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J Biol Chem, Vol. 274, Issue 31, 22081-22088, July 30, 1999

Calmodulin Binding to G Protein-coupling Domain of Opioid Receptors^*

Danxin Wang, Wolfgang Sadée^§, and J. Mark Quillan

From the Departments of Biopharmaceutical Sciences and Pharmaceutical Chemistry and the Center for the Neurobiology of Drug Addiction, University of California San Francisco, San Francisco, California 94143-0446

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The ubiquitous intracellular Ca²⁺ sensor calmodulin (CaM) regulates numerous proteins involved in cellular signaling of G protein-coupled receptors, but most known interactions between GPCRs and CaM occur downstream of the receptor. Using a sequence-based motif search, we have identified the third intracellular loop of the opioid receptor family as a possible direct contact point for interaction with CaM, in addition to its established role in G protein activation. Peptides derived from the third intracellular loop of the µ-opioid (OP₃) receptor strongly bound CaM and were able to reduce binding interactions observed between CaM and immunopurified OP₃ receptor. Functionally, CaM reduced basal and agonist-stimulated ³⁵S-labeled guanosine 5'-3-O-(thio)triphosphate incorporation, a measure of G protein activation, in membranes containing recombinant OP₃ receptor. Changes in CaM membrane levels as a result of overexpression or antisense CaM suppression inversely affected basal and agonist-induced G protein activation. The ability of CaM to abolish high affinity binding sites of an agonist at OP₃ further supports the hypothesis of a direct interaction between CaM and opioid receptors. An OP₃ receptor mutant with a Lys²⁷³  Ala substitution (K273A-OP₃), an amino acid predicted to play a critical role in CaM binding based on motif structure, was found to be unaffected by changes in CaM levels but coupled more efficiently to G proteins than the wild-type receptor. Stimulation of both the OP₁ (-opioid) and OP₃ wild-type receptors, but not the K273A-OP₃ mutant, induced release of CaM from the plasma membrane. These results suggest that CaM directly competes with G proteins for binding to opioid receptors and that CaM may itself serve as an independent second messenger molecule that is released upon receptor stimulation.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Calmodulin (CaM)¹ is a ubiquitous Ca²⁺-sensitive regulatory protein found in virtually every class of living organism from plants to alga, from humans to protozoa (1). Originally identified as the Ca²⁺-dependent factor responsible for activating 3',5'-cyclic-nucleotide phosphodiesterase (2-5), the role of CaM as a regulator of cytoplasmic enzymes has broadened to include adenylate cyclases, Ca²⁺/CaM-dependent kinases and phosphatases, ion channels, Ca²⁺-ATPases, and others (1, 6, 7). Mapping relationships between the multitude of enzymes regulated by CaM is essential to the understanding of biochemical cascades linked to activation of G protein-coupled receptors (GPCRs).

While previous studies have revealed broad involvement of CaM in downstream signaling pathways initiated by G protein-coupled receptors, progress is also being made toward uncovering interactions more proximal to the receptor. Recent examples include the discovery of a CaM binding site on a G protein -subunit (8) and another report that describes a CaM binding site on the metabotropic glutamate subtype 5 receptor (9). The CaM binding domain found on the metabotropic glutamate subtype 5 receptor is located in the extended C-terminal region and appears unique to this receptor. Whereas core regions of the receptor involved in G protein coupling have not been previously implicated in CaM interactions, circumstantial evidence suggests such an involvement of CaM at the receptor level. The ability of certain CaM-binding peptides, like mastoparan and mellitin, to activate G as well, in essence substituting for the receptor (10-12), leads to suspicion that GPCRs may contain core regions of structural similarities to CaM-binding domains.

In this report, we identify an i3 loop region within receptors of the opioid family, in particular OP₁ and OP₃, as a potential site for CaM interaction. We also demonstrate that CaM inversely affects G protein coupling by OP₁ and OP₃ and propose that CaM itself could function as an independent second messenger molecule.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Synthesis of OP₃ Receptor i3 Loop Peptides-- Peptides corresponding to sections of the i3 loop of the OP₃ receptor (see Table I) were prepared on an Applied Biosystems 433A automated peptide synthesizer (Foster City, CA) using para-methylbenzylhydramine resin and standard Fmoc (N-(9-fluorenyl)methoxycarbonyl) solid phase methods. Peptides were purified on a Dynamax SD-200 high pressure liquid chromatograph (Varian Chromatography Systems, Walnut Creek, CA) using a reverse-phase C-18 column and eluting across a gradient of 10-50% acetonitrile in water containing 0.1% (v/v) trifluoroacetic acid.

Cell Culture and Transfections-- HEK293 cells were maintained at 37 °C and under 5% CO₂ in DME/H16/F12 medium supplemented with 10% fetal calf serum, 100 µg/ml streptomycin, and 100 IU/ml penicillin. Stably transfected HEK293 cell lines were established as described by Arden et al. (13) containing the following opioid receptor subtypes: (i) the rat µ-opioid receptor (rOP₃) (14); (ii) an N-terminally FLAG-labeled mouse µ-opioid receptor (FLAG-mOP₃) (15); (iii) a mouse -opioid receptor (mOP₁) (16, 17); (iv) an N-terminal FLAG-labeled human µ-opioid receptor (FLAG-hOP₃), and (v) a Lys²⁷³  Ala FLAG-hOP₃ mutant receptor (K273A-OP₃) (described below). Cell lines established with cDNA encoding mouse, rat, and human OP₃ receptors (HEK-mOP₃, HEK-rOP₃, and HEK-hOP₃, respectively) were used interchangeably, since their i3 loop sequences are identical. The receptor content of the HEK-OP₁ and HEK-OP₃ cell lines was approximately 1-2 pmol/mg of total protein determined by [³H]diprenorphine (2 nM) binding in intact whole cell monolayers as described by Arden et al. (13).

Site-directed Mutagenesis of the hOP₃ Receptor-- The FLAG epitope was attached to N terminus of the hOP₃ receptor (18) using Pfu DNA polymerase (Stratagene, La Jolla, CA) and the oligonucleotides 5'-GCTCTAGAGCTTAGGGCAACGGAGCAGTTTC-3' and 5'-CCCAAGCTTGGGATGGACTACAAGGACGATGATGACAAGGACAGCAGCGCTGCCCCCACGAAC-3'. The amplified PCR product was subcloned into the pcDNA3 (Invitrogen, Carlsbad, CA), using HindIII and XbaI. Mutation of Lys²⁷³ to Ala in the i3 loop region of the FLAG-hOP₃ receptor was accomplished using the QuikChange^TM site-directed mutagenesis kit (Stratagene, La Jolla, CA) and confirmed by DNA sequencing (Biomolecular Resource Center, University of California San Francisco).

Gel Mobility Shift Assays-- Gel mobility shift assays involving CaM and test peptides were performed using 12% nondenaturing polyacrylamide gel electrophoresis essentially as described by Liu et al. (19). Briefly, 60 µM of purified CaM (Calbiochem) was incubated for 30 min at room temperature with test peptides dissolved in 10 mM HEPES, pH 7.0, containing 1 mM CaCl₂ or 2 mM EGTA. Proteins were visualized with Coomassie Brilliant Blue staining.

Measurement of Peptide-CaM Interactions Using Fluorescence Assays-- Dansylation of CaM was performed as described by Kincaid et al. (20). Fluorescence emission spectra of dansylated CaM (dansyl-CaM) were measured from 400 to 600 nm using a Fluorolog Series 2 spectrofluorometer (SPEX, Edison, NJ) and an excitation wavelength of 340 nm. Test peptides were added to 0.6 µM dansyl-CaM in the absence or presence of 200 µM Ca²⁺ in 10 mM HEPES pH 7.0 buffer.

Interactions between i3 loop peptides and dansyl-CaM were also examined indirectly by measuring test peptide-induced changes in fluorescence intensity of dansyl-CaM complexed with Ca²⁺/CaM-KII-(290-309) (21), in a Fluostar '97 fluorimeter, equipped with microinjectors (BMG Lab Technologies, Durham NC). Samples were divided evenly into solid white 96-well microtiter plates (135 µl/well) containing 150 nM dansyl-CaM, 100 nM Ca²⁺/CaM-KII-(290-309), and test peptide. Fluorescence intensity was recorded at 100-ms intervals using an excitation wavelength of 340 nM and emission of 490 nm. Ten consecutive steady-state base-line observations were averaged for each peptide using Ca²⁺-free buffer (10 mM HEPES, pH 7.0, 100 µM EGTA), and replicates of each averaged value were combined. Concentration-response curves were plotted using a logistic curve-fitting equation as described by De Lean et al. (21). Subsequently, the effect of adding Ca²⁺ was determined on the competition between test peptides, Ca²⁺/CaM-KII-(290-309), and dansyl-CaM. Using the same starting conditions outlined above, base-line measurements were then followed by an injection of 15 µl of a 0.11 mM CaCl₂ solution yielding a final free [Ca²⁺] of ~10 µM in 150 µl. Changes in fluorescence intensity were monitored at 100-ms intervals for 18 s. In the absence of dansyl-CaM, no fluorescence was observed under any condition. In the presence of dansyl-CaM alone, Ca²⁺ injection caused a reproducible increase in fluorescence that reached maximum intensity within 200 ms and then remained at that new plateau level. When a mixture of Ca²⁺/CaM-KII-(290-309) and dansyl-CaM was injected with Ca²⁺ (in the absence of test peptides), there was an increase in fluorescence intensity that peaked within 200 ms, followed by an exponential decay in fluorescence intensity, which reflects the kinetic interaction of Ca²⁺/CaM-KII-(290-309) with dansyl-CaM in its altered Ca²⁺-bound form. The ability of peptides to inhibit the decay in fluorescence intensity was used as an indication of the test peptide's ability to disrupt formation of this new complex between dansyl-CaM and Ca²⁺/CaM-KII-(290-309), providing an indirect measure of relative binding potency in the presence of Ca²⁺ and plotted as percentage change in fluorescence intensity (F_f/F_i × 100), where F_i is the average fluorescence intensity between 200 and 700 ms after injection of Ca²⁺ and F_f is the average fluorescence intensity between 15.8 and 17.7 s after injection.

Binding of Purified FLAG-mOP₃ Receptor to CaM Affinity Gels-- HEK-FLAG-mOP₃ cells or pRC/CMV empty vector-transfected HEK293 control cells were homogenized in phosphate-buffered saline (PBS; 1 mM KH₂PO₄, 10 mM NaHPO₄, 137 mM NaCl, 2.7 mM KCl) and centrifuged at 30,000 × g for 20 min, and the membrane pellet was solubilized for 30 min at 4 °C with 1% NP₄₀ in 20 mM Tris-HCl, 100 mM NaCl, pH 7.5 (Buffer A), containing a protease inhibitor mixture (Roche Molecular Biochemicals). This mixture was then centrifuged at 50,000 × g for 40 min at 4 °C, and the supernatant was concentrated to its volume using an Ultrafree centrifugal filter with a nominal molecular weight limit of 50 kDa. Concentrated supernatant was diluted 10-fold in buffer A to reduce the concentration of NP₄₀ to 0.1%, and solubilized material was incubated with volume of anti-FLAG mAb (M2) affinity gel (Eastman Kodak Co.) for 1 h at 4 °C with gentle agitation. The gel was then washed with buffer A plus 0.1% NP₄₀, and FLAG-mOP₃ receptor was eluted by adding 400 µM FLAG peptide and incubating at room temperature for 30 min. FLAG peptide was removed by three cycles of concentration and dilution. Immunopurified FLAG-mOP₃ receptor was then incubated with 20 µl of immobilized bovine CaM affinity gel (Calbiochem) for 1 h at 4 °C in buffer A plus 0.1% NP₄₀ and 1 mM CaCl₂. The resin was pelleted and washed three times with buffer A plus 0.1% NP₄₀ and 1 mM CaCl₂ for 10 min at 4 °C. Samples were then eluted by incubating with 20 µl of Laemmli sample buffer at 65 °C for 5 min. FLAG-mOP₃ receptor was separated on 8% SDS-PAGE and detected by Western blot using rabbit anti-OP₃ receptor C-terminal antiserum (Incstar, Stillwater, MN), followed by an AP color development kit (Bio-Rad).

Binding of Biotinylated CaM to FLAG-mOP₃ Receptor-- HEK-FLAG-mOP₃ cells or pRC/CMV vector-transfected HEK cells were solubilized in PBS, pH 7.4, containing 1% NP₄₀ and a protease inhibitor mixture. The reaction mixture was centrifuged at 50,000 × g for 40 min at 4 °C. The supernatant was then gently agitated for 9 h at 4 °C with 3 µg of anti-FLAG mAb (M2) (Sigma) and 75 µl of a 20% slurry of washed protein G-Sepharose beads (Amersham Pharmacia Biotech); total volume was 0.7 ml. Beads were pelleted by centrifugation, washed three times with PBS plus 0.1% NP₄₀, and gently mixed with 2.2 µg of biotinylated CaM (Calbiochem) in 300 µl of PBS and 0.1% NP₄₀ for 2 h at 4 °C, either in the presence of 1 mM CaCl₂ or 2 mM EGTA. Beads were then washed with PBS containing 0.1% NP₄₀ (with CaCl₂ or EGTA), dissolved in 20 µl of Laemmli sample buffer, and analyzed on 10% SDS-polyacrylamide gel electrophoresis. Biotinylated CaM was detected using an AP-conjugated avidin and an AP color development kit.

Expression of Sense and Antisense CaM Vectors in HEK-rOP₃ Cells-- cDNA encoding chicken type II CaM (from Dr. Anthony R. Means of Duke University (22)) was subcloned into pcDNA3, Zeo(+)/pcDNA3 (sense CaM), and Zeo()/pcDNA3 (antisense CaM) using BamHI and XbaI. (Amino acid sequences of chicken and mammalian CaMs are identical.) Transfection of HEK-rOP₃ cells was accomplished by using Superfect reagent (Qiagen, Santa Clarita, CA). The sense CaM-pcDNA3 construct was used for transient expression, and cells were harvested after 36 h. Stable cell lines were established with CaM-Zeo(+)/pcDNA3 (sense) or CaM-Zeo()/pcDNA3 (antisense) plasmids with 400 µg/ml Zeocin^TM (Invitrogen, Carlsbad, CA) for initial selection and then lowering the concentration of Zeocin^TM to 100 µg/ml once established. Expression levels of rOP₃ receptor, determined with [³H]diprenorphine as described earlier, were not measurably altered by changes in CaM expression.

Measurement of CaM Levels in Plasma Membranes-- Plasma membranes were prepared as described by Nehmad et al. (23). CaM was then extracted from the plasma membrane fractions, containing approximately 70% of total [³H]naloxone binding sites, and CaM levels were determined by measuring phosphodiesterase activity using [³H]cAMP as described previously (24). Standard curves were generated with known quantities of purified CaM (Sigma), with correlation coefficients ranging between 0.95 and 0.98. CaM recovery over the assay procedure was 65-68%. Results were confirmed by Western blot analysis.

[³⁵S]GTPS Binding Studies-- Membranes for [³⁵S]GTPS binding assays were prepared from cells washed with PBS and then homogenized in 40 mM Tris-HCl buffer, pH 8.0, in the absence (control membranes) or presence of 2 mM EGTA (EGTA-washed membranes), incubated on ice for 15 min, and centrifuged at 30,000 × g for 20 min at 4 °C. Thus, washing in pH 8/EGTA buffer serves to reduce the CaM content of the membranes (23). Pellets were then resuspended briefly in 10 mM HEPES buffer, pH 7.4, centrifuged, and used for the [³⁵S]GTPS binding assay as described by Burford et al. (25).

[³H]Naloxone Binding Experiments-- Membranes for binding studies, prepared as described above for [³⁵S]GTPS binding assays, were suspended in 10 mM HEPES buffer containing 10 mM MgCl₂ and 100 mM NaCl, pH 7.4. Morphine (final concentrations ranging from 0 to 10 µM) and 1 nM [³H]naloxone (specific activity 69 Ci/mmol; Amersham Pharmacia Biotech) were added to each membrane sample (containing ~20 µg of protein/sample), incubated for 2 h at room temperature, filtered, and washed at 4 °C, and filter-bound [³H]naloxone was determined by scintillation counting (13). Where indicated, membranes were preincubated for 30 min at room temperature with 60 µM CaM, and then diluted 4 times before [³H]naloxone binding was determined. Binding curves were analyzed using one- or two-site models by GraphPad Prism (San Diego, CA).

cAMP Measurements-- cAMP measurements were performed by radioimmunoassay as described elsewhere (13). Cell suspensions were incubated with 10 µM forskolin for 10 min at 37 °C in the presence of various concentrations of morphine (0-1 µM).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

CaM-binding Motif Search-- Binding of CaM to CaM-binding proteins does not involve a universal, conserved sequence motif (26); therefore, it remains difficult to define CaM binding domains in distinct protein families using sequence alignment data. To identify possible CaM binding domains in GPCRs, we used a series of sequence motifs derived from distinct protein families with known CaM binding domains for a PatScan (27) pattern search of the SwissProt protein data base. Scanning for possible binding motifs produced matches within a number of known CaM-binding proteins and a limited number of additional proteins, including GPCR sequences located in the i3 loop. Among these, the opioid receptors emerged with several motif searches showing similarities to myosin light chain kinases, Ca²⁺/CaM kinases, and phosphodiesterases. Shown in Fig. 1 are representative alignments, with key motif residues underlined, suggesting the possibility that the i3 loop might interact not only with G proteins (10, 19, 28, 29) but also with CaM.

View larger version (38K): [in this window] [in a new window]   Fig. 1.   Sequence alignments between known CaM binding domains and i3 loop sequences of GPCRs. Alignments were identified from pattern scan searches of the Swiss-Prot data base. Recurrent alignments at the same position are underlined: Ser/Thr; Arg/Lys; Val. Additional similarities in the motif structures include the location of lipophilic amino acids (e.g. Val, Leu, Ile, and Met) interspersed with positively charged residues. Sequences shown include phosphodiesterase (CN1B); Ca2+/CaM kinases (KCC); myosin light chain kinases (KML); opioid receptors (OPR); an orphan receptor (GPR2); and adrenergic receptor (A1AA) (Swiss-Prot nomenclature as follows: OPRD, OP1; OPRK, OP2; OPRM, OP3; OPRX, orphanin FQ/nociceptin receptor).

Binding of OP₃ Receptor-derived Peptides to CaM-- The proposed site for interaction with CaM in the i3 loop of opioid receptor was first examined by observing mobility shifts of CaM using nondenaturing gel electrophoresis (8). CaM was incubated in varying molar ratios with selected peptides derived from the sequence of the OP₃ receptor i3 loop (Table I; Fig. 2A). Three peptides containing the C-terminal portion of the i3 loop predicted to interact with CaM, namely i3-1, i3-2, and i3-3, caused a notable shift in the electrophoretic mobility of CaM, whereas the N-terminal peptide i3-4 did not (Fig. 2A). The small gel shift caused by the low mass C-terminal i3-1 peptide is better visualized in Fig. 2B. These results indicate that the C-terminal portion of the OP₃ receptor i3 loop is a suitable candidate for interaction with CaM. Mobility shifts were not observed in the absence of Ca²⁺ (not shown), suggesting that peptide-CaM interactions are strengthened in the presence of Ca²⁺.

View larger version (47K): [in this window] [in a new window]   Fig. 2.   Binding of OP3 receptor i3 loop-derived peptides to CaM using a nondenaturing gel shift assay. Calmodulin was incubated with peptides derived from the OP3 receptor i3 loop in molar ratios of 1:1, 1:10, and 1:100 (CaM/peptide as indicated) in the presence of Ca2+ and separated by nondenaturing polyacrylamide gel electrophoresis including Ca2+. The addition of EGTA to chelate Ca2+ suppressed the gel shift (not shown). For peptide designations see Table I. A, lane 1, CaM alone; lanes 2-13, CaM plus the indicated peptides, i3-1, -2, -3, and -4. B, lane 1, CaM alone; lanes 2-4, CaM plus peptides i3-1, -5, and -6.

In contrast to mobility shifts observed with Ca²⁺/CaM kinase II-(290-309) or -(281-309), which were complete at molar ratios of 1:1 (Table I), shifts in CaM mobility caused by i3 loop peptides were incomplete even at substantially higher ratios. The i3-2 peptide, which lacks the N-terminal portion of the i3 loop, for example, produced a visible gel shift only at a 100-fold excess over CaM (Fig. 2A). The inability of i3 loop peptides to cause a complete shift in CaM under these conditions is similar to results reported for peptides derived from the CaM binding region of the metabotropic glutamate subtype 5 receptor (9).

Since motif analysis indicated that Lys²⁷³ of the OP₃ receptor may be important for binding interaction with CaM, two modified i3-1 peptides (one in which the N-terminal Lys is removed (i3-5) and another in which Lys is replaced with Ala (i3-6)) were also synthesized and tested (Fig. 2B, Table I). Both of these modified peptides failed to induce mobility shifts of CaM, demonstrating that modifications to this critical residue can affect the ability of i3 loop peptides to interact with CaM.

Shifts in the mobility of Ca²⁺/CaM kinase II peptides reflect the strong interaction that occurs with CaM, consistent with previous reports (30). In contrast, several peptides representing mostly the autophosphorylation substrate domain of Ca²⁺/CaM kinases failed to shift CaM. Moreover, autocamtide-2 and mastoparan, two peptides with sequence motifs and predicted secondary structures resembling those of CaM binding domains also failed to shift the CaM band (Table I), although mastoparan is known to bind to CaM with high affinity when determined by fluorescence shifts of dansylated CaM (11). This suggests that unlike the i3 loop peptides, binding stability of mastoparan to CaM appears insufficient to induce a detectable shift in this gel assay.

Interaction of i3 Loop Peptides with Dansyl-CaM-- CaM binding of i3 loop peptides was also examined by measuring changes in the dansyl-CaM fluorescence spectrum (20). The i3-3 peptide, which represents the amino acid sequence of the entire OP₃ receptor i3 loop, caused the largest change of dansyl-CaM fluorescence among the i3 loop peptides tested, and changes in fluorescence intensity occurred both in the presence and absence of Ca²⁺ (Fig. 3A). This suggests that i3-3 is capable of inducing a substantial conformational change in CaM (12) and that its interaction is at least partially Ca²⁺-independent. Each of the other i3 loop peptides was also able to induce detectable, although smaller, changes in dansyl-CaM fluorescence at higher concentrations, although some of these peptides failed to induce a CaM gel shift, and no response was observed with the control FLAG peptide.

View larger version (36K): [in this window] [in a new window]   Fig. 3.   Emission fluorescence spectra of dansyl-CaM. A, emission fluorescence of dansyl-CaM (0.6 µM) in 10 mM HEPES (pH 7.0) buffer, in the absence and presence of 200 µM Ca2+, respectively, was determined with and without the i3 loop peptide i3-3 added (2 µM). Excitation was at 340 nm. The i3-3 peptide alone did not afford measurable fluorescence at 400-600 nm. B, C, and D, competition between Ca2+/CaM Kinase II-(290-309) peptide (100 nM) and various concentrations of i3 loop-derived peptides for binding to dansyl-CaM (150 nM), measured by fluorescence excitation/emission at 340/490 nm in a fluorescence plate reader. B, in the presence of 10 µM free Ca2+; C and D, in the absence of Ca2+.

To estimate the relative potency of the i3 loop peptides, changes in fluorescence intensity were measured when Ca²⁺/CaM kinase II-(290-309) is complexed with dansyl-CaM, an interaction that has an approximate binding K_d of 50 nM (30), in the presence of test peptides. In the presence of Ca²⁺, i3 loop peptides altered changes in fluorescence intensity that were dose-dependent and of different magnitude. The entire i3 loop peptide, i3-3, was most potent and efficacious with a relative IC₅₀ of 42 (S.D. = 3) nM followed in rank order by i3-1, i3-2, and then (of similar low efficacy) i3-4, -5, and -6 (Fig. 3B; curves for i3-1, -3, -4, 5, and -6 shown). A similar rank order of relative potency was obtained in the absence of Ca²⁺ (Fig. 3, C and D; curves for i3-1 and i3-6, i3-3, and i3-4 shown). Thus, substituting or deleting Lys (Lys²⁷³ of the OP₃ receptor) in the i3 loop reduced the interaction of the C-terminal peptides with CaM. The relative affinity of i3-3 appeared to be lower in the absence of Ca²⁺ (IC₅₀ ~300 nM), indicating that Ca²⁺ enhances CaM binding. The binding curve of the i3-3 peptide appeared to be biphasic (Fig. 3D), suggesting that more than one binding interaction may be involved.

CaM Binding to Solubilized OP₃ Receptor-- FLAG-mOP₃ receptor, immunopurified using FLAG monoclonal antibody and analyzed by Western blotting with OP₃ receptor antiserum, was visible as a main band at ~65 kDa, a band absent in mock-transfected cell extracts (Fig. 4, A, B, and C). In the presence of Ca²⁺, FLAG-mOP₃ receptor was retained on CaM affinity gels but not control gels (Fig. 4A). (Binding in the absence of Ca²⁺ with EGTA or BAPTA added could not be tested because of excessive nonspecific background binding to control gels.) C-terminal i3 loop peptides, i3-1, -2, and -3, reduced receptor binding observed to CaM affinity gels, whereas the N-terminal peptide, i3-4, did not (Fig. 4, A and B). A reduction in binding was also observed with Ca²⁺/CaM-II-(290-309) but not with the control FLAG peptide (Fig. 4A). The Lys²⁷³ truncated peptide, i3-5, and the K273A peptide, i3-6, were less effective at inhibiting OP₃ receptor binding to CaM gels (Fig. 4C). These results indicate, therefore, that OP₃ receptor binding to CaM is mediated predominantly by the C-terminal portion of the i3 loop, and this parallels the results observed in gel shift assays.

View larger version (47K): [in this window] [in a new window]   Fig. 4.   Binding of CaM to OP3 receptor. A, B, and C, FLAG-mOP3 binding to a CaM affinity gel and inhibition by i3 loop peptides. Immunopurified FLAG-mOP3 was detected by Western blot using anti-OP3 receptor C-terminal antiserum. A, lane 1, FLAG-mOP3 receptor; lane 2, FLAG-mOP3 receptor bound to the CaM gel; lanes 3-8, 50 µM concentration of the indicated peptides added; lane 9, FLAG-mOP3 receptor binding to a control gel lacking CaM. B, lane 1, FLAG-mOP3 receptor bound to the CaM gel; lane 2, i3-3 peptide (150 µM) added; lane 3, HEK293 vector transfected control cell extracts treated identically. C, lane 1, FLAG-mOP3 receptor bound to the CaM gel; lanes 2-4, the indicated peptides (100 µM) were added. Similar results were obtained in two additional experiments. D, binding of biotinylated CaM to FLAG-mOP3 receptor. Solubilized FLAG-mOP3 receptor was absorbed onto G-Sepharose beads using anti-FLAG antibody and incubated with biotinylated CaM (0.5 µM), which was eluted and detected by Western blot using AP-avidin. Lane 1, pRC/CMV vector-transfected HEK293 cells as a control, in the presence of 1 mM Ca2+; lane 2, binding of biotinylated-CaM to FLAG-mOP3 receptor in the absence of Ca2+ (plus 2 mM EGTA); lane 3, binding of biotinylated CaM to FLAG-mOP3 receptor in the presence of 1 mM Ca2+. This experiment was repeated twice with the same results.

Binding of CaM to OP₃ receptor was also tested using solubilized FLAG-mOP₃ receptor, which was immunoabsorbed to Sepharose G beads with an anti-FLAG monoclonal antibody. Neither CaM nor the G_i3 subunit of heterotrimeric G proteins (which is preferentially activated by OP₃ receptor (25)) was detectable by Western blot analysis when extracts of HEK-FLAG-mOP₃ cells were applied (data not shown). This suggests that if these proteins were associated with OP₃ receptor in the intact cells, they had dissociated during the solubilization process. On the other hand, externally added biotinylated CaM did bind to immobilized FLAG-mOP₃ receptor in the presence of Ca²⁺ (Fig. 4D). In contrast, CaM was not recovered when using extracts from vector-transfected cells treated in the same fashion. Moreover, in the absence of Ca²⁺, CaM was not retained by FLAG-rOP₃ receptor, indicating that the binding is Ca²⁺-sensitive (Fig. 4D).

CaM Blocks G Protein Activation by i3 Loop Peptide i3-3-- Peptides derived from both the N and C terminus of the i3 loop of various GPCRs have been shown to activate G proteins directly (29). Therefore, the ability of peptides derived from the i3 loop of OP₃ receptor to stimulate [³⁵S]GTPS binding to membranes of HEK-rOP₃ cells was examined. Each of the peptides tested, i3-1, -2, -3, and -4 (30 µM each), caused a significant 10-20% increase in [³⁵S]GTPS binding to membranes. The full-length i3 loop peptide, i3-3, produced the largest effect of 22% (SD = 2%, n = 6, p < 0.001, Student's t test, unpaired). The addition of an equimolar CaM concentration completely blocked G protein activation by i3-3, reducing [³⁵S]GTPS incorporation to the same level observed with CaM alone (~93% of control). This result indicates that CaM binding to i3-3 prevents G protein activation.

Effects of CaM on Opioid Receptor-G Protein Coupling-- The effect of CaM on G protein activation was examined in membranes prepared from HEK-mOP₁ (-opioid) and HEK-rOP₃ (µ-opioid) cells. Washing in Ca²⁺-free pH 8/EGTA buffer reduced CaM levels in plasma membranes (expressed as CaM activity/mg of protein) to 58% (S.D. = 14%, n = 9, p < 0.001, t test, unpaired) of its original level. [³⁵S]GTPS binding activity of washed membranes, on the other hand, was significantly elevated over unwashed controls (Fig. 5). The increase in [³⁵S]GTPS binding activity (of washed versus unwashed membranes) was observed in both absence (basal activity) and presence of agonists (OP₁ and OP₃ membranes stimulated with 1 µM DPDPE and morphine, respectively) (Fig. 5). Washing membranes containing OP₃ receptors, therefore, caused an increase in both basal and stimulated levels of [³⁵S]GTPS incorporation that is reflected in an upward shift of concentration-response curves illustrated in Fig. 6. Both basal and stimulated increases in [³⁵S]GTPS binding activity were abolished by pertussis toxin pretreatment in opioid receptor-containing membranes, and no change in activity was observed in mock-transfected (pRC/CMV) membranes under any of the conditions described above (Fig. 5). This suggests that CaM may interfere with or reduce G protein coupling in membranes containing OP₁ and OP₃ receptors. Furthermore, elevated [³⁵S]GTPS binding activity is observed in membranes containing either opioid receptor when compared with mock-transfected control under agonist-free conditions (Fig. 5). This enhanced activity was abolished by pertussis toxin, indicating that it reflects an increase in basal activity caused by the presence of opioid receptors.

View larger version (34K): [in this window] [in a new window]   Fig. 5.   Effect of CaM washout on basal and agonist-stimulated [35S]GTPS binding to HEK-rOP3 and HEK-mOP1 cell membranes. Cells were homogenized in 40 mM Tris buffer (pH 8.0) either without EGTA (control) or with EGTA (EGTA wash or W). Some cells were also pretreated with pertussis toxin (100 nM for 18 h) (PTX pre) before membrane preparation. [35S]GTPS binding to membranes was determined in the presence of 1 µM GDP, with or without 10 µM morphine or 10 µM DPDPE. HEK-Vec, vector mock-transfected control cells. Values are means ± S.D., n = 6. **, p < 0.01 versus HEK-Vec control; ##, p < 0.01 versus control in the same cell membrane without EGTA wash (unpaired two-tailed Student's t test).

View larger version (34K): [in this window] [in a new window]   Fig. 6.   Effect of CaM levels on morphine dose-response curves for [35S]GTPS binding to cell membranes. A, experiments were performed with untreated (without EGTA washing) HEK-rOP3 membranes (OP3), after pH 8 EGTA washing (OP3 + W), and after readdition of 10 µM CaM (OP3 + W + CaM). [35S]GTPS binding was determined in the absence of GDP. Vec, Basal [35S]GTPS binding in vector mock-transfected control cells. B, membranes prepared from HEK-rOP3 cell after mock transfection with the empty CaM vector (OP3/Vec) or transient transfection with CaM (OP3/CaM) or after washing the latter with pH 8/EGTA medium (OP3/CaM + W). Shown as a control is basal [35S]GTPS binding activity in mock-transfected HEK293 cells (Vec-Vec, transfected with both empty vectors). C, membranes prepared from HEK-rOP3 cell (OP3) or HEK-rOP3 cell stably transfected with antisense CaM (OP3/as-CaM) without or with pH 8/EGTA wash (+W). Basal OP3 [35S]GTPS binding differs significantly from that of OP3 + W, OP3/as-CaM and OP3/as-CaM + W (p < 0.001). D, membranes prepared from HEK-FLAG-hOP3 cell (F-hOP3) and K273A-FLAG-hOP3 (KA-F-hOP3) without or with pH 8/EGTA washing (+W). Values are means ± S.D., n = 3-6 with triplicate measurements (t test, unpaired).

The effect of adding CaM to washed membranes was also considered and is illustrated in Fig. 6A. The addition of CaM to washed HEK-rOP₃ membranes caused a lowering of [³⁵S]GTPS binding activity to levels observed before washing, across the entire concentration range of morphine, without significant changes in EC₅₀ values (0.84-1.94 nM). These results reinforce the contention that CaM is responsible for the changes observed and inversely affects G protein coupling. Guanine nucleotides (GDP) were omitted in the experiment illustrated in Fig. 6A to facilitate [³⁵S]GTPS binding and possible competition between CaM and G proteins. In the presence of 1 µM GDP, pH 8/EGTA washing similarly enhanced [³⁵S]GTPS binding activity over the entire concentration range of morphine used, but the addition of CaM was unable to fully reverse this enhanced activity (data not shown). This suggests that a rather stable OP₃ receptor-G protein interaction may have formed in the membranes (31).

Effect of Alteration of CaM Levels in Intact Cells on Opioid Receptor G protein Coupling-- CaM levels were also altered in intact cells by expressing plasmids encoding sense and antisense CaM cDNA. Membranes prepared from HEK-rOP₃ cells transiently transfected with sense CaM cDNA, expressing CaM at levels of 186% (CaM activity/mg protein; S.D. = 24%, n = 9, p < 0.001, t test, unpaired) over mock-transfected cells, had a significantly reduced level of basal and agonist-stimulated [³⁵S]GTPS binding activity (illustrated in Fig. 6B) This was reversed upon washing cell membrane with pH 8/EGTA buffer (Fig. 6B), which lowered CaM levels by 46% (S.D. = 8%, n = 9, p < 0.001, t test, unpaired). EC₅₀ values for morphine did not change significantly under any condition (3.5-4.3 nM). (Higher EC₅₀ values observed in Fig. 6B compared with Fig. 6A are due to the presence of GDP in the assay incubations.) CaM transfection not only lowered the entire dose-response curve but also blunted the maximal morphine effect on [³⁵S]GTPS binding activity (E_max from 120 ± 12 to 94 ± 9% over basal, means ± S.D., p < 0.05, t test, unpaired); this effect was also reversed upon washing (E_max 126 ± 7%).

In contrast to cell lines with sense CaM vectors, stable expression of antisense CaM reduced CaM levels to 73% (S.D. = 13%, n = 9, p < 0.001, t test, unpaired) of the control, and these cells were found to have significantly elevated basal and morphine-activated [³⁵S]GTPS binding activity (Fig. 6C). Importantly, washing of the antisense CaM cell membranes failed to affect the morphine concentration-response curve of [³⁵S]GTPS binding activity (Fig. 6C). Moreover, the maximal effect of morphine was enhanced by antisense CaM expression from 110 ± 11 to 147 ± 21% over basal (means ± S.D., n = 9, p < 0.05, t test, unpaired), and the EC₅₀ value was significantly reduced from 5.5 ± 1.4 to 1.7 ± 0.2 nM (means ± S.D., n = 9, p < 0.05, t test, unpaired). The activity of mock-transfected cell membranes was equivalent to untransfected controls (data not shown), ruling out a nonspecific effect of plasmid transfection.

G Protein Coupling of the Mutant K273A-FLAG-hOP₃ Receptor-- Since the motif search and experimental results with i3 loop-derived peptides indicated that Lys²⁷³ of OP₃ receptor could play a role in the proposed interaction with CaM, a K273A-OP₃ receptor mutant was constructed, and its ability to stimulate [³⁵S]GTPS binding activity was compared with wild-type FLAG-hOP₃ receptor. The FLAG-K273A-hOP₃ receptor was still retained on a CaM affinity gel, as determined by immunoblotting, but retention was relatively small and not easily quantifiable (data not shown). Thus, CaM interaction with this mutated receptor does not appear to be completely abrogated, in agreement with the i3-6 peptide's partial ability to block FLAG-mOP₃ binding to CaM (Fig. 4C).

Compared with the wild-type FLAG-hOP₃ receptor, basal and morphine-activated [³⁵S]GTPS binding activity for the FLAG-K273A-hOP₃ receptor were elevated regardless of washing with pH 8/EGTA buffer (see Fig. 6D). These results indicate that substitution of Lys²⁷³ with Ala induces elevated levels of basal G protein stimulation and that the receptor is no longer responsive to changes in CaM content.

CaM Affects High Affinity Binding of Morphine to the OP₃ Receptor-- The addition of CaM to HEK-rOP₃ membranes had no significant effect on binding of the antagonist tracer, [³H]naloxone, which is thought to bind to the G protein-coupled and -uncoupled states of the receptor equally well. In contrast, morphine-[³H]naloxone competition curves in HEK-rOP₃ membranes revealed the presence of low and high affinity sites (Fig. 7A). The displacement curve is fitted better by a two-site, rather than a one-site, receptor binding model, indicating a distribution of 34 ± 2% high affinity and 64 ± 8% (means ± S.D.) low affinity sites.

View larger version (18K): [in this window] [in a new window]   Fig. 7.   Morphine displacement curves of 1 nM [3H]-naloxone bound to HEK-OP3 (A), HEK-OP3 stably transfected with a sense CaM vector (B, HEK-OP3/CaM), and HEK-K273A-FLAG-hOP3 cell membranes (C). Binding assays were performed in the absence (control) or presence of 15 µM CaM (+CaM) and/or 50 µM GTPS (+GTPS) and in the presence of 50 µM Ca2+. A, control curve. C, control and +CaM curves fit a two-site model significantly better than a one-site model (p < 0.01), whereas the other curves are adequately described by a one-site model (p > 0.2). Estimated EC50 values for the high affinity site were 0.18-0.79 nM, and values for the low affinity site were 130-190 nm (two-site model). Each binding curve was determined in three separate experiments, each performed in duplicate per point. Data were analyzed by GraphPrism.

If the high affinity site were to represent the G protein-coupled state, one would expect displacement of G proteins by CaM to abolish the high affinity site. Indeed, incubation of the membranes with CaM (final concentration, 15 µM) in the presence of 50 µM CaCl₂ largely eliminated the high affinity site (Fig. 7A), and the resultant data fit a one-site model (Fig. 7A). The binding curve in the presence of CaM is nearly identical to that obtained in the presence of GTPS, which uncouples the OP₃ receptor from its G proteins (Fig. 7A). Chelation of Ca²⁺ by EGTA prevented the effect of CaM on the high affinity binding site, another indication that CaM-OP₃ interactions are Ca²⁺-sensitive.

We also measured [³H]naloxone-morphine displacement curves in membrane obtained from HEK-rOP₃ cell that overexpressed CaM (HEK-rOP₃/CaM, stably transfected, in which membrane CaM activity was 134 ± 16% of that of mock-transfected cells). Overexpressing CaM eliminated the high affinity binding site (Fig. 7B). Moreover, the addition of GTPS had no further effect on the morphine displacement curve (Fig. 7B). Thus, elevated CaM levels abolished the high affinity agonist binding site.

To address the question of whether CaM effects on OP₃ receptor arise from direct binding to the receptor or indirectly to another protein attached to the receptor such as G, we determined ligand binding of the mutant receptor K273A-FLAG-hOP₃, which effectively couples to G proteins but is functionally insensitive to CaM. Shown in Fig. 7C, the high affinity site of the K273A-FLAG-hOP₃ receptor remained unaffected by CaM addition, although the addition of GTPS eliminated this high affinity site. This supports the view that the changes in high affinity morphine binding to OP₃ receptors were mediated by direct CaM-OP₃ receptor interactions.

Morphine-induced CaM Release from Plasma Membranes-- To determine whether OP₃ receptor activation can cause dissociation of CaM from the plasma membrane, we measured changes of CaM content in plasma membranes prepared from HEK-rOP₃ that had been exposed to 1 µM morphine (Fig. 8A). There was a significant reduction of membrane CaM content within 1 min, leveling off at 15 min. A similar effect was observed with HEK-FLAG-hOP₃ and with HEK-mOP₁ cells stimulated with 1 µM DPDPE (Fig. 8B). Moreover, treatment with pertussis toxin (100 ng/ml for 18 h) failed to affect the morphine-induced loss of CaM from plasma membranes in HEK-rOP₃ cells, whereas morphine had no effect on CaM levels in K273A-FLAG-hOP₃ cells (Fig. 8B). These results suggest that G proteins do not play a role in CaM translocation and, further, that CaM depletion from the membrane might be a direct result of CaM dissociation from the receptor.

View larger version (15K): [in this window] [in a new window]   Fig. 8.   Effect of agonist stimulation of intact cells on the CaM content in HEK293 plasma membranes. A, time course of CaM content in plasma membranes of HEK-rOP3 cells stimulated with 1 µM morphine. ***, p < 0.001 versus time 0 (n = 6, analysis of variance). B, CaM content in plasma membranes of HEK-rOP3, HEK-mOP1, HEK-FLAG-hOP3, and HEK-K273A-FLAG-hOP3 cell membrane after stimulation for 15 min with 1 µM morphine or DPDPE (for mOP1), respectively. The two bars on the far right show data for HEK-rOP3 with pertussis toxin pretreatment, 100 ng/ml for 18 h. *, p < 0.05; ***, p < 0.001 versus control, n = 6, paired t test.

Effect of CaM Levels on OP₃ Receptor Regulation of cAMP in Intact Cells-- The ability of morphine to lower forskolin-stimulated cAMP levels was measured in HEK-rOP₃ and in sense and antisense CaM-expressing HEK-rOP₃ cells. Changing the CaM content failed to affect the EC₅₀ value of morphine significantly (range between 4 and 8 nM). However, in HEK-rOP₃/as-CaM cells with reduced CaM content, maximum inhibition of cAMP accumulation by morphine increased from 73 ± 3% (normal CaM level) to 97 ± 4% (means ± SD, n = 6, p < 0.05, t test, unpaired). Similarly, morphine was slightly but significantly more efficacious in HEK-K273A-FLAG-hOP₃ than in HEK-FLAG-hOP₃ cells (maximum inhibition of cAMP accumulation was 82 ± 3 versus 68 ± 2%; means ± S.D., n = 6, p < 0.01, t test, unpaired), again without a significant change in the EC₅₀ (2 nM in each case). These results parallel those observed with [³⁵S]GTPS binding to cell membranes.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

This study provides evidence in support of the hypothesis that CaM interacts with the i3 loop of OP₃. We have demonstrated that solubilized OP₃ interacts with CaM in a Ca²⁺-sensitive manner. Moreover, peptides derived from the i3 loop of OP₃ bound to CaM and suppressed interactions between CaM and solubilized OP₃. As expected from these results, CaM content of plasma membranes inversely affected G protein coupling by OP₃ and suppressed high affinity binding sites of morphine to OP₃, thought to represent the G protein-coupled state of the receptor. However, these results taken alone did not rule out the possibility that CaM could modulate receptor-G protein coupling indirectly, for example, by binding to the  subunit of G proteins (8). However, the solubilized and immunopurified OP₃ appeared devoid of associated G protein, since we were unable to detect any G_i3 (a major G-coupling protein of OP₃) in the immunopurified rOP₃ preparation; therefore, G proteins are unlikely to mediate the observed CaM binding to purified OP₃. To resolve the question whether a direct interaction occurs, we screened a number of OP₃ mutants with single residue substitutions in the i3 loop for loss of CaM interaction, but with preserved G protein coupling. Importantly, the OP₃ mutant K273A (selected on the basis of the role of this Lys residue in motif structure and i3-loop peptide binding to CaM) displayed enhanced G protein coupling, but it was insensitive to CaM. This result strongly supports a direct interaction between OP₃ and CaM.

Taken together, our results suggest that CaM and G proteins compete for a shared binding domain in the i3 loop of OP₃. As proposed for G protein binding domains, both the N terminus and the C terminus of i3 may play a role in CaM binding, although the C terminus is the dominant factor. While the binding mechanism between CaM and the i3 loop remains to be resolved, it is possible that a distortion of the i3 loop upon receptor activation could lower the affinity to CaM and cause CaM dissociation. CaM binding to OP₃ was enhanced by Ca²⁺, but studies with i3 loop peptides indicate that Ca²⁺ is not essential. At resting conditions with low intracellular Ca²⁺ levels, OP₃ may thus be occupied either by CaM or by G proteins. OP₃ activation could cause dissociation of CaM so that the unoccupied receptor can subsequently rebind G proteins. Therefore, elevated CaM levels failed to abolish agonist-induced G protein coupling.

This study further establishes a significant level of basal G protein coupling in cell membranes for both OP₃ and OP₁, as suggested previously (31-33). Basal G protein signaling was highly sensitive to CaM content; thus, overexpression of CaM obliterated, while down-regulating CaM enhanced, basal G protein signaling. This is consistent with the proposed model that CaM binding to OP₃ suppresses G protein coupling, and that agonist activation is needed to dislodge CaM and initiate G protein signaling. This appears to provide a novel mechanism of regulating basal G protein signaling. Accordingly, the CaM-insensitive mutant receptor K273A-hOP₃ displayed increased basal activity over that of wild-type hOP₃.

The results in this study further raise the possibility that CaM, like G proteins, may serve directly as a receptor-mediated second messenger molecule, translocating signals upon release from receptor. Subcellular redistribution of CaM upon morphine administration had already been reported to occur in intact animals and in tissue culture (23, 34). Similarly, enkephalins and opiate antagonists were shown to control a parallel CaM-opioid receptor redistribution in neuroblastoma × glioma NG108-15 cells (expressing OP₁), suggesting a tight regulatory, and possibly structural, relationship between opioid receptors and CaM (34). Since no CaM dissociation from plasma membranes was observed with K273A-OP₃ in our study, the loss of CaM from the plasma membrane appeared to be a direct result of CaM-OP₃ dissociation upon activation of the receptor rather than a secondary effect of OP₃-G protein signaling. This finding is further supported by the observation that uncoupling OP₃ from G proteins with pertussis toxin failed to affect morphine-induced CaM loss from the plasma membrane.

CaM dissociation from the plasma membrane can account for previous findings that opioid receptors activate Ca²⁺/CaM-dependent cAMP phosphodiesterases in a G protein-independent fashion (35, 36). Other candidate signaling pathways for CaM include adenylyl cyclases, NO synthases, CaM kinases, and ion channels. In some tissues, including HEK-OP₃ cells, morphine activation of OP₃ causes a sustained increase in intracellular Ca²⁺ levels (37). Together with CaM mobilization, this could lead to enhanced OP₃ phosphorylation by Ca²⁺/CaM kinase-II (38) as a possible regulatory pathway.

Motif searches of CaM binding domains revealed a number of GPCRs as possible candidates for interacting with CaM. Among these, the opioid receptors all share nearly identical i3 loop sequences (Fig. 1). Thus, G protein coupling of OP₁ was similarly sensitive to CaM content in the plasma membrane, and it is plausible that CaM could bind to yet other GPCRs, such as the adrenergic (Fig. 1) and muscarinic receptors, that also contain a putative CaM motif. Similar to the opioid receptors, muscarinic receptors regulate phosphodiesterases by a Ca²⁺-dependent mechanism distinct from G protein-coupled regulation of adenylyl cyclases (39), possibly involving CaM.

In conclusion, we present evidence in support of the hypothesis that CaM binds to the i3 loop of opioid receptors and that CaM may serve as a second messenger molecule upon receptor activation.

	ACKNOWLEDGEMENTS

We thank Dr. Mark von Zastrow for FLAG-mOP₃ receptor cDNA, Dr. Lei Yu for rOP₃ receptor cDNA, Dr. C. J. Evans for mOP₁ receptor cDNA, and Dr. C. K. Surratt for hOP₃ receptor cDNA, and we thank Dr. Katharine Winans for assistance in preparing i3 loop peptides and editing the manuscript.

	FOOTNOTES

^* This work was supported by National Institute on Drug Abuse Grant DA04166 and by a grant from the University of California San Francisco Center for the Neurobiology of Drug Addiction.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Supported in part by an international National Institute on Drug Abuse-INVEST fellowship grant.

^§ To whom correspondence should be addressed: School of Pharmacy, University of California, San Francisco, CA 94143-0446. Tel.: 415-476-1947; Fax: 415-476-0464; E-mail: sadee@cgl.ucsf.edu.

	ABBREVIATIONS

The abbreviations used are: CaM, calmodulin (protein); CaM, calmodulin (cDNA); dansyl-CaM, 5-(dimethylamino)naphthalene-1-sulfonyl-calmodulin; OP₃ receptor, µ-opioid receptor; rOP₃, mOP₃, and hOP₃ receptor, rat, mouse, and human OP₃ receptor, respectively; i3 loop, third intracellular loop; G protein, heterotrimeric GTP binding protein; GPCR, G protein-coupled receptor; HEK, human embryonic kidney cells; FLAG-mOP₃ and FLAG-hOP₃ receptor, FLAG epitope-tagged mouse and human µ-opioid receptor, respectively; HEK-rOP₃ and -hOP₃, HEK cells stably transfected with rat or human OP₃ receptors, respectively; HEK-FLAG-OP₃, HEK cells stably transfected with FLAG epitope-tagged rat or human OP₃ receptors; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; GTPS, guanosine 5'-3-O-(thio)triphosphate; DPDPE, [D-Pen^2,5]-enkephalin; BAPTA, 1,2-bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, sodium.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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