jpet fast forward. published on march 27, 2003 as doi:10...

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Characterization of the Binding of [3H][Dmt1]DALDA, a Highly Potent Opioid Peptide1

Claire L. Neilana, Adam J. Janveya, Elizabeth Bolana, Irena Berezowskab, Thi M-D. Nguyenb,

Peter W. Schillerb, Gavril W. Pasternaka

Laboratory of Molecular Neuropharmacology, Department of Neurology, Memorial Sloan

Kettering Cancer Center, New York, NY 10021 (CLN, AJJ, EB, GWP) and the Laboratory of

Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, Montreal,

Canada H2W 1R7 (IB, TM-DN, PWS).

23-Mar-03

Copyright 2003 by the American Society for Pharmacology and Experimental Therapeutics.

JPET Fast Forward. Published on March 27, 2003 as DOI:10.1124/jpet.103.049742This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on March 27, 2003 as DOI: 10.1124/jpet.103.049742

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Running title: [3H][Dmt1]DALDA binding

Corresponding author: Dr. Gavril Pasternak

Dept. of Neurology

1275 York Ave

New York, NY 10021

Tel: (212) 639 7046

Fax: (212) 794 4332

E-Mail: [email protected]

KEY WORDS: receptor binding, mu opioid receptor, Divalent cations,

splice variant, GTPγS, DALDA, opiate, MOR-1, MOP-1

Text pages 26 Abstract 257

Tables 3 Introduction 463

Figures 6 Discussion 1409

References 36

Abbreviations:

[Dmt1]DALDA H-Dmt-D-Arg-Phe-Lys-NH2

Dmt 2’,6’-dimethyltyrosine

DAMGO [D-Ala2,MePhe4,Gly(ol)5]enkephalin

CHO Chinese Hamster Ovary

M6G morphine-6β-glucuronide

RT-PCR Reverse Transcriptase-Polymerase Chain Reaction

MOR-1 Mu Opioid Receptor clone-1

This article has not been copyedited and formatted. The final version may differ from this version.JPET Fast Forward. Published on March 27, 2003 as DOI: 10.1124/jpet.103.049742

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ABSTRACT

The dermorphin-derived peptide [Dmt1]DALDA (H-Dmt-D-Arg-Phe-Lys-NH2;

Dmt=2',6'-dimethyltyrosine) labels mu opioid receptors with high affinity and selectivity in

receptor binding assays. In previous studies [Dmt1]DALDA displayed a mechanism of action

distinct from that of morphine, as evidenced by its insensitivity to antisense probes reducing

morphine analgesia and incomplete cross tolerance to morphine. In an effort to further elucidate

the unusual mechanism of action, [3H][Dmt1]DALDA has been synthesized and its binding

profile studied. [3H][Dmt1]DALDA binding was high affinity (KD 0.22nM) and showed a

regional distribution consistent with mu receptors with highest levels in calf striatal membranes.

[3H][Dmt1]DALDA binding was far less sensitive than [3H]DAMGO to the effects of divalent

and sodium cations and guanine nucleotides, although NaCl and Gpp(NH)p together reduced

specific [3H][Dmt1]DALDA binding levels by almost 75%. Competitions studies confirmed the

mu-selectivity of the binding, with Ki values that were not appreciably different from those seen

against [3H]DAMGO. In [35S]GTPγS binding assays in brain and spinal cord membranes,

[Dmt1]DALDA was more potent than DAMGO, but showed plateaus suggestive of a partial

agonist. [Dmt1]DALDA bound to MOR-1 and its splice variants with high affinity. Unlike

[3H]DAMGO, [3H][Dmt1]DALDA appeared to label both agonist and antagonist conformations

of MOR-1 expressed in CHO cells. In [35S]GTPγS assays [Dmt1]DALDA showed high efficacy

with all the MOR-1 variants, but its potency (EC50) varied markedly among some of the splice

variants despite similar affinities in receptor binding assays. Although [3H][Dmt1]DALDA is a

very potent mu-selective analgesic, its binding characteristics and its ability to stimulate GTPγS

binding differed from that of the classical mu opioid peptide DAMGO.


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Mu opioids continue to be widely used for the treatment of moderate to severe pain. Mu

opioids and opioid peptides comprise a large, diverse group of ligands, typically classified by

their ability to bind with high affinity and selectivity to the µ-opioid receptor and to elicit such

pharmacological actions as analgesia, inhibition of gastrointestinal transit, respiratory depression,

and the induction of tolerance and dependence. There are several lines of evidence from both

clinical and animal studies to suggest that mu opioid analgesics vary considerably in their actions

(Pasternak, 2001; Wolozin and Pasternak, 1981; Rossi et al., 1995; Payne and Pasternak, 1992).

Mu opioids, such as heroin, morphine-6β-glucuronide (M6G), 6-acetylmorphine, and fentanyl

retain their analgesic potencies in CXBK mice, a strain that is insensitive to morphine (Rossi et

al., 1996) and they display limited cross-tolerance to morphine in animal studies (Lange et al.,

1980) (Rossi et al., 1996) and clinically (Cherny et al., 2001). The antagonist

3-methylnaltrexone selectively blocks the actions of heroin, 6-acetylmorphine and M6G more

potently than morphine (Brown et al., 1997a; Walker et al., 1999). Antisense mapping studies on

the cloned mu opioid receptor MOR-1 find different sensitivity profiles for morphine as

compared to heroin and M6G (Rossi et al., 1995; Rossi et al., 1996). In addition, M6G, heroin

and 6-acetylmorphine retained their analgesic activity in a knockout mouse with disruption of

exon 1 of the MOR-1 receptor that was totally insensitive to morphine (Schuller et al., 1999).

Although exon 1 has been deleted, there is still expression of MOR-1 splice variants in this

knockout mouse, as demonstrated by both RT-PCR and immunohistochemistry (Schuller et al.,

1999; Pan et al., 2001).

The dermorphin analog [Dmt1]DALDA (H-Dmt-D-Arg-Phe-Lys-NH2; Dmt = 2',6'-

dimethyltyrosine) (Schiller et al., 2000) displays a very unique pharmacology that differs from

that of morphine, as evidenced by its insensitivity to antisense probes that reduce morphine


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(exons 1, 4, 5, 6, 7, 8, 9, 10) or M6G (exon 2) analgesia, its incomplete cross tolerance to

morphine (Neilan et al., 2001; Riba et al., 2002) and persistent analgesia in a MOR-1 knockout

mouse (Neilan et al., 2003). This peptide also is an effective analgesic in morphine-insensitive

CXBK mice (Neilan et al., 2001). [Dmt1]DALDA has a long duration of action, and is

metabolically stable with limited penetration of the placental barrier (Szeto et al., 2001).

Radioligand binding studies have proved to be a valuable tool in investigating the mechanisms of

action of mu opioids. Detailed binding experiments reported over twenty years ago first

proposed multiple classes of mu binding sites (Wolozin and Pasternak, 1981). More recent

studies using [3H]M6G also support the concept of mu receptor heterogeneity (Brown et al.,

1997b). In an attempt to further investigate the more unusual aspects of its pharmacology, we

now have characterized the receptor binding profile of [3H][Dmt1]DALDA.


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MATERIALS AND METHODS

Drugs

For the preparation of tritiated [Dmt1]DALDA, a precursor peptide containing 2',6'-

dimethyl-3',5'-diiodotyrosine [Tyr(2',6'-Me2-3',5'-I2] needed to be synthesized. Fmoc-Dmt-OH

was iodinated by treatment with I2 in the usual manner to yield Fmoc-Tyr(2',6'-Me2-3',5'-I2)-OH.

This protected amino acid was then used in the solid-phase synthesis of H-Tyr(2',6'-Me2-3',5'-I2)-

D-Arg-Phe-Lys-NH2 according to a protocol published elsewhere (Schiller et al., 2000). The

peptide was purified by preparative reversed-phase chromatography, and its structure was

confirmed by fast atom bombardment mass spectrometry. Catalytic tritiation of this precursor

peptide was performed at the Institute of Isotopes (Budapest, Hungary), resulting in the tritiated

peptide [3H][Dmt1]DALDA (H-Dmt[3',5'-3H2]-D-Arg-Phe-Lys-NH2) with a specific activity of

47Ci/mmol. [3H]DAMGO, specific activity 50 Ci/mmol, and [3H]naloxone, specific activity 60

Ci/mmol, were purchased from DuPont NEN (Boston, MA). Unlabeled [Dmt1]DALDA was

synthesized in the laboratory of P.W.S. All other ligands were obtained from the Research

Technology Branch of the National Institute of Drug Abuse (Rockville, MD).

Receptor binding

Membranes were prepared as previously reported (Clark et al., 1989; Clark et al., 1988).

Tissue or cells were homogenized in 50 volumes of 50 mM Tris buffer, pH 7.7, containing 10

µM phenylmethylsulfonyl fluoride, 100 mM NaCl and 1 mM K+ EDTA. The homogenate was

incubated for 15 min at 25°C, centrifuged at 39,000 X G for 45 min, resuspended in 0.32 M

sucrose and frozen at -80°C. Binding was performed in potassium phosphate buffer (50 mM; pH

7.4), containing MgSO4 (either 1 mM or 5 mM, respectively) for assays with [3H][Dmt1]DALDA


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or [3H]DAMGO. Reactions were incubated at 25°C for 60 min, with the exception of

dissociation and association binding which used the indicated time points. Assay volumes were

3 ml for calf thalamic and frontal cortex membranes, 2 ml for guinea-pig cerebellum membranes

and calf striatal membranes, and 1 ml for spinal cord, whole mouse brain, and CHO cell

membranes. Protein concentration was 3 mg/ml wet weight of tissue for all calf brain and GP

cerebellar membranes and 150 µg for binding assays using CHO cell membranes. Reactions

were terminated by rapid filtration over glass fiber filters, that then were subjected to liquid

scintillation counting. For assays using [3H][Dmt1]DALDA, filters were soaked in 0.5% w/v

polyethylimine (PEI) solution for 5 min prior to using. Non-specific binding, which typically

represented 15% of total binding, was determined using levallorphan (1 µM) or [Dmt1]DALDA

(1 µM) for assays using [3H]DAMGO and [3H][Dmt1]DALDA respectively, with no appreciable

difference in levels of non-specific binding. Only specific binding is reported. Saturation and

competition studies were analyzed using nonlinear regression analysis with the Prism program

(GraphPad Software, San Diego, CA).

[35S]GTPγS assays were performed in Tris buffer (50 mM, pH 7.4) containing EGTA

(0.2 mM), NaCl (100 mM), and MgCl2 (3 mM). Membranes (25-50 µg) were incubated for 1 hr

at 30°C with GDP (30 µM), [35S]GTPγS (0.05 nM), and unlabeled ligand at varying

concentrations. Reactions then were terminated by rapid filtration and subject to liquid

scintillation counting as above. All values were normalized as a percentage of the stimulation by

DAMGO at 10 µM.


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RESULTS

General characterization of [3H][Dmt1]DALDA binding

The highest levels of specific [3H][Dmt1]DALDA (0.1 nM) binding were seen at pH 7.4

(Fig. 1) and this pH was used in all experiments. Binding was linear with increasing amounts of

tissue up to 5 mg of wet weight tissue/ml (data not shown). Hence, all subsequent experiments

used 3 mg of wet weight of tissue/ml or less.

Association experiments using calf striatal membranes showed that [3H][Dmt1]DALDA

binding (0.1 nM) reached maximum levels within 60 min (Fig. 2a). The observed half-life of

dissociation (τ1/2) was 16.1 ± 3.3 min. A semi-log plot of the [3H][Dmt1]DALDA dissociation

(Fig. 2b) revealed a linear relationship consistent with the labeling of a single population of

receptors. Saturation binding in striatal membranes under equilibrium conditions showed that

the peptide labeled a single population of receptors affording a KD value of 0.22 ± 0.02 nM and a

Bmax value of 255 ± 14 fmol/mg protein (Fig. 3).

Sensitivity towards ions and nucleotides

Mu opioid receptor binding is modulated by a variety of ions and nucleotides, with the

binding of agonists and antagonists generally affected in opposite ways (Pert et al., 1973;

Childers and Snyder, 1980; Pasternak et al., 1975). In general divalent cations enhance mu

opioid agonist binding, leaving antagonist binding unaffected. Conversely, sodium ions and

guanine nucleotides markedly reduce agonist, but not antagonist, binding.

Compared to [3H]DAMGO binding, [3H][Dmt1]DALDA binding was relatively

insensitive to any of the divalent cations (Fig. 4), although MgCl2 (1 mM) did modestly enhance

binding by approximately 15 % (Fig. 3; P < 0.01). We observed no difference between MgCl2


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and MgSO4 and therefore routinely included MgSO4 (1mM) in all [3H][Dmt1]DALDA binding

assays. Manganese ions potentiate the binding of the mu opioid agonist [3H]dihydromorphine

(Pasternak et al., 1975). Similarly, MnCl2 (1 mM) increased [3H]DAMGO binding. However,

[3H][Dmt1]DALDA binding was unaffected. Similarly, [3H][Dmt1]DALDA binding was not

influenced by CaCl2, despite the modest lowering of [3H]DAMGO binding.

Sodium ions diminish the binding of [3H]dihydromorphine while enhancing the binding

of the antagonist [3H]naloxone (Pert et al., 1973). Guanine nucleotides also selectively diminish

agonist binding. In our studies, we observed a similar decline in [3H]DAMGO binding (Fig. 5).

Yet, neither Na+ ions alone nor the GTP analog Gpp(NH)p alone affected [3H][Dmt1]DALDA

binding to an appreciable degree, although the combination of NaCl (50 mM) and Gpp(NH)p

(0.1 mM) reduced binding by almost 75% (P < 0.01). The sensitivity of [3H][Dmt1]DALDA

binding to the combination is consistent with the established agonist nature of the drug (Childers

and Snyder, 1980; Selley et al., 2000). However, its insensitivity to sodium ions or the GTP

analog alone is somewhat unusual and sets it apart from other mu opioid agonists.

Binding Selectivity Profile and regional distribution of [3H][Dmt1]DALDA and

[3H]DAMGO binding

We next compared the binding selectivity profile of [3H][Dmt1]DALDA to that of

[3H]DAMGO. Full competition curves were generated and Ki values determined for both

radioligands (Table 1). All compounds show similar potencies against both radioligands.

[3H][Dmt1]DALDA binding showed a selectivity typical for mu radioligands, with the traditional

mu opioids DAMGO, morphine, methadone and fentanyl all potently lowering binding as

effectively as against [3H]DAMGO. The delta-selective peptide DPDPE

([D-Pen2,D-Pen5]enkephalin) and the kappa-selective drug U50,488H displayed low affinities,


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consistent with the mu selectivity of the radioligands. None of the Hill slopes for any of the

ligands tested differed significantly from unity.

Functional assessment of [Dmt1]DALDA and DAMGO in [35S]GTPγS binding assays

The efficacy and potency of [Dmt1]DALDA was tested in vitro in an attempt to further

understand the highly potent nature of the compound in vivo. [35S]GTPγS binding was carried

out in calf striatal membranes, whole mouse brain and mouse spinal cord membranes following

stimulation with either [Dmt1]DALDA or DAMGO (Fig. 6). [Dmt1]DALDA (EC50 = 12.2 ± 2.7

nM) was 26-fold more potent than DAMGO (EC50 = 322 ± 21 nM) in calf striatal membranes,

34-fold more potent in mouse spinal cord (EC50 = 10.1 ± 1.3 nM compared to EC50 = 343 ± 49

nM for DAMGO), and 80-fold more potent in whole mouse brain (EC50 = 2.9 ± 0.2 nM

compared to EC50 = 228 ± 32 nM for DAMGO). Despite its greater potency, [Dmt1]DALDA

had a ceiling effect in striatal membranes, implying that it was a partial agonist. This was more

pronounced in the whole brain membrane assays, where the maximal value was only

approximately 75% that of DAMGO stimulation at 10 µM. Only the spinal cord assay revealed

agonist activity for [Dmt1]DALDA that was similar to that of DAMGO.

[Dmt1]DALDA interactions with the cloned MOR-1 Splice Variants

A number of MOR-1 splice variants have recently been isolated and cloned (Pan et al.,

1999; Pan et al., 2000; Pan et al., 2001; Zimprich et al., 1995; Bare et al., 1994). First, we

determined the affinity of [3H][Dmt1]DALDA for a series of murine MOR-1 variants expressed

in CHO cells (Table 2). With the MOR-1 clone, [3H][Dmt1]DALDA was about 3-fold more

potent than [3H]DAMGO in receptor binding assays. Yet, with the MOR-1C and MOR-1D


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clones, the two radioligands were equipotent. Their equivalent potency in the MOR-1C and

MOR-D clones also contrasted with their relative potencies in calf striatal membranes, where

[Dmt1]DALDA was 5- to 10-fold more potent in the competition studies (Table 1).

In saturation studies using membranes from cells transfected with the MOR-1 clone,

[3H][Dmt1]DALDA labeled approximately twice as many receptors as [3H]DAMGO (P < 0.05)

(Table 3). Interestingly, [3H][Dmt1]DALDA labeled the same number of receptors in these

membranes as the opioid antagonist [3H]naloxone, suggesting that [3H][Dmt1]DALDA may label

both agonist and antagonist conformations of the receptor.

We next compared [Dmt1]DALDA and DAMGO in GTPγS binding assays (Table 2).

All [Dmt1]DALDA values were normalized to DAMGO at 10 µM. The potency of DAMGO for

the variants was similar, ranging from about 50-70 nM. In contrast, [Dmt1]DALDA values

ranged over 10-fold among the clones. This was quite unexpected in view of the similar receptor

binding affinities of [Dmt1]DALDA for the clones and the small structural differences among the

variant receptors. The relative potency of [Dmt1]DALDA to DAMGO for each variant, defined

as the ratio of their EC50 values also varied markedly. The MOR-1 cells displayed the greatest

difference, where [Dmt1]DALDA was almost 100-fold more potent than DAMGO (P < 0.05).

However, this may be an over-representation of the difference in efficacy between them since

[Dmt1]DALDA also has a higher binding affinity. To compensate for this, we also calculated

the relative potency of stimulation of the drugs in which the EC50 ratio is divided by the ratio of

the KD values. Although only an estimate, the value takes into consideration differences between

the binding affinities of the drugs for the receptors in assessing their relative potencies in the

stimulation of GTPγS binding. [Dmt1]DALDA still was almost 30-fold more effective in

stimulating GTPγS binding in MOR-1 than DAMGO after taking into consideration the


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differences in binding affinities. We also observed a similar enhanced relative potency for the

other clones, but they were more modest, ranging from only 3.5-fold for MOR-1E to 10.4-fold

for MOR-1D (Table 2). In all cases [Dmt1]DALDA activated GTPγS binding far more potently

than DAMGO. Together, these observations illustrate differences between the two drugs in

their activation of these receptors.


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DISCUSSION

Overall, [3H][Dmt1]DALDA shows high affinity and selectivity for the mu opioid

receptor, as well as a regional distribution typical for mu opioid binding, with highest levels of

binding in the striatum and thalamus. Yet, [Dmt1]DALDA displays unique characteristics in

both receptor and GTPγS binding assays that distinguish it from DAMGO.

Mu opioid receptor binding has been extensively characterized over the past thirty years.

A variety of ions and treatments have documented the ability to distinguish between agonist and

antagonist binding, findings were then generalized to other G-protein coupled receptors. For

example, the ability of sodium ions to selectively enhance antagonist and inhibit agonist binding

was first described with mu opioid receptors (Pert et al., 1973), as were the ability of a number of

divalent cations to selectively enhance agonist binding (Pasternak et al., 1975). Guanine

nucleotides also distinguish between agonist and antagonist binding, lowering the binding of

agonists but not antagonists (Childers and Snyder, 1980).

Our current studies confirmed these effects with [3H]DAMGO. However,

[3H][Dmt1]DALDA binding was different. All of the previous studies with [Dmt1]DALDA

clearly show that the peptide is a mu opioid agonist whose analgesic responses in vivo are

inhibited by the mu-selective antagonist β-funaltrexamine. Although a reduction in specific

[3H][Dmt1]DALDA binding was observed following addition of both NaCl and Gpp(NH)p to the

binding buffer, its insensitivity to sodium ions or Gpp(NH)p alone clearly distinguished it from

most other mu opioid agonists. Nor was it increased to the same degree as [3H]DAMGO by

divalent cations. Although these differences are subtle, they imply differences in the mode of

binding/activation of mu opioid receptors by [Dmt1]DALDA.


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Differences between [Dmt1]DALDA and DAMGO also were observed functionally in the

GTPγS binding studies. [Dmt1]DALDA is far more potent in vivo than DAMGO (Neilan et al.,

2001; Shimoyama et al., 2001; Riba et al., 2002). Thus, it was not surprising that it remained

more potent in the GTPγS binding studies. However, the presence of ceiling effects compared to

DAMGO in both mouse brain and calf striatum imply that it is a partial agonist in these tissues

with an efficacy below that of DAMGO, whereas it retained full agonist activity in the spinal

cord. Although its maximal stimulation varied among the tissues, its EC50 value remained the

same.

The increased spinal cord efficacy observed for [Dmt1]DALDA is consistent with its

extraordinary intrathecal analgesic potency in vivo. However, other factors may also be

important. Synergistic interactions between µ-opioid and α2-agonists at the level of the spinal

cord have been widely reported (Fairbanks et al., 2002; Ossipov et al., 1990; Ossipov et al.,

1989), and in a recent study the α2-adrenergic antagonist yohimbine significantly attenuated

[Dmt1]DALDA-mediated intrathecal analgesia in rats (Shimoyama et al., 2001). Therefore it is

interesting to speculate whether the increased efficacy of [Dmt1]DALDA in spinal cord

membranes is due in part to an interaction with other receptors, such as the α2-receptor.

However, in preliminary studies yohimbine at concentrations as high as 100 nM failed to

compete [3H][Dmt1]DALDA binding, leaving open how these receptors may be interacting.

The Oprm gene, which encodes MOR-1 and its variants, is large (>250 kb) and has a

complex pattern of alternative splicing (Bare et al., 1994; Zimprich et al., 1995; Pan et al., 2000;

Pan et al., 1999; Pan et al., 2001). In vivo and in vitro studies using brain and/or spinal cord

membranes, the overall actions of [Dmt1]DALDA presumably reflect the summation of its

interactions with a variety of splice variants of the mu receptor. Therefore, we also examined the


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effects of [Dmt1]DALDA on a series of variants individually expressed in CHO cells. These

variants differ from MOR-1 itself and each other only at the tip of the intracellular COOH tail of

the receptor. All these variants selectively bind mu opioids with high affinity, but show subtle

differences in affinity for certain compounds across the variants, particularly the endogenous

ligands β-endorphin and dynorphin A (Pan et al., 1999; Pan et al., 2000; Pan et al., 2001). These

variants also differ functionally among themselves. Internalization has been well described for

MOR-1 (Burford et al., 1998; Keith et al., 1998). In these studies, DAMGO, but not morphine,

internalizes MOR-1. When expressed in HEK293 cells, however, MOR-1D and MOR-1E

internalize in response to either DAMGO or morphine treatment, whereas MOR-1 and MOR-1C

only internalize in response to DAMGO treatment and not with morphine (Koch et al., 2001). In

vivo, MOR-1 internalizes in response to DAMGO, but not morphine, whereas MOR-1C

internalizes with both (Abbadie and Pasternak, 2001). The ability of morphine to internalize

MOR-1C in neurons in the brain and not HEK293 cells may be due to a variety of issues, such as

the overexpression of the protein in the HEK293 cells, the different environment of the receptor

and the repertoire of associated proteins.

In binding studies with the splice variants [3H][Dmt1]DALDA displayed modest

differences from DAMGO. Looking at the ratios of KD values for the two radioligands,

MOR-1C and MOR-1D showed virtually no difference in affinity while [3H][Dmt1]DALDA was

almost 4-fold more potent than [3H]DAMGO against MOR-1 itself. The most intriguing

difference between the binding of the two radioligands involved the number of sites each

labeled. G-protein coupled receptors are thought to have both agonist and antagonist

conformations, with agonists labeling only the agonist conformation and antagonists labeling

both. Thus, it was not surprising to see [3H]naloxone labeling about twice as many sites as

[3H]DAMGO in membranes from MOR-1-expressing CHO cells. We did not anticipate seeing


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[3H][Dmt1]DALDA label the same number of receptors as the antagonist [3H]naloxone. This

suggests that [3H][Dmt1]DALDA was labeling both agonist and antagonist states of the receptor.

The ability of [Dmt1]DALDA to label the antagonist conformation of the receptor may help

explain the relative insensitivity of [3H][Dmt1]DALDA binding to either sodium or divalent

cations and to GTPγS and is consistent with its apparent partial agonist actions in the brain

GTPγS binding studies.

The functional actions of [Dmt1]DALDA with the various MOR-1 variants further

illustrated differences among them. In the transfected cell lines, [Dmt1]DALDA appeared to

have full agonist activity, giving maximal responses equivalent to those of DAMGO. The

difference in structure among the variants is limited to the terminal amino acids of the

intracellular COOH tail, far away from the binding pocket formed by the transmembrane

domains. Although prior work from our laboratory has shown subtle differences in affinity of

several endogenous opioid peptides among the variants, most mu drugs show little difference

(Pan et al., 1999; Pan et al., 2000; Pan et al., 2001). Therefore, we anticipated similar affinities

of the variants for [3H][Dmt1]DALDA in receptor binding assays, but we did not expect the

major differences in its stimulation of [35S]GTPγS binding. [Dmt1]DALDA stimulated GTPγS

binding about 10-fold more effectively in the MOR-1 expressing cell membranes than in those

expressing either MOR-1C or MOR-1E. While these studies help with our understanding of

[Dmt1]DALDA actions, they also provide important insights into the variants themselves. As

noted above, the structural differences among them are quite small, being restricted to the

terminal amino acids in the intracellular COOH tail. This variability in response to

[Dmt1]DALDA would suggest that the tip of the COOH tail is important in modulating the


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efficacy of mu opioids, possibly by influencing the association of the receptor with other

proteins.

The relative ability of [Dmt1]DALDA to stimulate [35S]GTPγS binding compared to

DAMGO also was interesting. In all cases [Dmt1]DALDA was more potent. The greatest

difference was seen with MOR-1 itself where the EC50 of [Dmt1]DALDA was almost 100-fold

lower than that of DAMGO. Direct comparisons can be somewhat misleading since

[Dmt1]DALDA has a higher binding affinity than DAMGO for MOR-1 sites. Even after taking

the relative affinity of the drugs to take into consideration, we estimated that [Dmt1]DALDA was

still almost 30-fold more effective than DAMGO in stimulating [35S]GTPγS binding. While

[Dmt1]DALDA also was more active with the other variants, the difference for MOR-1E was

only 3.5-fold. Although these are only rough estimates of the relative potencies of the two drugs,

they do raise the possibility of activation differences for the two drugs and among the various

variants.

The pharmacology of [Dmt1]DALDA is complex and not easily explained. Binding

studies imply that it is a partial agonist and capable of labeling both agonist and antagonist

receptor conformations. Yet, [Dmt1]DALDA activates G-proteins far more potently than

DAMGO, even when taking their relative receptor binding affinities into consideration. Further

studies into the pharmacology of [Dmt1]DALDA may provide valuable insights into the action of

mu analgesics.


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Footnote

1This work was supported, in part, by grants (DA02615 and DA07242) and a Senior Scientist

Award (DA00220) to GWP from the National Institute on Drug Abuse, a core grant from the

National Cancer Institute (CA08748) to MSKCC and a Program Project Grant (P01-DA08924)

to PWS.


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Figure Legends

Fig. 1: Effect of buffer pH on specific [3H][Dmt1]DALDA and [3H]DAMGO binding in calf

striatal membranes.

[3H][Dmt1]DALDA (0.1nM) and DAMGO (1 nM) binding was performed in calf striatal

membranes as described in Materials and Methods at the indicated pH. Results are mean ±

S.E.M of at least three independent determinations.

Fig. 2: Association and dissociation of [3H][Dmt1]DALDA binding in calf striatal membranes.

(a) Association of [3H][Dmt1]DALDA binding (0.1nM) was determined at the stated

times after the addition of radiolabel. (b) Dissociation was initiated by the addition of

[Dmt1]DALDA (1 µM) after letting the binding equilibrate with [3H][Dmt1]DALDA (0.1 nM)

for 45 min and was then filtered at the stated times after addition of 1µM unlabeled. All values

are the means ± S.E.M. of at least three independent replications.

Fig. 3: Saturation studies of [3H][Dmt1]DALDA binding to calf striatal membranes.

Saturation studies were performed using the indicated concentration of

[3H][Dmt1]DALDA. The data shown is from a representative experiment that has been

independently replicated three times. The insert shows a Scatchard transformation. The mean ±

S.E.M. values from the three replications gave a KD value of 0.22 ± 0.02 nM and a Bmax value of

255 ± 14 fmols/mg protein.


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Fig. 4: Effect of divalent cations on specific [3H][Dmt1]DALDA (0.1nM) and [3H]DAMGO (1.0

nM) binding.

Striatal membranes were incubated with radioligand in either 50 mM K2(PO4) buffer

alone (control, normalized to 100%) or with indicated concentration of divalent cation. All

values represent the mean ± S.E.M. *P < 0.05, **P < 0.01, Students t-test.

Fig. 5: Effect of sodium ions and guanine nucleotides on specific [3H][Dmt1]DALDA (0.1 nM)

and [3H]DAMGO (0.1 nM) binding.

Striatal membranes were incubated with radioligand in either 50 mM K2(PO4) buffer

alone (control, normalized to 100%) or with indicated concentration of ion and/or guanine

nucleotide. All values represent the mean ± S.E.M. *P < 0.05, **P < 0.01, Students t-test.

Fig. 6: Stimulation of [35S]GTPγS binding

The binding of [35S]GTPγS was determined in (a) calf striatal membranes, (b) whole

mouse brain membranes and (c) spinal cord membranes. All stimulations were normalized to the

values seen with maximal stimulation with DAMGO (10 µM), which was assigned the value of

100%. EC50 values and normalized maximal stimulations calculated using GraphPad Prism.

Each value represents the mean ± S.E.M for at least three independent determinations.


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Table 1. Selectivity of [3H][Dmt1]DALDA and [3H]DAMGO binding in calf striatal

membranes.

Ki Values (nM)

[3H][Dmt1]DALDA [3H]DAMGO

[Dmt1]DALDA 0.12 ± 0.01 0.14 ± 0.03

DAMGO 1.23 ± 0.43 0.71 ± 0.16

Morphine 2.51 ± 0.37 1.56 ± 0.37

Methadone 5.89 ± 1.22 4.9 ± 0.2

3-Methoxynaltrexone 113 ± 14 132 ± 58

Fentanyl 1.37 ± 0.13 2.25 ± 0.80

M6G 8.84 ± 0.93 15.6 ± 4.2

Naltrexone 0.18 ± 0.03 0.29 ± 0.01

Codeine > 500 > 500

Naloxone benzoylhydrazone 0.12 ± 0.01 0.18 ± 0.02

CTAP 0.64 ± 0.06 0.90 ± 0.16

Diprenorphine 0.13 ± 0.02 0.14 ± 0.07

DPDPE > 500 > 500

U50,488H >400 > 350

Binding was performed with [3H][Dmt1]DALDA (0.1 nM) and [3H]DAMGO (1 nM) as

described in Material and Methods. Competitions were performed with at least three

concentrations of the stated drug. IC50 values were determined and converted to Ki values

according to the Cheng and Prussoff equation (Cheng and Prusoff, 1973). Results are mean ±

S.E.M of at least three independent Ki determinations. Two-way ANOVA reveals significant

differences (P<0.001). The differences among the competitors for each radioligand were

significant, but no significant differences were seen for a specific competitor between the

radioligands.


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Table 2. Receptor binding and stimulation of [35S]GTPγS binding by [Dmt1]DALDA and

DAMGO in CHO cells transfected with MOR-1 splice variants

_ DAMGO _ _ [Dmt1]DALDA _

KD (nM) EC50

(nM)

KD (nM) EC50

(nM)

Stimulation (% DAMGO)

EC50

Ratio Relative

potency of stimulation

MOR-1 0.63 ± 0.03 68 ± 4 0.19 ± 0.06 0.7 ± 0.2 120 ± 15 97 29

MOR-1C 0.57 ± 0.14 62 ± 4 0.51 ± 0.14 7.2 ± 2.9 90 ± 9 9 7.7

MOR-1D 0.35 ± 0.07 62 ± 6 0.34 ± 0.18 5.8 ± 1.6 96 ± 9 11 10.4

MOR-1E 0.74 ± 0.11 48 ± 4 0.41 ± 0.19 7.6 ± 2.5 110 ± 23 6 3.5

MOR-1F 1.04 ± 0.04 50 ± 6 0.44 ± 0.15 2.8 ± 0.6 79 ± 12 18 7.6

KD values for [3H][Dmt1]DALDA were determined using saturation analysis on

membranes prepared from CHO cells stably transfected with the indicated clone. Each value

represents the mean ± S.E.M of three independent replications. The KD values for [3H]DAMGO

are from the literature (Pan et al., 1999)(Pan et al., 2000), with the exception of MOR-1 which

was assayed in the same membranes [3H][Dmt1]DALDA. Stimulation of [35S]GTPγS binding by

[Dmt1]DALDA was performed as described in Materials and Methods on membranes prepared

from CHO cells stably transfected with the indicated clones. Values for DAMGO were from the

literature (E.A. Bolan and G.W. Pasternak, submitted). All stimulations were normalized to

DAMGO (10 µM), which was assigned the value of 100%. The stimulation by DAMGO at 10

µM was 180% of basal levels in the spinal cord and 150% basal levels in brain. Values in the

transfected cell lines varied from 135% to 240% basal levels. This variation may be due to the

different levels of expression from line to line. EC50 values and normalized maximal

stimulations calculated using GraphPad Prism. Each value represents the mean ± S.E.M for at

least three independent determinations. The EC50 Ratio represents the ratio between the two

drugs. The Relatively Potency of Stimulation normalizes the EC50 value for differences in

affinity in binding assays by dividing the EC50 Ratio by the ratio of the KD values).


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Table 3. [3H][Dmt1]DALDA, [3H]DAMGO, and [3H]naloxone binding in MOR-1 transfected

cells

Bmax value (fmol/mg protein)

[3H][Dmt1]DALDA 142 ± 26*

[3H]DAMGO 62 ± 17

[3H]naloxone 134 ± 28*

Bmax values were obtained from saturation studies using CHO cells stably transfected

with MOR-1. The same preparation of cell membranes was used for each experiment. Values

represent the mean ± S.E.M for three independent experiments, each performed in triplicate.


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5 6 7 8 9 100

500

1000

1500

2000 [3H][Dmt1]DALDA[3H]DAMGO

pH

[3 H][

Dm

t1 ]DA

LD

Abi

ndin

g (c

pm)

Figure 1


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0 60 120 1800

1000

2000

3000

4000AssociationA

Time (mins)

[3 H][

Dm

t1 ]DA

LD

Abi

ndin

g (c

pm)

0 30 60 90 120100

1000

10000B

Time (mins)

Spec

ific

[D

mt1 ]D

AL

DA

bind

ing

(cpm

)

Figure 2


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0.0 0.1 0.2 0.3 0.4 0.50

50

100

150

200

[Dmt1]DALDA (nM)

Bou

nd (f

mol

s/m

g)

0 100 200 3000.0

0.5

1.0

1.5

B

Bou

nd/F

ree

Figure 3


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0.1 1 10

80

100

120

140

160*

*

*

[3H]DAMGO[3H][Dmt1]DALDA

*

**

*

*

*

**

1 1

CaCl2 MnCl2 MgCl2 (mM)

Spec

ific

Bin

ding

(% o

f con

trol

)

Figure 4


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10 100 1000

25

50

75

100

*

** **

NaCl(50 mM)

**

+

B

0

Gpp(NH)p (µ M)

Spec

ific

Bin

ding

(% o

f con

trol

)

10 1000

25

50

75

100

*

*

*[3H][Dmt1]DALDA

[3H]DAMGO

A

0

NaCl (mM)

Spec

ific

Bin

ding

(% o

f con

trol

)

Figure 5


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[Dmt1]DALDA

DAMGO

-10 -9 -8 -7 -6 -50

25

50

75

100

A

log[Drug] (M)

-9 -8 -7 -6 -50

25

50

75

100

B

log[Drug] (M)

-11 -10 -9 -8 -7 -6 -50

25

50

75

100

C

log[Drug] (M)

Per

cent

Max

imal

DA

MG

O S

tim

ulat

ioin

Figure 6


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