JournalofNeurochemistryLippincott—Raven Publishers, Philadelphia© 1997 International Society for Neurochemistry

Rapid Communication

Site Mutation in the Rat ~u-OpioidReceptor Demonstrates theInvolvement of CalciumlCalmodulin-Dependent Protein

Kinase II in Agonist-Mediated Desensitization

Thomas Koch, Thomas Kroslak, Peter Mayer, Evelyn Raulf, and Volker Höllt

Department of Pharmacology and Toxicology, Otto-von-Guericke University, Magdeburg, Germany

Abstract The rat 1z-opioid receptor (rMOR1), expressed in hu-man embryonic kidney 293 (HEK293) cells, showsa desensitiza-tion to the inhibitory effect of the js agonist DAMGO on adenylatecyclase activity within 4 h of DAMGO preincubation. To investi-gate the role of calcium/calmodulin-dependent protein kinase Il(CaM kinase Il) on 1s-opioid receptor desensitization, we coex-pressed rMOR1 and constitutively active CaM kinase Il inHEK293 cells. This coexpression led to a faster time course ofagonist-induced desensitization of the ~-opioid receptor. Theincrease ofdesensitization could not be observed with a te-opioidreceptor mutant (S261A!S266A) that lacks two putative CaMkinase Il phosphorylation sites in the third intracellular loop. Inaddition, injection of CaM kinase Il in Xenopus oocytes led onlyto desensitization of expressed rMOR1, but not of an S261 A!S266A receptor mutant. These results suggest that phosphoryla-tion of Ser

251 and Ser266 by CaM kinase II is involved in thedesensitization of the

1s-opioid receptor. Key Words: ~t-Opioidreceptor — Desensitization — Calcium! calmodulin - dependentprotein kinase phosphorylation—G protein-activated channel—Xenopus oocytes.J. Neurochem. 69, 1767—1770 (1997).

Extensive physiological, behavioral, and pharmacologicalstudies have defined three major types of opioid receptors,designated ~.t, 6, and K (Wood and Iyengar, 1988; Corbettet al., 1993). The activation of all three opioid receptor typescan inhibit adenylate cyclase. In addition, opiates have beenshown to open K~channels and to close Ca

2~channels(North, 1993).

In Xenopus oocytes expressing opioid receptors and theG protein-gated, inwardly rectifying K channel (known asKGA or GIRK1), application of the ~.t agonist [D-Ala2,MePhe4,G1y5-ol] enkephalin (DAMGO) evoked a dose-de-pendent increase in K + conductance (Chen and Yu, 1994).Chen and Yu (1994) demonstrated that repeated agoniststimulation of the ~i-opioid receptor coexpressed withGIRK 1 in Xenopus oocytes resulted in desensitization of the

u-activated current response. Using theXenopus expressionsystem, it has also been reported that phosphorylation byprotein kinase C and Ca2~/calmodulin-dependentproteinkinase II (CaM kinase II) modulates desensitization of thehuman p~-opioidreceptor (Mestek et al., 1995), whereasreceptor desensitization of the 6-opioidreceptor is influencedby protein kinase C (Ueda et al., 1994, 1995). Due to the

experimental conditions, it is not clearly shown in theseexperiments whether the target of these kinases is the opioidreceptor, the G protein, the K + channel, or some intermedi-ary factors. Moreover, Kovoor et al. (1995) suggested thatthe desensitization of the rat ~t-opioid receptor in the Xeno-pus oocyte system is receptor-independent and caused by aninactivation of the K + channel itself.

To investigate further the role of CaM kinase II in opioidreceptor desensitization, we used, in addition to theXenopusoocyte expression system, the human embryonic kidney 293(HEK293) cell line for expressing the rat ji-opioid receptortypesand determined theinhibition of forskolin-induced ade-nylate cyclase after DAMGO stimulation as a measure forreceptor activity. Putative CaM kinase phosphorylation sites(Ser26‘ and Ser266) in the rat ~-opioid receptor were changedinto alanines using in vitro mutagenesis. Receptor desensiti-zation was measured with and without coexpression of con-stitutively active CaM kinase II.

MATERIALS AND METHODS

In vitro mutagenesisThe rat p~-opioidreceptor rMOR1 cDNA subcloned into

the pRc/CMV expression vector was kindly provided by Dr.Lei Yu (Indianapolis, IN, U.S.A.). Point mutations of the ~-

opioid receptor were generated by oligonucleotide-mediatedsite-directed mutagenesis using a33-mer mutagenesis primerlA-Ala (S‘-A CTC AAG GCC GTT CGC ATG CTA GCGGGC TCC AA-3‘) and Altered Sites in vitro mutagenesiskit from Promega according to the manufacturer‘s directions.Primer lA-Ala matches at nucleotides 774—806 of the cod-ing sequence of rMOR1 and introduces two alanines instead

Resubmitted manuscript received June 24, 1997; accepted June24, 1997.

Address correspondence andreprint requests to Dr. V. Höllt at theDepartment of Pharmacology and Toxicology, Otto-von-GuerickeUniversity, 39120 Magdeburg, Leipziger Str. 44, Germany.

Abbreviations used: CaM kinase II, Ca2~/calmodulin-depen-dent protein kinase II; cAMP, cyclic AMP; DAMGO, [D-A1a2,MePhe4 ‚Gly~-ol]enkephalin; GIRK, G protein-gated, inwardly rec-tifying K~channel; HEK293, human embryonic kidney 293; HK,high K~rMOR, rat ~t-opioidreceptor.

1767

1768 T. KOCH ET AL.

of serines in amino acid positions 261 and 266 of the rMOR1protein. Nucleotide sequence was confirmed by double-strand DNA sequencing.

Generation of cell lines expressing rat ~.t-opioidreceptors and constitutively active CaM kinase

Transfection of HEK293 cells was performed by the cal-cium phosphate precipitation method as described by Chenand Okayama (1988). Approximately 1.5 X 106 cells weretransfected with 20 jtg of plasmid DNA. For coexpressionof active CaM kinase II in HEK293 cells, expressing eitherrMOR1 or mutated rat ~z-opioid receptor, we used plasmidRSV CaMKa l-290, a generous gift from Dr. Loeffler(Strasbourg, France). This plasmid has been described byKapiloff et al. (1991) and led to expression of a truncatedand constitutive active CaM kinase a subunit that is Ca2~/calmodulin-independent and needs no activation by auto-phosphorylation.

Measurements of cyclic AMP (cAMP) levelsCells (1.5 X l0~)were seeded in 22-mm 12-well dishes

with Dulbecco‘s modified Eagle medium nutrient mixtureF-12 Ham containing 10% fetal calf serum. On the day ofassay, media were removed from the individual wells andwere replaced with 0.5 ml of serum-free medium containing20

1iM 3-isobutyl-1-methylxanthine and forskolin (25 p~Mfinal concentration) or a combination of forskolin (25 iM)andDAMGO (1 1.tM). The cells were then incubatedat 37°Cfor 15 min. The reaction was terminated by removing themedium and sonicating the cells in 1 ml of ice-cold HC1/ethanol (1 volume of 1 M HC1/l00 volumes of ethanol).After centrifugation, the supematant wasevaporated, theres-idue dissolved in Tris-EDTA buffer, andcAMP content mea-sured using a commercial radioassay kit (Amersham,Braunschweig, Germany).

Oocyte injection and expression of the receptorsand the G protein-activated K + channel

Oocytes were prepared using standard methods (Gurdonand Wickens, 1983). Mature Xenopus laevis females(Nasco, U.S.A.) were anesthetized in 0.15% tricaine(Sigma), ovarian lobes were surgically removed, and indi-vidual oocytes were isolated manually at room temperature.Stage V and VI oocytes were injected with a total of 50 nlcontaining 1 ng of each mRNA and incubated for 3—4 daysat 20°C in ND96 (96 mM NaCl, 2.0 mM KC1, 1.8 mMCaC12, 1 mM MgCl2, 5 mM HEPES) supplemented with5% fetal bovine serum, 2.5 mM sodium pyruvate, 100 U!mlpenicillin, and 100 jsg/ml streptomycin. To generate cappedmRNA for injection, T7 RNA polymerase and mCAPmRNA Capping kit (Stratagene) were used.

CaM kinase II (Biomol) was activated by autophosphoryla-tion, to become Ca

2~‘7calmodulin-independent,as described byMestek et al. (1995). Fifteen minutes before recording highK~(HK)- and HK/DAMGO-induced current, oocytes wereinjected with 50 nl (2 fmol) of CaM kinase II.

ElectrophysiologyOocytes with a membrane resistance Rm 1 M1~lwere

voltage-clamped at —80 mV at room temperature using atwo-microelectrode voltage clamp (Turbo TEC-05 npi am-plifier), and the data were analyzedwith Eggworks software(Polder, Germany). The clamp electrodes were filled with3 M KC1 and had a tip resistance of 0.5—1 Mft Clampedoocytes were superfused with either ND96, HK solution (96mM KC1, 2.0 mM NaC1, 1.8 mM CaCl

2, 1 mM MgCl2, 5mM HEPES), HK solution containing the ‚u-opioid-specificagonist DAMGO (1 tiM), or HK/BaC12 solution (HK with300 p1%‘! BaCl2).

RESULTS AND DISCUSSION

Various cell surface receptors are phosphorylated uponbinding of their ligand, and this phosphorylation seems tobe involved in the signal transduction and!or the feedbackregulation of this signal. Kinases involved in receptor phos-phorylation are cAMP-dependent protein kinase, protein ki-nase C, and CaM kinase. CaM kinase II phosphorylationwas shown to involve regulation of NMDA, GABAA, andhuman ~.t-opioid receptor activity (McDonald and Moss,1994; Mestek et al., 1995; Yakel et al., 1995).

The third intracellular ioop, which has been shown tobe important for G-protein coupling and signal transduction(Dohlman et al., 1991), is highly conserved among all opioidreceptor types. In this region occur two strictly conservedserine residues that may serve as CaM kinase II phosphoryla-tion sites. In the rat ~s-opioid receptor, the putative CaMkinase sites were in positions Ser

261 and Ser266. Conversionof either Ser261 or Ser266 into alanines resulted in a partialblockade of DAMGO-induced ‚u-opioid receptor desensitiza-tion (P. Y. Law, personal communication). To increase theinhibitory effect on agonist-induced desensitization, wechanged both 5er26‘ and Ser266 into alanines. A schematicdrawing of the primary structure of the ~-opioid receptorand the positions of mutations is presented in Fig. 1. Thewild-type and mutated ~.t-opioidreceptor were transientlytransfected into HEK293 cells.

Two days after transfection of the ~i-opioidreceptor types,DAMGO, a ~s-opioidreceptor agonist, was used to determinethe agonist-dependent loss of the functional coupling of thereceptor by assaying for changes in levels of intracellularcAMP after stimulation. Therefore, HEK293 cells express-

FIG. 1. Schematic representation of the structureof the rat ji-opioid receptor. Putative CaM kinaseIl phosphorylation sites are shown by filled circles.The lower panel shows the third intracellular loopresidues 258—268 ofthe wild-type receptor in sin-gle-letter amino acid code. The asterisks indicatethe putative CaM kinase Il phosphorylation sites.The amino acid sequence of the mutant identicalto the wild type is represented by the dashed line.

J. Neurochem., Vol. 69, No. 4, 1997

CaM KINASE II AND p~-OPIOIDRECEPTOR DESENSITIZATION 1769

FIG. 2. Time courses of agonist-induced desensitization ofrMOR1 and S261A!S266A receptor mutant. TransfectedHEK293 cells were preincubated at 37°Cwith 1 1jM DAMGO forthe indicated time intervals. After removal of the preincubationmedium, cells were treated with forskolin (25 btM) or forskolin(25 ~tM)plus DAMGO (1 pM), and cAMP levels were determinedas described above (see Materials and Methods). Values repre-sent means ±SEM from four separate measurements performedin triplicate. The asterisks indicate significant differences (p<0.05) between wild type and receptor mutant (ANOVA, fol-lowed by Bonferroni test). a: The effect of mutation of putativeCaM kinase Il phosphorylation sites on the p-opioid receptordesensitization. b: The effect of coexpression of CaM kinase Ilon the desensitization of p-opioid receptor and 5261A!S266Areceptor mutant.

ing wild-type or mutant receptors were pretreated with 1 iMDAMGO for O mm, 30 mm, 1 h, or 4 h, after which theDAMGO-induced inhibition of forskolin-stimulated adenyl-ate cyclase was determined. Forskolin treatment resulted ina fivefold increase of cAMP levels as compared with thosein untreated HEK293 cells. DAMGO mediated an inhibitionof forskolin-stimulated cAMP accumulation by -—40% inHEK293 cells expressing either therMOR1 wild type or theS261A/S266A receptor mutant. In HEK293 cells lackingopioid receptors, no agonist-caused inhibition of forskolin-stimulated cAMP levels could be observed (data notshown). Preincubation of rMORI wild type or 526lAIS266A receptor mutant-expressing cells with DAMGO (1tiM) produced a significantly decreased sensitivity in acuteinhibition of the forskolin-stimulated cAMP levels. Attenua-tion of the ability of DAMGO to inhibit the intracellularcAMP level from —40% (no pretreatment) to —4% (4-hpretreatment) was observed for both examined receptors.

Thetime course of agonist-dependent loss ofreceptor activ-

ity of rMOR1 or the S261A1S266A receptor mutant is illus-trated in Fig. 2a. For rMOR1, this decrease in DAMGO (1jtM) -induced inhibition of cAMP level was from 38.3 ~ 1.2%in cells without pretreatment to 37.4 ± 5%, 13 ±4.7%, and2 ±4.2% following 30 mm, 1 h, and 4 h of chronic DAMGOadministration, respectively. For the receptor mutant, the de-crease in inhibition of cAMP level was from 41.5 ±5.9% incells without pretreatment to 41 ±5.1%, 33 ±4.3%, and 6±5.8% following the respective intervals after chronicDAMGO administration. These data demonstrated that therMOR1 receptor desensitizes faster than the S2611S266 re-ceptor mutant during DAMGO preincubation, indicating thatthese putative CaM kinase II consensus sequences in the thirdintracellular 1oop are phosphorylated during receptor desensi-tization and play an important role in functional coupling ofthe receptors to G proteins and signal transduction. This issupported further by our studies in HEK293 cells expressingeither rMOR1 wild type or receptor mutant, coexpressed withconstitutively active CaM kinase II. Despite the absence ofagonist, ~.t-opioidreceptor rMOR1 is completely desensitizedwhen the CaM kinase II a subunit is coexpressed in HEK293cells (Fig. 2b). In contrast, coexpression of CaM kinase II asubunit has no effect on the desensitization rate of the 526lAIS266A receptor mutant.

In Xenopus oocytes expressing either rMORI or S26 lA/S266A mutant, and G protein-activated K~ channel(GIRK1), DAMGO superfusion led to an inward K~cur-rent. This current through an inwardly rectifying K + channelcan be blocked completely by the K~-channel blocker Ba

2~(300 ‚iM) (Fig. 3). Oocytes injected with the receptormRNAs alone did not produce the current upon DAMGOstimulation (data not shown). Due to variation in mRNAexpression among different oocytes, the amplitude of the K +

current can vary from oocyte to oocyte. Therefore, receptoractivity was defined as a ratio of DAMGO-induced K + cur-rent (IHK/OIRK~ctivated)to basal K~current throughthe GIRK1channel (iHK/GLRKha~aj), both measured under extracellularHK concentration (HK superfusion) (Fig. 3).

FIG. 3. A: Coupling of the rat p-opioid receptor tothe G protein-activated inwardly rectifying K~channel, GIRK1. Oocytes wereinjected with mRNAs for the p-opioid receptor wild type or mu-tant and the GIRK1 channel. A representative current trace re-corded at a holding potential of —80 mV shows the change incurrent following solution changes. DAMGO (1 pM) and BaCI

2(300 pM) were applied at the times indicated. ‘HK/GIRK-bas~I

represents the GIRK-mediated HK-activated current and‘HK/GIRK-ao5~ated the GIRK1 -mediated DAMGO-induced current.Both GIRK-specific currents were blocked completely byBaCl2. B; After injection of CaM kinase Il, the DAMGO-induced‘F-1K/GIRK-~ctivatedcurrent is reduced, whereas the ‘HK/GIRK-basa currentis not influenced.

J. Neurochem., Vol. 69, No. 4, /997

1770 T. KOCH ET AL.

fying other protein kinases and precise sites of phosphoryla-tion involved in opioid receptor desensitization.

Acknowledgment: The study was supported by the LandSachsen-Anhalt, SFB 426, and Fonds der Chemischen Industrie.

REFERENCES

FIG. 4. Modulation of p-opioid receptor coupling to GIRK1 inXenopus oocytes by CaM kinase Il. Oocytes were injected withboth the rat p-opiOid receptor wild-type or mutant receptor andthe GIRK1 mRNAs. Membrane currents were recorded at a hold-ing potential of —80 mV. Data are expressed as the ratio ofIHK/GIFtK-actuated to ‘HK/GIRK-basal and are presented as mean ±SDvalues. The asterisks indicate a significant difference (p <0.05)between receptor current ratios in oocytes with and without thecoinjection of CaM kinase Il (ANOVA, followed by Bonferroni test).

Previous studies have shown that repeated agonist stimula-tion desensitizes receptor-channel coupling in oocytes. Toinvestigate whether receptor phosphorylation is involved inthis process of receptor desensitization, we used direct injec-tion of CaM kinase II protein to inducereceptor desensitiza-tion in Xenopus oocytes without continuous agonist treat-ment. Receptor coupling in Xenopus oocytes expressingGIRK1 and rMOR1 or S261A/S266A mutant was measuredwith and without coinjection of activated CaM kinase. De-sensitization was defined as a decrease in the ratio of theDAMGO-induced current (‘HK/GIRK-activated) to the GIRK1-in-duced current (IHK/GLRK-basal). Figures 3 and 4 showthe effectof the CaM kinase II on ~-opioid receptor desensitization.Only in oocytes expressing therMOR1 receptor, but not theS261A/S266A mutant receptor, did injection of CaM kinaselead to desensitization of the receptor coupling (Fig. 4). Inreceptor-expressing oocytes coinjected with CaM kinase II,we found no difference in the activity of the GIRK1 channelunder HK (‘HK/GIRK-basal) compared with oocytes withoutCaM kinase coinjection (Fig. 3B). Therefore, the decreasein the ratio of IHK/GlRK.~etjvated/IHK/G1RK.basa~is due only to adecrease in ‘HK/GIRK-activated

These results from cell line and Xenopus oocyte studiesprovide direct evidence of the involvement of CaM kinaseII phosphorylation on agonist-induced ~t-opioid receptor de-sensitization, and they are also in good agreement with otherstudies demonstrating that, in transfected HEK293 cells, the~-opioid receptor was phosphorylated in the presence ofCa2~(Arden et al., 1995). The DAMGO-induced increaseof intracellular Ca2~concentration andinositol trisphosphateformation in Chinese hamster ovary cells stably expressingthe~.t-opioidreceptor (Zimprich et al., 1995) may be apossi-ble mechanism for CaM kinase activation causing receptordesensitization. Also, the K-opioid receptor has been shownto mobilize intracellular Ca2~following agonist treatment(Kaneko et al., 1994). As regulation of CaM kinase activityis also mediated by autophosphorylation resulting in a Ca2~/calmodulin-independent enzyme, theincrease of intracellularCa2~might not be the only mechanism, but it seems to bethe initial step in the CaM kinase activation.

Our data demonstrate that Ser261 and Ser266 in the thirdintracellular loop are important phosphorylation sites recog-nized by CaM kinase II. Future efforts will focus on identi-

Arden J. R., Segredo V., Wang Z., Lameh J., and Sadée W. (1995)Phosphorylation and agonist-specific intracellular trafficking ofan epitope-tagged p-opioid receptor expressed in HEK 293cells. J. Neurochem. 65, 1636—1645.

Chen C. A. and Okayama H. (1988) Calcium phosphate-mediated genetransfer: a highly efficient transfection system for stably trans-forming cells with plasmid DNA. Biotechniques 6, 632—638.

Chen Y. and Yu L. (1994) Differential regulation by cAMP-depen-dent protein kinase and protein kinase C of the p opioid receptorcoupling to a G protein-activated K~channel. J. Bio!. Chem.269, 7839—7842.

Corbett A., Paterson S., and Kosterlitz H. (1993) Selectivity ofligands for opioid receptors, in Handbook of ExperimentalPharmacology, Vol. 104: Opioids I (Herz A., cd), pp. 645—679. Springer, Berlin.

Dohlman H. G., Thomer J., Caron M. G., and Lefkowitz R. J. (1991)Model systems for the study of seven-transmembrane-segmentreceptors. Annu. Rev. Biochem. 60, 653—688.

Gurdon J. B. and Wickens M. P. (1983) The use of Xenopus oocytesfor the expression of cloned genes. Methods Enzymol. 101,370—386.

Kaneko S., Nakamura S., Adachi K., Akaike A., and Satoh M.(1994) Mobilization of intracellular Ca2 and stimulation ofcyclic AMP production by kappa opioid receptors expressed inXenopus oocytes. Brain Res. Mol. Brain Res. 27, 258—264.

Kapiloff M. S., Mathis J. M., Nelson C. A., Lin C. R., and RosenfeldM. G. (1991) Calcium/calmodulin-dependent protein kinasemediates a pathway for transcriptional regulation. Proc. NaIl.Acad. Sci. USA 88, 3710—3714.

Kovoor A., Douglas J. H., and Chavkin C. (1995) Agonist-induceddesensitization of the p opioid receptor-coupled potassiumchannel (GIRK1). J. Biol. Chem. 270, 589—595.

McDonald B. J. and Moss S. J. (1994) Differential phosphorylationof intracellular domains of gamma-aminobutyric acid type Areceptor subunits by calciumlcalmodulin type 2-dependent pro-tein kinase and cGMP-dependent protein kinase. J. Bio!. Chem.269, 18111—18117.

Mestek A., Hurley J. H., Bye L. S., Campbell A. D., Chen Y., TianM., Liu J., Schulman H., and Yu L. (1995) The human muopioid receptor:modulation offunctional desensitizationby cal-cium/calmodulin-dependent protein kinase and protein kinaseC. J. Neurosci. 15, 2396—2406.

North R. (1993)Opioid actions on membrane ion channels, in Hand-book ofExperimental Pharmacology, Vol. 104: Opioids I (HerzA., cd), pp. 773—797. Springer, Berlin.

Ueda H., Miyamae T., Fukushima N., and Misu Y. (1994) Proteinkinase inhibitor potentiates opioid delta-receptor currents inXenopus oocytes. Neuroreport 5, 1985—1988.

Ueda H., Miyamae T., Hayashi C., Watanabe S., Fukushima N.,Sasaki Y., Iwamura T., and Misu Y. (1995) Protein kinaseC involvement in homologous desensitization of delta-opioidreceptor coupled to G,-phospholipase C activation in Xenopusoocytes. J. Neurosci. 15, 7485—7499.

Wood P. L. and Iyengar S. (1988) Central actions of opiates andopioid peptides: in vivo evidencefor opioid receptor multiplic-ity, in The Opioid Receptors (Pastemak G. W., cd), pp. 307—356. Humana, Clifton, New Jersey.

Yakel J. L., Vissavajjhala P., Derkach V. A., Brickey D. A., andSoderling T. R. (1995) Identification of a Ca2~/calmodulin-dependent protein kinase II regulatory phosphorylation site innon—N-methyl-D-aspartate glutamate receptors. Proc. Nat!.Acad. Sci. USA 92, 1376—1380.

Zimprich A., Simon T., and Höllt V. (1995) Transfected rat muopioid receptors (rMORI and rMOR1B) stimulate phospholi-pase C and Ca2~mobilization. Neuroreport 7, 54—56.

J. Neurochem., Vol. 69, No. 4, 1997