Bioorthogonal click chemistry to assay mu-opioid receptor palmitoylation using 15-hexadecynoic acid and immunoprecipitation

Bioorthogonal click chemistry to assay mu-opioid receptor palmitoylation using 15-hexadecynoic acid and immunoprecipitation
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  Notes & Tips Bioorthogonal click chemistry to assay mu-opioid receptorpalmitoylation using 15-hexadecynoic acid and immunoprecipitation Brittany Ebersole a,b, ⇑ , Jessica Petko b , Robert Levenson b a Program in Chemical Biology, Penn State College of Medicine, Hershey, PA 17033, USA b Department of Pharmacology, Penn State College of Medicine, Hershey, PA 17033, USA a r t i c l e i n f o  Article history: Received 25 November 2013Received in revised form 9 January 2014Accepted 11 January 2014Available online 23 January 2014 Keywords: PalmitoylationClick chemistryMu-opioid receptorImmunoprecipitation a b s t r a c t We have developed a modification of bioorthogonal click chemistry to assay the palmitoylation of cellularproteins. This assay uses 15-hexadecynoic acid (15-HDYA) as a chemical probe in combination with proteinimmunoprecipitation using magnetic beads in order to detect S-palmitoylation of proteins of interest. Herewedemonstratetheutilityofthisapproachforthemu-opioidreceptor(MOR), aG-protein-coupledreceptor(GPCR) responsible for mediating the analgesic and addictive properties of most clinically relevant opioidagonist drugs. This technique provides a rapid, non-isotopic, and efficient method to assay the palmitoyla-tion status of a variety of cellular proteins, including most GPCRs.   2014 Elsevier Inc. All rights reserved. Protein palmitoylation is a posttranslational modification that typicallyinvolves covalent attachment of palmitic acid to proteins, often resulting inlipid raft localization of membrane proteins [1–3]. Palmitate is attached toproteins via an enzymatic reaction that is catalyzed by a family of proteinacyltransferases (PATs). 1 Palmitoylation enhances the hydrophobicity of pro-teins, thereby contributing to their membrane association, subcellular traffick-ing between membrane compartments, and modulation of protein–proteininteractions [1,3–6]. S-palmitoylation is a specific type of lipid modificationthatinvolvestheadditionofaC16acylchaintocytosoliccysteinesviathioesterbonds and is unique among lipid modifications in that it is reversible [3–5]. Classically, determining the palmitoylation status of a protein has reliedon metabolic labeling with [ 3 H]palmitate, followed by autoradiographicdetection of the labeled protein on Western blots. However, due to the lowspecific activity of [ 3 H]palmitate, this type of analysis can require the use of large quantities of labeled palmitate, and detection may require weeks-longor even months-long exposure times. Recently, a number of non-isotopiclabeling methods, includingbioorthogonalclickchemistry, havebeendevel-oped that can be used to detect and quantitate protein palmitoylation. Inadditiontoofferingsignificantlygreatersensitivityandmorerapiddetectiontimes than metabolic labeling with radioactive palmitate, these assays canalsobeusedtodeterminewhichPATs areresponsiblefor thepalmitoylationof specific target proteins.Bioorthogonal click chemistry (BCC) is a non-isotopic labeling techniquethat often uses 17-octadecynoic acid (17-ODYA) as a chemical probe. ThisC18 lipid probe is taken up by living cells and incorporated into proteins viaPATs. Following uptake of the lipid probe, proteins are harvested from cellsand reacted with a bioorthogonal azide-labeled fluorescent chromaphore viaclickchemistry[7].OnelimitationofthistechniqueisthatsomePATsarelessefficient at attaching fatty acid chains that are larger than 16 carbons (i.e.,17-ODYA) to a target protein [8]. In this study, we investigated the use of 15-hexadecynoic acid (15-HDYA) as the chemical probe. The structure of 15-HDYAisidenticaltopalmitatewiththeexceptionthatitcontainsan x -ter-minalalkynenecessaryfortheclickreaction.Herewedemonstratetheefficacyof using BCC with 15-HDYA to interrogate the palmitoylation status of themu-opioid receptor (MOR), a G-protein-coupled receptor (GPCR) responsiblefor mediating the analgesic and addictive properties of opioid agonist drugs.The MOR has previously been reported to be palmitoylated via conventionalmetabolic labeling with [ 3 H]palmitate and another non-isotopic labelingmethod, acyl-biotinexchangechemistry[9,10]. Furthermore, BCCinconjunc- tion with magnetic bead immunoprecipitation should significantly reduceboth sample loss and the time required for protein purification, therebyimprovingthesensitivity of thesubsequent clickchemistry reaction.To determine whether 15-HDYA can be effectively used as a chemicalprobe in the BCC assay, HEK-293 cells were incubated for 24 h with varyingdoses of 15-HDYA. Cell lysates were then prepared using a sodium phos-phate-based lysis buffer. It is important to note that Tris-based lysis bufferswill not work with BCC because Tris can act as an inhibitory ligand for theCu(I) species used in the click chemistry reaction [11]. In this and subse-quent experiments, cells treated with dimethyl sulfoxide (DMSO) alone (atthe indicated concentrations) served as control. Click chemistry was per-formed as described previously [7,12,13] with the exception that we usedTAMRAazide (Lumniprobe) as the probe instead of alkyl-TAMRA (see onlineSupplementary material). Cell lysates (50 l g/well) were subjected to so-dium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), andthe gel was imaged using a Typhoon 9410 fluorescent imager (GE Amer-sham). Proteins were then transferred to a polyvinylidene fluoride (PVDF)membrane and analyzed via Western blotting with a chicken anti-GAPDH(glyceraldehyde-3-phosphatedehydrogenase)antibody(1:10,000;Millipore).   2014 Elsevier Inc. All rights reserved. ⇑ Corresponding author at: Department of Pharmacology, Penn State College of Medicine, Hershey, PA 17033, USA. E-mail address: (B. Ebersole). 1 Abbreviations used: PAT, protein acyltransferase; BCC, bioorthogonal clickchemistry; 17-ODYA, 17-octadecynoic acid; 15-HDYA, 15-hexadecynoic acid; MOR,mu-opioid receptor; GPCR, G-protein-coupled receptor; DMSO, dimethyl sulfoxide;SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; PVDF, poly-vinylidene fluoride; GAPDH, glyceraldehyde-3-phosphate dehydrogenase. Analytical Biochemistry 451 (2014) 25–27 Contents lists available at ScienceDirect Analytical Biochemistry journal homepage:  As shown in Fig. 1A, 15-HDYA was incorporated into a similar pattern of cellular proteins at all concentrations tested, whereas optimal incorporationof the lipid probe was obtained at a dose of 100 l M. It is important to notethat 125 l M15-HDYA was cytotoxic to the cells, whereas 100 l M15-HDYAdid not appear to appreciably affect cellular viability. These results are inagreement with previously published reports [13]. We next compared theability of 15-HDYA and 17-ODYA to label cellular proteins in HEK-293 cells.HEK-293 cells were treated for 24h with 100 l M15-HDYA or 17-ODYA. Ly-sates were prepared, labeled with TAMRA azide, and imaged as describedabove. Separated proteins were transferred to a PVDF membrane andanalyzed via Western blotting with the chicken anti-GAPDH antibody. Asshown in Fig. 1B, 15-HDYA and 17-ODYA were incorporated to similarextents into free palmitoylation sites in HEK-293 cells.To test the feasibility of using 15-HDYA as the lipid probe for detectingMOR palmitoylation, HEK–MOR cells were metabolically labeled for 24hwith 100 l M 15-HDYA or 17-ODYA. HEK–MOR cells are HEK-293 cells sta-bly expressing FLAG-tagged MORs [14]. Cell lysates were prepared, addedto Protein G MAG Sepharose (GE Healthcare) magnetic beads coated in rab-bit anti-FLAG antibody (Sigma), and allowed to immunoprecipitate for 1h.Magnetic beads were collected and washed. Bead-associated proteins werethen subjected to the click chemical reaction (see Supplementary material).Labeled immunocomplexes were eluted from the magnetic beads using 4  protein loading dye and analyzed via SDS–PAGE followed by in-gel fluores-cence imaging. Separated proteins were then transferred to a PVDF mem-brane and analyzed by Western blotting with a mouse anti-FLAG antibody(1:5000; Sigma). As shown in Fig. 1C, we detected a band, correspondinginsizetotheMOR,incellslabeledwitheither15-HDYAor17-ODYAmigrat-ing between 55 and 70kDa both in the in-gel Typhoon scan (upper panel)and on the Western blot (lower panel). This immunoreactive band is alsopresent in lysates prepared from control HEK–MOR cells and is consistentinsizewithour previous analysisof MOR [15]. Normalizationof the amountof palmitoylated MOR to the amount of immunoprecipitated MOR revealeda significant decrease (15%) in the amount of 17-ODYA incorporated intoMOR compared with 15-HDYA (Fig. 1D). Taken together, our results suggestthat BCC using 15-HDYA or 17-ODYA as the lipid probe, coupled with mag-netic bead immunoprecipitation, is an effective method to detect palmitoy-latedMORandthat15-HDYAismoreefficientlyincorporatedintoMORthanis 17-ODYA. Fig.1.  Palmitoylation of HEK-293 cell proteins analyzed by bioorthogonal clickchemistry.(A)Doseresponseusing15-HDYAasthelipidprobeonpalmitoylationof endogenous HEK-293 cell proteins. In panels A–C control cells were treated withDMSO (0.2%). (B) Palmitoylation of endogenous HEK-293cell proteins with 100 l M15-HDYA or 17-ODYA. In panels A and B, GAPDH served as a loading control. (C)Palmitoylation of MOR in HEK–MOR cells treated with 100 l M 15-HDYA or 17-ODYA. MOR was immunoprecipitated using rabbit anti-FLAG antibody. Afterfluorescent imaging (top), proteins were analyzed by Western blotting with amouseanti-FLAGantibody(bottom).BracketsindicatebandscorrespondingtoMOR that were used for quantitation. (D) Levels of palmitoylated MOR were normalizedto the amount of immunoprecipitated receptor. The bar graph represents theaverage pixel density from three experiments. IB, immunoblotting; IP, immuno-precipitation. Data were analyzed using a two-sided paired Student’s  t   test(expressed as±standard errors,  n  =3,  ⁄ P   <0.05). Fig.2.  Palmitoylation of the MOR. (A) zDHHC4 and GODZ interact with MOR. HEK–MOR cells were transiently transfected with myc-tagged zDHHC4 or GODZ, andMOR was immunoprecipitated using a rabbit anti-FLAG antibody. Rabbit IgG wasused as a mock control. Blots were probed for PATs using a mouse anti-mycantibody. Lysates (L) contain 5% of the total protein compared with mock (M) andimmunoprecipitation (IP) lanes. (B) zDHHC4 and GODZ can palmitoylate the MOR.HEK–MOR cells were transiently transfected with myc-tagged zDHHC4 or GODZ.MORwasimmunoprecipitatedwithrabbitanti-FLAGantibody.RabbitIgGwasusedas a control. The double-headed arrow indicates bands corresponding to MOR. Thesingle-headed arrows indicate palmitoylated PATs (upper: GODZ; lower: zDHHC4).(C) Western blotting was performed using either mouse anti-FLAG (top) or mouseanti-myc (bottom) antibodies. (D) Palmitoylated levels of MOR were normalized tothe amount of immunoprecipitated MOR. The bar graph shows average pixeldensity from three experiments. IB, immunoblotting; IP, immunoprecipitation;Untrans, Untransfected. Data were analyzed using a two-sided paired Student’s  t  test (expressed as ±standard errors,  n  =3,  ⁄ P   <0.05,  ⁄⁄ P   <0.005).26  Notes & Tips/Anal. Biochem. 451 (2014) 25–27   The results presented here using BCC and those presented previously byothers [9,10] indicate that the MOR is a palmitoylated GPCR. However,whichPATsareresponsibleforpalmitoylatingtheMORisnotknown.Atotalof 23 mammalian PATs have been identified, and substrates for many (butnot all) PATs have been characterized [6,16]. We chose to examine 2 mam-malianPATs, GODZ(zDHHC3) andzDHHC4, aspotential candidatesforMOR palmitoylation. GODZ (zDHHC3) is a PAT that is known to palmitoylate avariety of membrane proteins, including the GABA A c 2 subunit and GluR1/2 [16]. Studies we have carried out suggest that both GODZ and zDHHC4are capable of palmitoylating the D2 dopamine receptor (B. Ebersole et al.,unpublished work). To determine whether GODZ and/or zDHHC4 have thepotential to palmitoylate the MOR, we first asked whether either of thesePATs interacted with the receptor. To do this, we tested the ability of anti-FLAG antibodies to coimmunoprecipitate FLAG-tagged MOR and myc-tagged zDHHC4 or GODZ from transfected HEK–MOR (stably expressingFLAG-taggedMOR)cells.AsshowninFig. 2A,GODZandzDHHC4eachcoim-munoprecipitated with the MOR. These results suggest that these two PATSinteract with the MOR and, therefore, are candidate MOR palmitoylatingenzymes.To determine whether GODZ and zDHHC4 are capable of palmitoylatingthe MOR, we tested whether overexpression of each PAT caused an increasein the palmitoylation levels of the MOR. Overexpression of PATS in culturedcells has commonly been used to ascertain substrate specificity of a varietyofdifferentPATS[17–20].Forthisexperiment,GODZandzDHHC4weresep-arately transfected into HEK–MOR cells and BCC was performed as de-scribed above. The MOR was immunoprecipitated using a rabbit anti-FLAGantibody, and the palmitoylated receptor was visualized by in-gel fluores-cence imaging (Fig. 2B) and, after protein transfer, on a Western blot usingthe mouse anti-FLAG antibody (Fig. 2C). Palmitoylated MOR levels werequantitated and normalized to the amount of immunoprecipitated MOR (Fig. 2C, top panel). As shown in Fig. 2D, overexpression of zDHHC4 and GODZproducedanincreaseinMORpalmitoylationof113and246%, respec-tively, compared with untransfected cells. Interestingly, both in-gel imaging(Fig. 2B) and probing of the Western blot with anti-myc antibodies (Fig. 2C) revealed that the two PATs used to palmitoylate the MOR were themselvespalmitoylated in the BCC assay. These results suggest that zDHHC4 andGODZ contribute to the palmitoylation of the MOR in transfected cells. Itis interesting to note that zDHHC4 coimmunoprecipitates more efficientlywiththeMORthandoesGODZ,butitislessefficientthanGODZatpalmitoy-lating the receptor. Thus, GODZ may be more efficient than zDHHC4 atpalmitoylating the MOR in this cell system.Using bioorthogonal click chemistry, we have confirmed previous re-portsthat the MORispalmitoylated[9,10]. Thedatapresentedherearecon-sistent with the followingconclusions. First, non-isotopic labeling of a GPCR with a lipid probe, in combination with immunoprecipitation of the labeledprotein with magnetic beads, represents a rapid and sensitive means tocharacterize GPCR palmitoylation. Second, BCC can be used as an analyticaltool to determine which PATs are responsible for palmitoylating a specificreceptor. Our results suggest that two candidate PATs, GODZ and zDHHC4,are capable of interacting with and palmitoylating the MOR, at least withinthe context of transfected mammalian cells. However, the potential interac-tion of these (and other) PATs with the MOR in vivo is not known and willdependinlargepartonwhethertheMORandaparticular PATarecoexpres-sed within the same cell or cellular compartment within the nervoussystem.  Acknowledgments We thank Bernhard Lüscher and Casey Kilpatrick (Penn StateUniversity, University Park) for suggesting the use of 15-HDYA.This study was supported by a grant from the National Instituteon Drug Abuse (NIDA, DA025995) and a CURE grant to R.L. fromthe Pennsylvania Department of Health using Tobacco SettlementFunds. The funding agencies had no role in study design, datacollection and analysis, decision to publish, or preparation of themanuscript.  Appendix A. 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