. Lackman / Pharmacological Research xxx (2013) xxx–xxx

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1. Introduction The general well-being of cells is dependent on strict quality control (QC) mechanisms that maintain protein homeostasis. Newly synthesized proteins targeted to the secretory pathway enter the endoplasmic reticulum (ER) and are subjected to scrutiny by its quality control machinery. This system, which involves molecular chaperones, folding enzymes and other factors, helps the nascent proteins to fold and makes decisions of their final destination [1,2]. Whereas correctly folded and assembled proteins are exported out of the ER and transported to their site of action, misfolded ones are targeted for degradation in the cytosol via the ubiquitin–proteasome pathway or form aggregates in the ER [3,4]. The ERQC operates by distinguishing imperfections in protein structure, which can lead to inappropriate retention and degradation of potentially functional protein molecules. It has been estimated that about 30% of all synthesized proteins are degraded [5], and thus even normal wild-type proteins can be targeted to the seemingly wasteful disposal. This can also apply to mutant proteins that cause human loss-of function diseases, including those involving G protein-coupled receptors (GPCRs) [6,7]. As many of the disease-causing mutations do not lead to cross structural impairment that would impair function of the affected protein, they are amiable for rescue. The imperfections in protein structure are generally related to problems in protein folding. Promoting this critical step has been one of the major keys in the development of therapeutic approaches to diseases caused by ER retention [8–10]. As protein misfolding can result from reduced stability of folding intermediates, these approaches have generally aimed to improve the stability of the folding protein in either an indirect or direct manner. This has been achieved by changing the folding environment in the ER with compounds that do not directly interact with the affected protein molecule (chemical chaperones) or using compounds that can specifically bind to target proteins [11–14]. These latter compounds, called pharmacological chaperones (PCs), have gathered a lot of attention during the last fifteen years and have been extensively studied. The PCs are small-molecular lipophilic compounds that act as ligands or substrates for their respective targets, such as receptors, channels and enzymes [12,14,15]. By binding to their target proteins they can create stabilizing links within the protein molecule. The GPCRs are particularly amiable to this type of approach because of the large number of pharmacologically diverse small-molecular ligands that have been generated during drug development efforts. The PC concept was first applied to GPCRs in an attempt to improve the cell surface expression of ER-retained V2 vasopressin receptor mutants [16] that are responsible for the disabling disease, X-linked nephrogenic diabetes insipidus. Two cell-permeable receptor-selective antagonists, SR121463A and VPA-985, were found to convert the precursor form of a receptor mutant, lacking three amino acids in the first cytoplasmic loop, into a fully mature protein that was targeted to the cell surface in heterologous expression systems. At the plasma membrane, the mutant receptors were fully functional. The functional rescue could not be mediated by nor competed with an antagonist that is membrane impermeable, confirming the intracellular site of action of the tested ligands. Soon after this original finding, the PC action of receptor-selective membrane-permeable ligands was demonstrated for the ␦-opioid receptor [17] and the gonadotropin-releasing hormone receptor [18]. The number of GPCRs that have been rescued from ER retention and degradation by their respective PCs has increased steadily during the last ten years. Importantly, it has become increasingly evident that not only mutant GPCRs but also wild-type receptors can be targeted by PCs [19–25], opening up wide-ranging possibilities to use PCs as regulators of receptor function. This review

summarizes the current knowledge and recent developments in our understanding of PCs that target opioid receptors, emphasizing on the mechanism of action of opioid receptor PCs and their functional relevance in vivo.

2. The opioid receptor family Opioid receptors (ORs) belong to the ␥ subfamily of family A GPCRs together with a number of other receptors that bind peptide ligands [26]. There are three closely related OR subtypes, ␮OR, ␦OR and ␬OR (MOP, DOP and KOP receptors, respectively, according to the IUPHAR nomenclature) that are primarily coupled to the heterotrimeric Gi /Go proteins, and are activated by endogenous opioid peptides endorphins, enkephalins and dynorphins, respectively [27–29]. The ORs are widely distributed in the central nervous system and are mainly involved in the control of analgesia and mood modulation. They also have a role in the periphery e.g. in respiration and gastrointestinal, endocrine and immune functions [30,31]. The ORs have a fundamental role in the clinical management of pain and are important therapeutic targets for a variety of exogenous or synthetic compounds, such as morphine and its derivatives. The ORs are typical GPCRs with seven transmembrane domains that are connected by three extracellular and three intracellular loops. The three subtypes share ∼70% sequence identity within the seven-transmembrane segment, whereas the intracellular loops and the extracellular N terminus and the intracellular C terminus are more diverse [27,28,32]. The nociceptive/orphanin FQ receptor is now classified as an OR because of its reasonable sequence identity (∼50%) with the three ORs [33]. It does not, however, bind classical opioid ligands [34] and is therefore not discussed here. The crystal structures of all OR subtypes have been recently solved at high-resolution by X-ray crystallography, revealing the highly similar overall structure and ligand binding pocket within the seven-transmembrane segment [35–38]. The ORs are also known to engage in receptor–receptor interactions, forming homomers and heteromers. The heteromers have distinct functional and pharmacological properties [39–41], creating new challenges and opportunities for the discovery of novel opioid ligands. Unlike is the case for most GPCRs that are targeted by PCs, no disease-causing mutants exist for ORs. However, the genes for ORs show sequence variations, single nucleotide polymorphisms (SNPs). These SNPs have been shown to associate with drug and alcohol addiction, pain sensitivity, and they change individual responses to opioid drugs [42–46]. According to the GPCR Natural Variants database [47], a few of the SNPs reside in the coding region and are non-synonymous, resulting in amino acid replacements. They may thus have a direct effect on receptor structure and/or function. Only two of these polymorphisms have been characterized at the cellular level in more detail, namely the ␮OR-N40D and ␦OR-F27C variants. In both cases, the amino acid substitution resides in the receptor extracellular N-terminal domain. The variants are fairly common and the allelic frequencies vary among human populations [42,48]. Whereas the ␮OR-N40D polymorphism has been shown to alter receptor function and stability at the cell surface [49–51], the ␦OR-F27C polymorphism changes receptor behavior in a more fundamental way both at the cell surface and in the biosynthetic pathway [25,52,53]. The ␦OR-C27 variant (allelic frequency ∼10% in Caucasians [48]) shows faster constitutive internalization from the cell surface in heterologous expression systems. Furthermore, a substantial fraction of the ␦OR-C27 variant precursors accumulate in the ER and are degraded by the ER-associated degradation (ERAD) pathway [25,52,53]. The ␦ORC27 variant also forms heteromers with the ␦OR-F27 and acts in a dominant negative manner, impairing the cell surface expression




3

as listed in Table 1. In heterologous expression systems, a number of membrane-permeable opioid ligands have been found to enhance the cell surface targeting of wild-type or mutant ␮ORs and ␬ORs and/or cause up-regulation of receptor expression levels. This has been shown for human as well as for rat and mouse receptors using various techniques (e.g. [22,24,55,56]). In addition, long-term opioid antagonist treatments have been found to lead to up-regulation of endogenous ␮OR and ␦OR in SH-SY5Y neuroblastoma cells and in NG108-15 neuroblastoma-glioma cells [57–60].

4. The mechanism of action of ORPCs 4.1. ORPCs are membrane-permeable ligands that can bind to intracellular receptor precursors

Fig. 1. The opioid antagonist naltrexone facilitates ␦OR maturation. HEK293 cells constitutively expressing the ␦OR-C27 variant were labeled for 60 min with [35 S]methionine/cysteine and chased in the absence or presence of 10 ␮M naltrexone as indicated. Receptors immunoprecipitated with FLAG M2 antibody, recognizing the C-terminal Flag epitope, were analyzed by SDS-PAGE and fluorography. The precursor and mature receptor forms are indicated with open and closed symbols, respectively. The graphs in B describe the time course of appearance of mature receptors. The values obtained by densitometric scanning from four independent experiments were normalized to the maximum label detected in untreated cells chased for 4 h. The data shown in A has been originally published in [17] and is shown in a modified form.

of the latter by redirecting precursors to degradation shortly after synthesis [54]. 3. Opioid receptor pharmacological chaperones (ORPCs) enhance receptor maturation and cause up-regulation The concept of ORPCs was first described for the ␦OR, specifically for its ␦OR-C27 variant [17] (Table 1). This variant that is characterized by a slow folding rate and inefficient maturation [25,52] was found to respond to membrane-permeable opioid ligands, which enhanced the maturation and cell surface targeting of receptor precursors [17]. In metabolic pulse-chase labeling experiments using stably transfected HEK293 cells, the addition of a lipophilic opioid antagonist naltrexone to the culture medium increased the turnover rate of ␦OR-C27 precursors, leading to a 2-fold increase in their processing to the mature form (Fig. 1). This enhancement in receptor maturation was accompanied by a 1.4-fold increase in [3 H]bremazocine binding sites in membrane preparations following 24-h naltrexone treatment and a 1.8-fold increase in the amount of cell surface receptors detected by flow cytometry. A mutant D95A receptor form that has a more severe impairment in maturation responded to the same treatment by a 2.4-fold increase in cell surface expression [17]. Importantly, only membrane-permeable ligands were found to be effective and both agonists and antagonists were able to facilitate the cell surface targeting of intracellularly trapped receptors (Fig. 2). Subsequently, ORPC-mediated enhancement in receptor maturation has been shown for the more common ␦OR-F27 variant that is characterized by inherently more efficient maturation [25], and opioid antagonists were also able to spare the F27 variant from degradation in cells co-expressing the C27 variant [54]. The findings obtained for the ␦OR are in line with the subsequent observations on the two other OR subtypes, ␮OR and ␬OR,

The ability of ORPCs to facilitate the maturation and cell surface targeting of newly synthesized receptors is an indication that the ligands act intracellularly. This is further supported by the finding that the PCs enhance the turnover and change the behavior of receptor precursors [17,22,24] (see also Section 4.3). In addition, the ligands are able to stabilize and increase the amount of ER-localized receptors when protein transport to the cell surface is blocked with brefeldin A [17,22,24,60], a reagent that causes receptor accumulation in the ER and prevents their transport to the plasma membrane. Importantly, lipophilic membrane-permeable opioid ligands appear to be able to act as ORPCs, whereas membrane-impermeable ones are inactive, as has become apparent in a thorough comparison of structurally divergent compounds [17,22,24,55,56,60]. A few ligands that were thought to be membrane impermeable have, however, had clear PC activity: a peptide analog TICP(␺) [17] and a positively charged analog of naloxone, naloxone methiodide [24,61]. The mechanism by which these ligands are able to cross membranes is unknown. The membrane-impermeable ligands are not able to prevent the effects of the lipophilic compounds. For example, the naltrexonemediated enhancement of ␦OR-C27 maturation is not blocked by a 200-fold excess of Leu-enkephalin, although the peptide agonist is able to cause down-regulation once the receptors reach the cell surface [17]. The findings described above clearly indicate that the ORPCs need to cross cellular membranes in order to have an access to newly synthesized receptors in the early secretory pathway. The PC action also requires receptor occupancy. This is additionally supported by the fact that the ligand-mediated enhancement in receptor expression and maturation is time- and dose-dependent [17,22] and 50% of the maximal response (EC50 ) is achieved with a concentration that is similar to the estimated Ki of the used ORPC [17]. A similar relationship between ligand affinity and PC-mediated response has been shown for the V2 vasopressin receptor [16,62]. The response of ORs to ORPCs is also pharmacologically selective since unrelated GPCR ligands are unable to mimic the effects mediated by the opioid ligands [17,22]. Neither a ␤-adrenergic antagonist propranolol nor a V2 vasopressin receptor antagonist SR121463A is able to mimic the effects mediated by naltrexone on ␦OR-C27 maturation [17], although these ligands can act as PCs for their corresponding receptors, the ␤1 -adrenergic receptor [63] and the V2 vasopressin receptor [16,62,64], respectively. The subtype selectivity of opioid ligands has also a clear effect on their PC activity, as naloxonazine and naltrindole, selective for the ␮OR and ␦OR, respectively, do not significantly enhance ␬OR expression in contrast to non-selective opioid ligands and subtypeselective compounds [22]. The PC activity of membrane-permeable opioid ligands is thus dependent on their ability to bind to the respective receptors in the ER shortly after they have been synthesized. The ligand




4

Table 1 ORPC action in heterologous expression systems. Receptor

Species

Variant/mutant

Ligands

Cell models

References

Human

Wild type

Antagonist: Naltrexone1 Antagonist: Naloxone6 , CTOP6 Antagonist: Naltrexone1,3 , Naloxone1 , ␤-CNA1 Agonist: Buprenorphine1 Antagonist: Naloxone6 , Cyprodime6 , CTOP6 Antagonist: Naloxone6

HEK293 (transient) HEK293 (stable) HEK293 (transient)

[56] [108] [56]

HEK293 (stable) HEK293 (transient) CHO (stable) CHO (stable)

[108] [69]

HEK293 (stable)

[55]

HEK293 (transient) CHO (stable) CHO (stable)

[69]

HEK293 (stable)

[109]

HEK293 (stable) HEK293 (stable) HEK293 (stable)

[25] [54] [17]

HEK293 (stable)

[60]

HEK293 (stable)

[25]

Antagonist: Naltrexone Antagonist: Naltrexone2,7 , Naloxone2 , Naltriben2 , Naltrindole2 Agonist: SNC-802 Antagonist: Naltrexone7 , Naltriben5,7 Antagonist: Naltrexone2 , ICI-1748642 , RTI-5989-252 , RTI-5989-232

HEK293 (stable) HEK293 (stable)

[54] [17]

HEK293 (stable) HEK293 (stable)

[60] [110]

Antagonist: Naloxone5,7 , Norbinaltorphimine7 Agonist: Etorphine7 , Pentazocine7 , ICI 204,4885,7 , U50,488H5,7 , TRK-8205,7 , Bremazocine5,7 , Asimadoline5,7 , Ethylketocyclazocine5,7 Antagonist: Naltrexone5,6,7 , Naloxone2,6,7 , Naloxone methiodide2,6,7 Agonist: Etorphine7 , Cyclazocine7

CHO (stable)

[22]

HEK293 (stable)

[24]

␮OR

K190 (natural variant)

Rat

C348,353 A (mutant) Wild type

258 RLSKV262 344 KFCTR348 (mutants)

D164 Q (mutant)

Mouse

Wild type

Human

F27 (natural variant)

Antagonist: Naloxone4,6 , Naltrexone6 , Naloxone methiodide6 Agonist: DAMGO4 Antagonist: Naloxone2,3,6 , Naltrexone2 ,Naltrindole2 , Diprenorphine2 Agonist: Morphine2 , Etorphine2,3 , Buprenorphine2 , Nalorphine2 , L-Methadone2 , Oxymorphone2 , Levorphanol2 Antagonist: Naloxone6,7(CHO) Antagonist: Naloxone4,6,7 , Naloxone methiodide6 ,Naltrexone6 ,Diprenorphine6 Agonist: Morphine6 , Etorphine6 , DAMGO4,6 Antagonist: Naloxone2 , RTI-d2 Agonist: Buprenorphine2

[61]

[61]

␦OR

C27 (natural variant)

Antagonist: Naltriben5 Antagonist: Naltrexone5 Antagonist: Naltrexone2,5,6,7 , Naltriben2,5 , Naloxone2,5 , Naltrindole2,5 , TICP(␺)5 Agonist: Buprenorphine5 , SNC-805 , TAN-675 , Bremazocine5 , Tonazocine5 , Nalbuphine5 Antagonist: Naltrexone2,4,6,7 , Naloxone2,4 Agonist: SNC-804 Antagonist: Naltriben5 5

D95 A (C27) (mutant)

Mouse

Wild type

Human

Wild type

Rat

Wild type

␬OR

Assay methods used: 1 enzyme-linked immunosorbent assay (ELISA), 2 fluorescence-activated cell sorting (FACS), 3 immunofluorescence, 4 in vitro stabilization, 5 metabolic labeling, 6 radioligand binding, 7 Western blotting. Abbreviations: CHO, Chinese hamster ovary; HEK, human embryonic kidney; N2A, neuro2A neuroblastoma.

binding ability of immature intracellular ORs was directly demonstrated for the ␦OR-C27 variant in saturation ligand binding assays [60]. This required complete separation of ER-localized precursors from cell surface mature receptors, which was accomplished by using stably transfected inducible HEK293 cells and the transport blocker brefeldin A. The addition of brefeldin A to the culture medium together with tetracycline that induced receptor expression, resulted in the accumulation of newly synthesized receptors in the ER. The saturation binding assays using [3 H]diprenorphine as the radioligand revealed the high-affinity binding ability of the precursors (Kd ∼ 0.5 nM), closely resembling the binding characteristics of mature cell surface receptors [60]. 4.2. ORPCs induce structural stabilization of receptor precursors The ability of ORPCs to mediate structural stabilization of newly synthesized ORs has been shown in an in vitro inactivation assay

using ␦OR-C27 precursors [60]. This assay has been broadly used to establish structural instability and ligand-mediated stabilization of constitutively active GPCRs [65–68], including the wild-type and mutant forms of the rat ␮OR [69]. For the assay on the ␦OR-C27, cellular membranes prepared from brefeldin A treated stably transfected HEK293 cells were incubated at 37 ◦ C with or without 1 ␮M naltrexone for various periods of time, and the remaining [3 H]diprenorphine binding activity was assessed after removing the ligand. The binding of membrane-bound receptor precursors that decreases in time was slowed down significantly when the membrane samples were incubated in the presence of naltrexone, with no detectable change in the receptor protein level. The ligand binding activity was also preserved when the membranes were incubated in the presence of another opioid antagonist naloxone or an agonist SNC-80, but not in the presence of phentolamine, a ␣-adrenergic receptor antagonist [60].


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U.E. Petäjä-Repo, J.J. Lackman / Pharmacological Research xxx (2013) xxx–xxx

- BFA

A 3

2

4

5

Non-peptidic

6

7

2

4

Non-peptidic 1 Naltriben 2 Naltrindole 3 Naltrexone 4 Naloxone

5

6

7

7

9

ntr Co

9

ntr Co

Peptidic

ol

ntr Co 1

3

2

Peptidic

4

5

6

Non-peptidic

5 TIPP( 6 TICP ( 7 ICI-174864 8 Propranolol 9 SR121463A

8

ol

ntr

1 Buprenorphine 2 TAN-67 3 SNC-80 4 Bremazocine

7

8

Peptidic 5 Tonazocine 6 Nalbuphine 7 Leu-enkephalin 8 BUBU 9 DPDPE

F 6

15 - BFA + BFA

5

0 1

2

3

4

5

6

7

8

9

Antagonists

- BFA + BFA

4

arbitrary units

10

arbitrary units

6

+BFA

E

Co

9

8

C

-5

5

4

Non-peptidic

ol

3

ol

3

2

Peptidic

ol

ntr Co 1

ol ntr Co 1

ntr

Co

9

8

+ BFA

B

- BFA

D ol

ol ntr Co 1

5

2

0

-2

1

2

3

4

5

6

7

8

9

Agonists

Fig. 2. Both membrane-permeable opioid antagonists and agonists can act as ORPCs. HEK293 cells constitutively expressing the ␦OR-C27 variant were pulse-labeled with [35 S]methionine/cysteine and chased for 6 h in the presence of 10 ␮M of the indicated opioid antagonists (A–C) or agonists (D–F). Brefeldin A (BFA, 5 ␮g/ml) or the vehicle was added to the medium 60 min prior to labeling. Receptors were immunoprecipitated with FLAG M2 antibody and analyzed by SDS-PAGE and fluorography. The values shown in C and F were obtained by densitometric scanning of the fluorograms and were normalized to the corresponding control values. For the graph, the control values were set to zero. The PC activity of non-peptidic membrane-permeable agonists can be seen in brefeldin A treated cells. In the absence of brefeldin A, the same agonists, except buprenorphine, bremazocine and nalbuphine, led to receptor down-regulation. The data shown (A, B, D, E) has been originally published in [17] and is shown in a modified form.

The ORPC-mediated conformational stabilization of receptor precursors is likely to result from more stable packing of the receptor transmembrane ␣-helices in an analogous manner to other small molecular ligands that have been shown to induce changes in protein thermostability or flexibility [70–72]. The recent crystallographic data on the three OR subtypes have revealed the precise contacts between the antagonists that were part of the crystallized receptor–ligand complexes and specific amino acids in the receptor transmembrane domains [35,36,38]. The ligands occupy a common region in the binding pocket involving transmembrane domains 3, 5, 6 and 7 [73]. Thus, binding of membrane-permeable ORPCs to newly synthesized receptors induces conformational constraints within the ␣-helical bundle that do not exist in unoccupied receptors. Interestingly, all membrane-permeable opioid ligands, whether having antagonistic or agonistic properties have been found to enhance OR maturation and cell surface expression [17,22,24,55,56,60] (Fig. 2). This indicates that both the inactive

receptor conformation(s), stabilized by antagonists or inverse agonists, and the active one(s), stabilized by agonists, are recognized as competent forms for ER export. This is expected because the ERQC scrutinizes and discriminates protein substrates via their structural attributes and not via functional criteria [1,2,8]. The key feature of PCs thus appears to be their ability to create stabilizing links within the receptor seven-transmembrane segment, achievable with ligands having divergent signaling efficacy profiles. An important finding concerning the ORPCs is the fact there is a clear correlation between the magnitude of ligand-mediated enhancement in receptor expression and maturation and the binding affinity of the ligand [17]. A similar correlation has been observed for other GPCRs and their respective PCs [74] and e.g. for enzyme inhibitors and blockers that rescue mutant forms of the ␣-galactosidase A and the HERG potassium channel, respectively [75,76]. The relationship between the binding affinity of a ligand and its PC activity can be explained by assuming that the ligands




4.3. ORPCs enhance the dissociation of receptor precursors from the ER quality control machinery The folding of newly synthesized proteins, including GPCRs, does not occur in isolation but is monitored and assisted by ER molecular chaperones, folding enzymes and other factors, constituting the complex ERQC machinery [1,2]. The major component of this machinery is the glycoprotein quality control that relies on N-glycans that are co-translationally added to / P) sequons asparagine residues in the glycoprotein N-X-S/T (X = [77,78]. A far majority of GPCRs contain consensus sites for N-glycosylation in their extracellular domains, and are therefore obvious targets for the glycoprotein ERQC. An increasing number of GPCRs have been shown to interact with calnexin (e.g. [19,60,79–85]), an ER membrane-bound lectin chaperone that together with its lumenal counterpart calreticulin binds to monoglucosylated protein-bound oligosaccharides [77,78]. Calnexin is closely associated with oligosaccharyltransferase that adds the core N-glycan (N-acetylglucosamine2 -mannose9 -glucose3 ) to the nascent polypeptide chain. The glucoses are eventually removed by ER glucosidase I/II but if the protein has not reached its stable correct conformation, it is reglucosylated by the folding sensor, UDP-glucose:glycoprotein glucosyltransferase, allowing the protein to bind again to calnexin [77,78]. This so-called calnexin cycle thus retains incompletely folded and assembled glycoproteins in the ER. Whereas wild-type GPCRs interact with calnexin in a transient manner, mutant forms appear to remain in a calnexinbound form for extended periods of time [19,80,85]. A few studies have reported that PCs can lead to the dissociation of calnexin from wild-type or mutant ER-retained receptors, including rhodopsin [85], the V3 vasopressin receptor [19] and the ␦OR [60]. The ability of ORPCs to enhance the dissociation of the ␦ORC27 from calnexin was studied by assessing PC-mediated changes in the amount of calnexin-bound receptor precursors [60] (Fig. 3). The treatment of HEK293 cells stably expressing the poorly maturing D95A mutant ␦OR-C27 with the antagonist naltriben was found to lead to a significant decrease in the amount of calnexin that co-immunoprecipitated with the receptor. This decrease did not entirely result from reduction in the total amount of receptor precursors that follows from the facilitated ER export, as a similar decrease was detected in brefeldin A-treated cells, in which the amount of ER-localized precursors is increased because of PC-mediated stabilization (Fig. 3). No significant antagonistinduced dissociation of calnexin from the wild-type receptor was detected, however, most likely because of the more transient nature of the interaction and the Western blot assay used that only detects robust steady-state changes in protein amounts. In contrast, using metabolic labeling, we recently showed that naltrexone can enhance ␦OR-C27 maturation even when its dissociation from

A

B

+ BFA

100 FLAG M2 Ab

Calnexin Ab

Lysate

80 ns

60 40 20

-

+ -

+

NTX NTB

N TB

0 N TX

can bind to the receptors and stabilize their native structure either via the induced fit or the kinetic selection model. In the former model, the higher affinity interaction provides higher binding energy that promotes the native receptor conformation, whereas in the selection model, the higher affinity ligands having longer average binding time (>kon /koff ) allow more time to the ligandbound stabilized receptor conformation to gain the native form. The two models are not mutually exclusive and both mechanisms may contribute to the PC activity of opioid ligands. Nevertheless, it is important to note that the PCs enhance the efficiency by which the receptor precursors are converted to the mature form without changing the kinetics of maturation [17] (Fig. 1). This suggests that the PCs do not have a direct role in receptor folding as such, but have a more subtle indirect role, most likely affecting the way by which the ERQC participates in the folding of the receptors and decides their fate (see Section 4.3).

% of Control

6

Fig. 3. Naltriben enhances ␦OR dissociation from calnexin. Inducible HEK293 cells were induced to express the D95A mutant of the ␦OR-C27 variant with tetracycline. Brefeldin A (BFA, 5 ␮g/ml) was added after 60 min, and cells were incubated in the absence or presence of naltrexone (NTX, 10 ␮M) or naltriben (NTB, 10 ␮M) for 3 h. Receptors were immunoprecipitated with FLAG M2 antibody, and samples were analyzed by Western blotting with either FLAG M2 (A, first panel) or calnexin antibodies (A, second panel). An aliquot of the solubilized membranes was probed with calnexin antibody, indicating that the amount of calnexin in each sample was equal (A third panel). The data were quantified by densitometric analysis and the calnexin-bound precursor/total receptor precursor ratio was calculated (B). The ratio in control cells not treated with antagonists was set to 100%. Values given are the mean ± S.E. of three independent experiments. **p < 0.001; ns, not significant, Ab, antibody. The data has been originally published in [60] and is shown in a modified form.

calnexin is impaired with a glucosidase I/II inhibitor.1 This gives indirect support to the notion that the receptor is engaged in an active calnexin cycle that involves repeated dissociation and reassociation with the molecular chaperone. Altogether, these results indicate that ORPC-mediated stabilization of h␦OR-C27 precursors promotes their disengagement from the ERQC machinery. It can be hypothesized that the bound ligand stabilizes the receptor molecules in such a way that they are no longer recognized by the folding sensor of the calnexin cycle and are therefore able to exit the QC cycle. Further support to this notion is provided by the observation that naltrexone was found to enhance the trimming of receptor N-glycans in brefeldin A-treated cells [60]. Brefeldin A collapses the Golgi complex, resulting in relocation of Golgi enzymes, like mannosidases and glycosyltransferases, to the ER [86]. This allows partial trimming of the N-glycans of substrate proteins if they have dissociated from the ER lectin chaperones. 4.4. ORPCs rescue receptor precursors from premature ER-associated degradation When newly synthesized proteins are incapable of gaining their native conformation, the extended interaction with the ERQC machinery is eventually followed by targeting the permanently misfolded or incompletely folded molecules to ERAD via the ubiquitin–proteasome pathway [3,4]. This is the final destination of numerous GPCR mutants and the alternative fate, in addition to ER export, for wild-type GPCRs that fold inefficiently (e.g. [19,20,53,81,87–91]). The evidence suggesting that PC-mediated stabilization of receptor precursors prevents their targeting to ERAD was originally indirect, deduced from the fact that the ligands improve ER exit of ER-localized precursors. More direct evidence has been provided with the demonstration that the treatment with membrane-permeable receptor-selective ligands leads to a decrease in receptor polyubiquitination, such as is the case with the V3 vasopressin receptor [19] and rhodopsin [85]. This phenomenon

1

Lackman et al., manuscript in preparation.




was recently described also for the ␦OR-C27.1 Furthermore, we have shown that the treatment of cells with ORPCs can decrease the amount of receptors that are normally retrotranslocated to the cytosol.1 A large fraction of wild-type ␦OR-C27 precursors are targeted to ERAD and this pathway involves polyubiquitination of the precursors and their retrotranslocation from the ER membrane to the cytosol concomitantly with N-glycan removal [25,53,54]. Thus, in the presence of a proteasomal inhibitor, such as lactacystin, receptors accumulate in the cytosol in a deglycosylated polyubiquitinated form. When HEK293 cells stably expressing the ␦OR-C27 variant were treated simultaneously with lactacystin and naltriben during metabolic pulse-chase labeling, the amount of receptors in the cytosol decreased significantly.1 These results allow us to speculate that the PCs, by binding and stabilizing receptor precursors, do not only lead to the dissociation of receptors from the QC machinery but also prevent the ERAD factors from recognizing the ligand-stabilized receptors as ERAD substrates. Thus, receptors that fold slowly suffer from the vigorous kinetic competition that occurs between the ERQC and ERAD machineries. Some receptor molecules are targeted to ERAD prematurely, while they are still folding competent, making them apparent targets for PC-mediated rescue. Taken together, the results on OPRCs obtained so far indicate that membrane-permeable ligands, irrespective of their signaling efficacy, enhance cell surface targeting and expression of newly synthesized receptors by direct ligand-mediated stabilization and a consequent release from the ERQC and ERAD pathways. The mechanism of action of ORPCs is summarized in Fig. 4.

5. Wild-type opioid receptors respond to ORPCs A characteristic feature of ORs that distinguishes them from most other GPCRs is the fact that not only mutant receptors but also wild-type forms are substrates for PCs in heterologous expression systems (Table 1). Whether this reflects a genuine difference compared with other family A GPCRs or tells more about active research on wild-type ORs is an open question. There appears to be some differences between the subtypes, however, as the effects of PCs on the ␮OR have been more modest compared with the ␦OR and ␬OR. In addition, conflicting results have been published. Li et al. [61] discovered a 1.2–1.4-fold up-regulation of the wildtype rat ␮OR measured with [3 H] diprenorphine binding following long-term naltrexone or naloxone treatment. This was observed in both transiently transfected HEK293 cells [69] and in stably transfected CHO cells [61]. In contrast, another study [55], using flow cytometry and [3 H]diprenorphine binding assays, found no apparent effect on the wild-type receptor although the response to various membrane-permeable ligands was clearly detected for two mutant receptor forms. The reason for the observed difference between the OR subtypes is not known but may relate to inherent differences in biosynthesis. The ␦OR and ␬OR have a relatively slow maturation kinetics and low maturation efficiency [22,24,25,52], whereas ␮OR precursors appear to mature more swiftly and with higher efficiency [51]. More systematic comparison of the three OR subtypes needs to be performed in the future in order to confirm the subtype-dependent differences and to gain a definitive explanation for their divergent responses to PCs. Nevertheless, results on the two ␦OR variants show clear correlation between the efficiency of maturation and the magnitude of the response to ORPCs [25], suggesting that receptors that have a tendency for impaired folding/maturation are more prone to PC-mediated rescue. Studies on other family A GPCRs are also in line with this hypothesis. An increasing number of wild-type receptors have been shown to mature inefficiently, including the V3 vasopressin receptor [19], the D4 dopamine receptor [20], the

7

V1a vasopressin receptor [21] and the gonadotropin-releasing hormone receptor [23]. All these receptors respond to their respective receptor-selective membrane-permeable ligands with enhanced maturation and up-regulation of cell surface receptors.

6. Opioid receptor up-regulation in endogenous tissues Importantly, the PC action of opioid ligands on receptor regulation is not a peculiarity of heterologous expression systems and cultured cells but has physiological relevance also in natural tissues in vivo. The up-regulation of ORs induced by long-term antagonist administration was first described in the 1970s for mouse brain, applying the newly developed radioligand binding assay [92]. Since then, the phenomenon has been thoroughly characterized using both radioligand binding assays and quantitative in vitro autoradiography (e.g. [93–100]). The functional relevance of OR up-regulation in vivo that results from chronic treatment with antagonists is exemplified by the fact that it causes supersensitivity to subsequently administered agonists [93,100]. The antagonist-induced up-regulation was first demonstrated for the ␮OR but later on also ␦OR and ␬OR up-regulation has been shown to take place following chronic treatment with the antagonists naltrexone or naloxone or the partial agonist buprenorphine [94–97,99]. In contrast to cultured cells, the ligand-induced receptor up-regulation in vivo has been most prominent for the ␮OR. Lesscher et al. [99], using autoradiographic analysis, found that chronic naltrexone treatment led to a significant increase in the ␮OR and ␦OR levels in specific regions of the mouse brain, whereas the effects on the ␬OR were more modest (overall increase 80%, 39% and 11% for ␮OR, ␦OR and ␬OR, respectively). The obvious contradiction to the experiments performed in cultured cells can be explained, at least partially, by technical differences. The studies on cells analyze changes in total receptor levels, often at a subcellular level, and usually assess the effects on only one receptor subtype at a time. In contrast, those done on tissues rely on measuring ligand binding sites with radioligands that are not entirely receptor-selective and using samples expressing all OR subtypes. In this particular study [99], the antagonist-induced changes in receptor levels were measured 24 h after terminating the treatment, in which case differences in the turnover of receptor subtypes may have an effect on the results. Species-specific differences may also play a role, as it can be speculated that the efficacy of ORPCs on a particular OR subtype may vary depending on the species, in a similar manner as has been described for the mouse, rat and human gonadotropin-releasing hormone receptors [101]. There are some indications in the existing literature suggesting differences between human and rodent ␬ORs [22,24], but more systematic studies are needed in order to make more definite conclusions. The precise mechanism behind the antagonist-induced OR up-regulation in vivo has remained unsolved. It has been demonstrated that chronic antagonist treatment does not trigger changes in receptor mRNA levels or stability [102–104], indicating that receptor up-regulation is a post-transcriptional event. It has been suggested that antagonists might prevent the binding of endogenous opioid peptides, leading to the inhibition of normal receptor internalization and degradation, or that the treatment might unmask ‘silent’ receptors [103,104]. Interestingly, one of the suggestions has been that the chronic antagonist treatment might ‘facilitate the processing of latent or precursor receptors into active receptors’ [103]. A single explanation for the antagonistinduced receptor up-regulation is unlikely as the phenomenon is undoubtedly complex. Nevertheless, it is tempting to speculate that PC activity of the ligands is a significant contributing factor. An unequivocal demonstration of ORPC action (enhancement of maturation of immature intracellular receptors) in natural


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Fig. 4. Schematic presentation of the mechanism of action of ORPCs. (1) Membrane-permeable opioid ligands (blue) that act as pharmacological chaperones (PC) can diffuse through the lipid bilayer into the cytosol and into the lumen of the endoplasmic reticulum (ER). (2) Opioid receptors are translated at the rough ER and translocated into the lipid bilayer. Co-translational addition of N-linked glycans takes place. (3) Folding intermediates of the receptor are scrutinized by the ER quality control (ERQC) machinery that includes molecular chaperones such as calnexin (CNX). The interaction with CNX is mediated by receptor N-glycans when they are in the monoglucosylated form. (4) A PC can bind to the receptor and stabilize its correct conformation, thus promoting its release from the quality control. (5) Receptors that fail to attain proper conformation will, in time, be targeted to the ER-associated degradation (ERAD) and subsequently degraded in the cytosol via the ubiquitin–proteasome pathway. The PCs rescue receptors from this pathway. (6) Receptors with correct conformation are exported to the ER exit sites and are transported to the Golgi apparatus and finally to the plasma membrane. (7) Once the bound PC dissociates and the receptor has reached the cell surface, it can be activated by agonists, many of which are membrane-impermeable peptides (red). Membrane-permeable agonists can act as PCs but they also induce down-regulation as soon as the receptor has been transported to the plasma membrane.

tissues expressing very low levels of endogenous receptors is, however, a formidable challenge with the methodology available today. 7. Conclusions and future perspectives Among GPCRs, the PC activity of receptor-selective membranepermeable ligands has been generally seen as a means to enhance the cell surface targeting of mutant receptor forms, especially disease-causing mutants. In contrast, PC-induced facilitation of OR maturation and expression is characteristic for wild-type receptors and common naturally occurring variants. The PC activity of opioid ligands is therefore likely to have general clinical relevance and it can be envisioned that the PC activity will be a useful parameter in

the design of better therapeutic drugs. The last decade has seen considerable advances in our understanding of ORPC action but many open questions still remain. These include the unknown effects of PCs on receptor heteromers and the potential PC activity of bivalent opioid ligands that bind to specific OR heteromers [105]. There is a wealth of evidence to suggest that ORs, like other GPCRs, form homomeric and heteromeric complexes already in the ER [54,106,107]. It can be envisioned that targeting these complexes early in the biosynthetic pathway with lipophilic opioid ligands, possibly also membrane-permeable bivalent ligands, might lead to changes in the homomer/heteromer ratio and therefore in function. Future years will give answers to these important questions and will also reveal the full potential of PCs in the opioid field.




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Pharmacological Profiles of Oligomerized μ-Opioid Receptors.

Opioid receptors and their pharmacological profiles.

Using pharmacological chaperones to restore proteostasis.

Pharmacological properties of newly synthesized derivatives of (-)-6 beta-acetylthionormorphine and their interactions with opioid receptors.

Pharmacological folding chaperones act as allosteric ligands of Frizzled4.

Drug repositioning can accelerate discovery of pharmacological chaperones.

Azasugar inhibitors as pharmacological chaperones for Krabbe disease.

Pharmacological chaperones stabilize retromer to limit APP processing.

Pharmacological chaperones in the age of proteomic pathology.

A thermodynamic assay to test pharmacological chaperones for Fabry disease.

Characterization of pharmacological receptors.

Design, synthesis and biological evaluation of multifunctional ligands targeting opioid and bradykinin 2 receptors.

Cyclic endomorphin analogs in targeting opioid receptors to achieve pain relief.

Bifunctional compounds targeting both D2 and non-α7 nACh receptors: design, synthesis and pharmacological characterization.

Targeting Hsp90 and its co-chaperones to treat Alzheimer's disease.

Molecular characterization of opioid receptors.

Kappa-opioid receptors and analgesia.

Opioid receptors and their biochemistry.

Targeting growth factor receptors with fusion toxins.

Chaperoning proteins for destruction: diverse roles of Hsp70 chaperones and their co-chaperones in targeting misfolded proteins to the proteasome.

Targeting interleukin-17 receptors.

Design, synthesis, pharmacological evaluation and molecular dynamics of β-amino acids morphan-derivatives as novel ligands for opioid receptors.

Simultaneous targeting of multiple opioid receptor types.

Why targeting? Physiological, pharmacological, and economic aspects.