Molecular cloning, stable expression and cellular localization of human α1-adrenergic receptor subtypes: effect of charcoal/dextran treated serum on expression and localization of α 1 D -adrenergic receptor

Molecular cloning, stable expression and cellular localization of human α1-adrenergic receptor subtypes: effect of charcoal/dextran treated serum on expression and localization of α 1 D -adrenergic receptor

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  Abstract  The cDNAs encoding for three sub-types of adrenergic receptors,  a 1A -,  a 1B - and  a 1D -ARs, were cloned and expressed in HEK 293cells. Expression of   a 1A - and  a 1B -AR subtypes inHEK 293 cells was stable even with increasedpassages but that of   a 1D -AR was not. Cellularlocalization studies using immunofluorescenceand flow cytometry revealed that expression of  a 1A - and  a 1B -ARs was primarily localized on thecell membrane whereas expression of   a 1D -ARwas predominantly intracellular. Our studiesclearly demonstrated that the culturing of therecombinant cell lines expressing  a 1D -AR incharcoal/dextran treated fetal bovine serum(FBS) resulted in targeting of   a 1D -AR to the cellmembrane and thus, significantly improving itsstability and availability for ligand bindingstudies. Keywords  Adrenergic receptors  Æ Cellular localization  Æ  Charcoal/dextran treatedfetal bovine serum  Æ  Stable cell lines Introduction The  a 1 -Adrenergic receptors ( a 1 -ARs) are mem-bers of the seven transmembrane G protein-cou-pled receptors (GPCRs) superfamily mediatingvarious sympathetic nervous system responsessuch as smooth muscle contractions, arterialblood pressure and cell growth (Terzic et al. 1993;Zhong et al. 1999). Alterations in normal  a 1 -ARfunctions may contribute to the pathophysiologyof diseases such as hypertension, congestive heartfailure and benign prostatic hyperplasia. Thus far,three  a 1 -AR subtypes,  a 1A ,  a 1B  and  a 1D , havebeen cloned and pharmacologically characterized(Cotecchia et al. 1988; Schwinn et al. 1990;Lomasney et al. 1991; Perez et al. 1991, 1994). a 1 -AR subtypes can exist as homo- and hetero-dimers.  a 1B -AR interacts with  a 1A  or  a 1D -ARs(Uberti et al. 2003). The hetero-dimerization of  a 1B -AR with  a 1D -AR is associated with increased Sunil M. Khattar, Roop Singh Bora and PriyankaPriyadarsiny contributed equally to this work.S. K. Khattar ( & )  Æ  R. S. Bora  Æ  P. Priyadarsiny  Æ A. Gautam  Æ  D. Gupta  Æ  K. S. SainiDepartment of Biotechnology, Ranbaxy ResearchLaboratories, Plot No. 20, Sector 18, Udyog ViharIndustrial Area, Gurgaon 122001 Haryana, Indiae-mail: sunil.khattar@ranbaxy.comA. Tiwari  Æ  K. Nanda  Æ  R. Singh  Æ  A. Chugh  Æ V. BansalDepartment of Pharmacology, Ranbaxy ResearchLaboratories, Plot No. 20, Sector 18, Udyog ViharIndustrial Area, Gurgaon 122001 Haryana, IndiaK. MookhtiarNew Drug Discovery Research, Ranbaxy ResearchLaboratories, Plot No. 20, Sector 18, Udyog ViharIndustrial Area, Gurgaon 122001 Haryana, IndiaBiotechnol LettDOI 10.1007/s10529-006-9148-x  1 3 ORIGINAL PAPER Molecular cloning, stable expression and cellularlocalization of human  a 1-adrenergic receptor subtypes:effect of charcoal/dextran treated serum on expressionand localization of   a 1 D -adrenergic receptor Sunil K. Khattar   Roop Singh Bora   Priyanka Priyadarsiny   Aarti Gautam   Dikshi Gupta   Atul Tiwari   Kamna Nanda   Rahul Singh   Anita Chugh   Vinay Bansal   Kasim Mookhtiar   Kulvinder Singh Saini Received: 31 May 2006/Accepted: 26 June 2006   Springer Science+Business Media B.V. 2006  surface expression and coupling efficiency of   a 1D -AR (Uberti et al. 2003; Hague et al. 2004a).Further, heterologous expression of recombinant a 1 -ARs in various cell lines has shown that cel-lular localization of   a 1 -ARs is complicated:  a 1A -and  a 1B -ARs are predominantly localized on thecell surface, whereas  a 1D -AR is mainly intracel-lular (Hirasawa et al. 1997; Hrometz et al. 1999;McCune et al. 2000; Piascik and Perez 2001).Hague et al. (2004b) have shown that  N  -terminaltruncation of   a 1D -AR increases its translocationto the cell surface.In order to address the above mentionedissues, we have cloned and expressed the full-length human  a 1A -,  a 1B - and  a 1D -ARs in humanembryonic kidney-293 (HEK-293) cells. Weexamined the stability of recombinant cell linesexpressing these receptors at higher passages.We report here for the first time thatrecombinant HEK-293 cells expressing  a 1D -ARcultured in the presence of charcoal/dextrantreated FBS lead to increased stability andhigher expression of the receptor on the cellmembrane as compared to the cells grown in thepresence of FBS where it was predominantlyintracellular. Materials and methods Cell lines and plasmidsDU 145, SK-N-MC and HEK-293 cells wereobtained from ATCC. pIRESpuro vector wasobtained from Stratagene.Cloning of human  a 1 -AR subtypesTotal RNA was extracted from DU145 (for iso-lation of human  a 1A - and  a 1B -ARs) or SK-N-MC(for isolation of human  a 1D -AR) cells using Trizol(Invitrogen). First strand cDNA was synthesizedusing Superscript II reverse transcriptase (Invi-trogen) with oligo dT/random primer. The full-length coding region of each gene was amplifiedby PCR using gene specific primers. The cDNAswere cloned into the suitable restriction sites inpIRESpuro expression vector.Cell culture and transfectionsHEK-293 cells were propagated in DMEM sup-plemented with 10% (v/v) FBS either heat inac-tivated or charcoal/dextran treated, 100  l gstreptomycin ml –1 and 100 U penicillin ml –1 in ahumidified atmosphere with 5% (v/v) CO 2 . Stablecell lines were obtained by transfection of expression vector pIRESpuro containing thecDNA constructs of each of the human  a 1 -ARsinto HEK-293 cells using Lipofectamine 2000reagent. Stable clones were selected for resistanceto 3  l g puromycin ml –1 . Cells were harvested andmembrane preparations were analyzed by Wes-tern blotting and radioligand binding assays.Western blottingThe cell pellets were lysed with lysis buffer[50 mM Tris/HCl pH 7.8,150 mM NaCl, 1%(v/v)Nonidet P40] and membrane fractions were sol-ubilized with 2% (w/v) n-dodecyl- b - D -maltoside(DM). The protein samples were separated on7.5% SDS-PAGE, transferred to nitrocellulosemembranes and blotted with goat polyclonal IgGspecific for  a 1A  or  a 1B  (1:500) or rabbit polyclonalIgG specific for  a 1D  (1:1000) (Santa Cruz Bio-technology, Inc.). Each of the  a 1 -AR subtypespecific proteins was detected using a chemilum-iniscence assay kit (GE Healthcare).Radioligand binding assaysTransfected cells from culture flasks weredetached with 5 ml PBS pH 7.4 containing 5 mMEDTA. Cells were centrifuged at 2000  g for 5 minand re-suspended in 4 ml homogenizing buffer(5 mM Tris/HCl, 5 mM EDTA pH 7.4) supple-mented with protease inhibitors and homoge-nized for 30 s with a polytron homogenizer PT1300D (Kinematica AG) at 14,000 to 20,000 rpm.The homogenate was centrifuged at 15,000  g  for30 min at 4  C. The membrane pellet was re-sus-pended in 500 (l assay buffer (50 mM Tris/HCl,1 mM MgCl 2  pH 7.4). The protein in homoge-nized membranes was estimated using a proteinassay kit (Bio-Rad). Radioligand binding assayswere performed as described previously (Khattaret al. 2006). The data was analyzed by non-linear Biotechnol Lett  1 3  one site binding hyperbola curve using GraphPadPrism software.ImmunocytochemistryStable HEK-293 cell lines expressing ( a 1 -ARwere grown in four well Lab-Tek chamber slidesfor 24 h at 37  C. The cells were fixed with 2%(w/v) paraformaldehyde/0.1% (v/v) Triton X-100for 20 min at room temperature. The cells wereblocked in 10% (v/v) FBS for 20 min at roomtemperature. The cells were incubated with 1:100dilution of appropriate primary antibody followedby incubation with 1:500 diluted Alexa-conju-gated secondary antibody (Molecular Probes). Tostudy the effect of phenylephrine on the cellularlocalization of ( a 1D -AR, cells were incubated with100  l M phenylephrine for 30 min at 37  C. Cellswere analyzed under a fluorescent microscope TE2000-E (Nikon Instech Co. Ltd.).Flow cytometryStable HEK-293 cells expressing ( a 1 -AR weredetached with 5 ml of PBS pH 7.4, containing5 mM EDTA. Cells were centrifuged at 2000  g for 5 min and washed twice in PBS pH 7.4. Cellswere incubated for 30 min at room temperaturein 100  l l of blocking buffer containing BODIPY-FL labeled prazosin [1% (w/v) BSA, 0.1% (w/v)Pluoronic F127, 1  l M BODIPY prazosin in PBS).Cells were washed, resuspended in 500  l l of PBSand analyzed by cell quest program of the FACSCalibur flow cytometer (BD Biosciences).Measurement of intracellular free Ca 2+ Confluent HEK293 cell lines expressing  a 1D -ARwere detached with 1 ml PBS pH 7.4, containing5 mM EDTA. Cells were washed, resuspended inculture medium and incubated for 45 min at 37  Cin a CO 2  incubator. Cells were washed with KrebsHEPES buffer pH 7.4 (118 mM NaCl, 4.7 mMKCl, 1.2 mM MgSO 4 Æ 7H 2 O, 1.2 mM KH 2 PO 4 ,4.2 mM NaHCO 3 , 11.7 mM  D -Glucose, 1.3 mMCaCl 2 , 10 mM HEPES). Cells were loaded withfluorescent Ca 2+ indicator Fura-2AM (3  l M) for30 min at 37 o C. Cells were washed with KrebsHEPES buffer, containing 0.5% (w/v) bovineserum albumin, to remove unincorporated Fura-2AM and transferred to 96-well black clear bottomplate. The agonist, phenylephrine, was injected tothe wells in increasing concentration. Ca 2+ responses were measured as the fluorescenceemission ratio at 510 nm of Fura-2 alternatelyexcited at 340 nm and 380 nm (F 340 /F 380 ), usingbottom optics in Flexstation (Molecular Devices).Each experiment was run in triplicate and eachvalue represents the mean ± SEM from threeindependent experiments. The results wereexpressed in terms of percent response to maxi-mum. The agonist concentration, which produces50% response to maximum (EC 50  value), wasdetermined from dose response curve throughnon-linear regression analysis using Graph PadPrism software (version 4.02). Results and discussion Generation of stable cell lines expressing a 1 -AR subtypesThe cDNAs encoding for three subtypes of   a 1A -, a 1B - and  a 1D -ARs were isolated by PCR cloning.The receptors were cloned in to pIRESpuromammalian expression vector. All the threecloned receptor cDNAs were confirmed byrestriction mapping and DNA sequencing. Thesequences of   a 1A -,  a 1B - and  a 1D -ARs were foundto be same as per the published reports.HEK 293 cells were transfected with thepIRESpuro plasmid harboring  a 1 -AR cDNAs( a 1A ,  a 1B  and  a 1D ). For obtaining stable trans-fectants, several Puromycin resistant clones foreach receptor cDNA were isolated. Expression of adrenergic receptors in stable recombinant celllines, termed as HEK- a 1A , HEK- a 1B  and HEK- a 1D -AR, was analyzed by Western blotting usingsubtype specific antibodies. Specific protein bandsof   a 1A -,  a 1B - and  a 1D -ARs at 55 kDa, 65 kDa and70 kDa, respectively, were detected in theimmunoblot (Fig. 1, panel A, B and C). All thethree subtypes showed expected molecular weightwith the subtype specific antibodies without anycross reactivity and no signal was detected in thecontrol HEK 293 cells. Biotechnol Lett  1 3  The pharmacological properties of all the threesubtypes were studied by radioligand binding as-say using [ 3 H]Prazosin as non-selective ligand for a 1 -AR. Non-specific binding was estimated using10  l M terazosin. The binding site densities ( B max )for  a 1A -AR in the recombinant cell line was2030 fmol/mg of protein with a  K  d  value of 0.28 nM, for  a 1B -AR was 2007 fmol/mg of proteinwith a  K  d  value of 0.14 nM and for  a 1D -AR was1003 fmol/mg of protein with a  K  d  value of 0.7 nM at passage 5 (Table 1).Heterologous expression of GPCRs in mam-malian cell lines is generally unstable, as with thesubsequent passaging and cell division, expressionof these proteins tends to decrease. In order tocheck the stability of our recombinant clones,expression of   a 1 -AR subtypes in cell lines wasmonitored by Western blotting and radioligandbinding assay for up to 35 passages. Western blotanalysis revealed that there was no change in theexpression level of   a 1A  and  a 1B -ARs even at thepassage 35 (data not shown). The binding sitedensity for  a 1A  and  a 1B -ARs in recombinant cellline at passage 35 was 3933 and 3400 fmol/mg of protein, respectively, as revealed by radioligandbinding assay (Table 1). These data suggest thatrecombinant cell lines expressing  a 1A  and  a 1B -ARs are highly stable. Unlike these two subtypes,expression of   a 1D -AR was found to be veryunstable. Radioligand binding studies revealedthat there was decrease in binding site density inthe recombinant cell line expressing full-length a 1D -AR with  B max  of 110 fmol/mg of protein atpassage 15. A recent study by Pupo et al. (2003)also highlighted that the expression of recombinant  a 1D -AR results in a much lowerbinding site density as compared to the expressionof   a 1A  and  a 1B -ARs. They also showed thatdeletion of the  N  -terminal 79 amino acids caused Fig. 1  Western blot analysis of (1-AR subtypes expressedin HEK 293 cells. Membranes from the cells expressingeach subtype were solubilized with 2% (w/v) DM sampleswere run on SDS-PAGE, transferred to nitrocellulosemembranes and Western blotted with anti goat polyclonal a 1A  IgG (panel A) or  a 1B  IgG (panel B) or anti rabbitpolyclonal  a 1D  IgG (panel C). Signals were detected usingchemiluminiscence assay kit. Arrows indicate apparentmonomers and probably the dimeric forms of each subtype Table 1  Maximum binding site density ( B max ) andequilibrium density constant ( K  d ) values of specific [ 3 H]Prazosin binding to HEK-293 cell membranes expressing a 1 -ARsReceptorsubtypes K  d  (nM)  B max  (fmol/mg protein)Passage 5 Passage 35 a 1A -AR 0.28 ± 0.06 2030 ± 195 3933 ± 227 a 1B -AR 0.14 ± 0.02 2007 ± 186 3400 ± 265 a 1D -AR 0.7 ± 0.07 1003 ± 83 355 a ± 14Rat liver b 0.09 ± 0.03 196 ± 34 168 ± 39Rat submaxillary b 0.15 ± 0.007 146 ± 40 152 ± 35Saturation analysis of specific binding was used to deter-mine Bmax and Kd values for [3H] Prazosin. Each valuerepresents mean ± S.E.M. of at least three experimentsperformed in triplicate a This value was obtained after growing recombinant celllines in presence of charcoal/dextran treated FBS b Rat liver and submaxillary membranes were used as anassay controlBiotechnol Lett  1 3  a 6- to 13- fold increase in receptor binding sitesas compared to the full-length  a 1D -AR. Earlierstudies have also demonstrated that the full-length  a 1D -AR does not undergo significant N  -linked glycosylation in HEK 293 cells despitethe presence of two consensus sites in its  N  -ter-minus (Vicentic et al. 2002). The lack of glyco-sylation indicates that  a 1D -AR is not properlyprocessed in HEK 293 cells. Pupo et al. (2003)observed that N  -terminal deletion mutant showedsubstantial glycosylation in HEK 293 cells,despite the elimination of one of the two con-sensus glycosylation sites, indicating that full-length and N  -terminal deletion mutant of   a 1D -ARare processed differently in these cells.Cellular localization of   a 1 -AR subtypesExpression and localization of   a 1 -AR subtypes inHEK 293 cells was analysed by immunocyto-chemical analysis at different passages. Usingcommercially available antibodies,  a 1A and  a 1B -ARs were found to be predominantly localized onthe cell membrane in recombinant HEK 293 cellsas shown in Fig. 2, panels B and C, whereas theexpression of   a 1D -AR (Fig. 2, panel D) was pri-marily intracellular as shown by punctate staining.Several recent reports have suggested that theexpression of   a 1D -AR is primarily intracellular,clearly indicating that this receptor protein doesnot translocate to the cell membrane due toimproper folding (Pupo et al. 2003). Hague et al.(2004b) indicated that  N  -terminus of   a 1D -ARprevents its cell surface expression and deletionof the  N  -terminal 79 amino acids resulted in itslocalization on the plasma membrane.Expression of all the three subtypes was alsoanalysed at different passages by fluorescence-activated flow cytometry (FACS). Previous stud-ies showed that BODIPY-FL prazosin can beused as a specific fluorescent ligand for  a 1-AR,although the K  d  value of BODIPY-FL prazosin isapproximately 100 times higher than that of theoriginal unlabelled prazosin (Hirasawa et al.1996; Sugawara et al. 2002). To detect the cellsurface expression of adrenergic receptors inmammalian cells, recombinant cell lines i.e HEK- a 1A , HEK- a 1B  and HEK- a 1D  were stained withBODIPY-FL prazosin as ligand and analysed byFACS. As shown in Fig. 3, all the recombinantcell lines expressing  a 1-AR subtype were posi-tively stained by BODIPY-FL prazosin. In case of recombinant cell lines HEK- a 1A , HEK- a 1B  andHEK- a 1D , 60%, 60% and 40% cell populationshowed a significant shift in fluorescence inten-sity, respectively, as compared to wild type HEK293 cells (Fig. 3, panels A, B and C).Effect of charcoal/dextran treated FBSon the localization of   a 1D -ARIn order to improve the localization and expres-sion of   a 1D -AR, HEK- a 1D -AR recombinant cellline was cultured under different conditions.Tsang et al. (2001) have demonstrated that cysticfibrosis transmembrane conductance regulator(CFTR), a cAMP-mediated Cl(–) channel foundin most epithelia, could be regulated by variousculture conditions including growth in the pres-ence of charcoal/dextran treated FBS. Their datasuggested that steroid hormones present in FBSmight regulate CFTR expression in vitro. Char-coal/dextran treatment of FBS is known to absorbfree hormones, free fatty acids, and various otherserum factors from their protein bound counter-parts (Lindquist and de Alarcon 1987). Therefore,HEK 293 cells expressing  a 1D -AR were grownin the presence of 10% (v/v) charcoal/dextran-treated FBS viz-a-viz 10% (v/v) FBS andimmunocytochemical analysis was performed.Interestingly after culturing cells in the presenceof 10% charcoal/dextran-treated FBS, there wasdramatic change in the localization of   a 1D -AR inHEK 293 cells. As shown in Fig. 4, panel B, mostof the  a 1D -AR protein was translocated to the cellmembrane. Binding site density of   a 1D -AR atpassage 35 was 355 fmol/mg protein (Table 1).HEK- a 1D  cells cultured in the presence of char-coal/dextran-treated FBS, when analyzed byFACS, showed an increase in the fluorescentintensity (around 60% shift) as compared to thecells cultured in the presence of FBS (40% shift)(Fig. 3, panel D).These results were further corroborated by afunctionalassaytodeterminetheagoniststimulatedintracellular Ca 2+ mobilization. Figure 5 illustratesthe concentration dependent response for phenyl-ephrine-inducedelevationinintracellularfreeCa 2+ Biotechnol Lett  1 3
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