Biochimlca et Biophysica.4c:a, 1134( I ~ ) 169-177 © 1992ElsevierSciencePubli~ker~B.V. AI! rights reserved0167.4~/92/$05.00


BBAMCR 13122

Stable expression of recombinant human a2-adrenoceptor subtypes in two mammalian cell lines: characterization with [3H]rauwolscine binding, inhibition of adenylate cyclase and RNase protection assay A n n e M a r j a m S k i ~, S a d A l a - U o t i l a ", Kirsti L u o m a l a ", M a r i a Per~lii e, C h r i s t i a n J a n s s o n ~, M a r k k u J a l k a n e n b, J o h n W . R c g a n d a n d M i k a S c h e i n i n " Departments of ~ Pharmacalogy and h Medical Biochemistry, Univerd~y of Tud:u, Tarku (Finland), "Department of Biocl~emis~ryand Pharmacy, ,~bo Aleaa~r~ SF-2~Y~OTurk~ F~nlana~and ¢ Deparvnent of Pharmacolosy and Taxicology, Collegeof Pharmacy, Onhrerdty of Adzom~ Tucson, AZ (USA)

(Received30 October 1991)

Keywords: a2-Adrenoceptorsubtype;Stabletransfeclion;Cell line;[3HJRauwoDcin~h~nding;Aden~la~ts~lasea¢livil,j; R N ~ I~Otectionassay Cloning of the genes encoding distinct sublypes of human o2-adrcncrgic receptOrS (¢z-AR) allows 1he sop,ante recombinant expression of carla individual subt3rp¢ in hetemlosous systems. We report here the transfecliou, ~leetion and prelim/naP/ pharmacological characterization of two mammalian ceil lines, -".dhegentSh/onogi $115 muse mammary,lumonr (:re]Isand hulnan B-]ymphoblasloid IBW4 cells 8rowing in suspension, expressin~ the human az-AR subtypes a2-CA and az-CI0 at densities of approx. 2.10 5 receptorS/celL Transfcetion of the subtype genes was verifgd using a specific llNas¢ gmt¢,.'lion ansa'/. Pharmacological characterization was carried oat with [3H]rauwols¢ine binding, whkh was inh1~led by ~ a z o l i n e and prazoxin in a subtylm-suleetiv¢ manner. The scnsiti-A~ of (-)-noradtenaline binding to the GTP-analogtte $'-geanylylimidodiphosphate suggested that the receptors are oau~ed to G-proteins. This was verified m Sll$ cells by eft'relent inhibition of forskolin-stit~ulatcd cA./MPproduction by the tz2+ARagonists, ( - )-noradrenaline a,tl r~nidiae. "rhes¢ cell ~ thus appear to be suitable for pharmacological studks on receptor funetiorl and ligand binding.

lntmductioa a2+Adrenergic receptors (a2-AR) convey signals cartied by the ncurotransmitter noradrenaline and the hormone adrenaline to intracellular second messengers via pathways involving 8canine nud¢otide binding proteins (G-proteins). az-AR located in the central nervous system mediate the therapeutic effect of tke antihyperteltsive drug clonidi~e, and are intricately involved in the ~hysio!osical regulation of sympathetic outflow aud ca.diovascolar control m.-chanisms, endocrine functions, and in the modulation of vigilance,

Abbrcvlstlens; PMSF, ISeayhnethylsulfonyl fluoride; IBMX, i~obutylmetlfflxanthiue; (3pl~lqH)p, §'*gusaylyliraidodiphosphate; DMEM,Dulbecgo'smodifiedEagle'smeditnn;FE:S,fetal calf serara; PBS, p,~m~ph~tc-b',dfe~!~,,l~ne:~z-AR,~,-adhtcncrl~creceptom. Correspondence:A. Ma~jem~ti,Departmento1 P~tmacolo~, Unive~ty elfTurku, giinamyllFaga~u10, 5F-20520Turke, Finland.

cognition, emotions and nociception [1]. ]n peripheral tissue.% prcsynaptic a2-AR mediate feedback inhibition of x]oradre~aline release from sympathetic nerve endin~. Both postwnaptic and extrasynaptie az-AR are involved in contraction o f smooth muscle, metaUolio regulation in liver and adipose tissue, endocrine regulation, control of exocrine secretion in the f~stromtes. final tract, control of i n ~ pressure and platctet aggregation [1,2]. Radioligand binding expermnents have previously demonstrated ~='e3istence of pharmacological a2-AR suMypes (azA, a2a, a~c) with distinct seiectivity profiles for some model compounds [3,4], but the roles of these subtypes in mediating the various a2-AR functions listed above are not clear, with the exception of the aT.~-AR on blood piatelets. Three human az-AR subtype genes have been cloned to date, d e s / p a l e d a2-C10, a2-C4 and ,%-C2 according to their chromosomal location [5--7]. The deduced peptide sequences o f the receptors identify them as members of the rhodolpsin-like receptor f a n fly, which arc integral celt membrane proteins witk

170 seven putative transmembrane domains [8]. The a2-Cl0 gone product has been identified as the pharmacologic,ally defined platelet-type azA-AR [5]. The a2-C4 eDNA was cloned from a human kidney eDNA library, and is thus expected to be expressed in this organ [6]. In rat tissues, expression of the receptor subtype genes corresponding to the human a : C 4 and a2-Ct0 was observed in marly brain areas, whereas expression of a2-C:2 was found only i~k some peripheral tissues Giver mid kidney) [9]. Genetic subtypes of a2-AR thus appear to have unique tissue distributions [9]. in addition, they may couple differently to intra¢¢llular signalling mechanisms [10,1t] and mediate distinct physiological functions, and are therefore possible targets for the development of subtype-selective pharmacological agents. The compounds most often employed to discriminate the pharmacological a2-AR sub~pes, oxymetazoli~e (which bitlds preferentially tn ~zA-AR) and prazosin (which has relatively high affinity for the ¢rze and a2c subtypes) [4], have also been found to show rclath'e selectivity towards the genetic otz-AR subtypes. These agents are not, however, specific for u2-AR; they also have significant effects at a,-AIL and are therefore .or suitable for pharmacological studies on the a : A R subtypes in rive or in isolated organ preparations. The search for specific and subtype-selective a2-AR iigands benefits greatly from the cloning of different ot2-AR genes, since recombinant techniques may now be employed to generate stable cell lines expressing single receptor subtypes. In addition to screening of new pharmacological agent~, such heterolngous expression systems may prove to be very useful in the elucidation of receptor-coupled signalling mechanisms. The target cells for receptor gone transfer may also be selected taking into account the expre~ion of different G-proteins and/or enzymes and ion channels required for specific second messenger functions in different cell types [12]. In this communication we report the generation, selection and preliminary pharmacological ¢haracterizat/on of stable recombinant lines of Shiunogi 115 mouse mammary mmour cells (SI15) and transformed human 3-1ymphoblastoid cells (IBW4) expressing the a ' : A R subtypes a2-CA and ~2-C10. Rod]el]Rand binding studies demonstrate the expected o~der of potency of prazosin and ox3,metazoline at these recombinant receptors. Both receptor subtypes are inhibitory to adenylate cyclase in $115 cells.

['~H]Rauwolscine was from DuPont New England Nuclear, and [~H]adenine, ['4C]cAMP and [a2P]UTP were from Amersham, Prezosin, oxymetazoline, e[onidine, forskolin, (-)-noradrenaline, d,.~xamethasone, testosterone, PMSF, propranolol, quinacrlne, IBMX, Gpp(NH)p and the neomycin analogue Geneticin ~ ((3418) were from Sigma, Phentolantine wa,~ front Ciba-Goigy. Cell culture reagents were supplied by Gibed, Other reagents were of analytical grade, and were purchased from commercial sources. Cell culture dishes and flasks were from Nunc. The expression vector pMAMneo was front Clootech Laboratories, Paid Alto, CA. The p[asmids pSPa2C4, clone 8 and pBCa2-C10 [6] as well as the stably transfoctod az-adreaergi¢ coil lines PC4 and PC10 [10] were l~enerously provided by Dr. P,J. [~tkowitz (Duke University Medical Center, Durham, NC). $115 cells were originally from Drs. P.D. Darbre and R.B.J. King, The Imperial Cancer Research Fund, London, UK [13]. IBW4 cells were provided by Dr. S. Jalkanen, University of Turku, Finland [14].

Construction of expr¢$siot~ ~ctor8 The plasmid pBCo¢2-CI0 was cleaved with Noel and ///nil, was blunt-ended with Klenow, and the 1A kb fragment containing the entire coding region of a : C 1 0 was isolated, pSP65 (Promega)was digested with Sinai, was phosphatased, and was ligated to the 1.4 kb a2-Cl0 fragment to yield pSP65a2-C10NH, pSP65a2-CIONH was linear]zeal with EeoRI~ blunt-ended with Klenow and cleaved with Sail. The EcoRI-Salf fragment (1.4 kb) containing the ~z~CI0 coding sequence was ligated to pMAMneo which had been linearized with Nhel, blunt-ended, and digested ,~ith Sail The resulting expression construct called pAUazCI0 is shown in Fig. 1. The plasmid pSPa2-C4 was cut with BstXl and /-/indIll and the 3 kb fragment containing the amino

az.ClO ~d.~ng

Materials and Methods

Materials Restriction enzymes, T4 DNA polymerase and lig. ase. RNase A and RNase TI were purchased from Boehringer-Mannheim.

rig. I. 1"heexpre~ionconstrue!pAUa2CI0.

171 terminal coding region of az+C4 was isolated. A partial done containing the t a r b e l l terminal portion of az-C4 (clone 8), was digested with BstXI and Hindlll and the 1.2 kb fragment was isolated _~odwas ligated ~o the aforementioned 3 kb fragment to yield pSP65az-C4BH. pSP65a2-C4BH was cut with EcoR! and the 1.6 kb fragment containing the a2-C4 coding sequence was isolated, blunt ended with Klenow, and ligated into the blunted Sail site of pMAMneo. The resulting expression construct was called pMAU~2C4.

Cell culture Adherent S115 cells were cultured in DMEM suppietflellted with 20 mM Hope'+, 20 mM NaHCO 3, 5% heat-inactivated FCS, penicillin (100 U/ml), streptomycin (59 pg/ml), sodium pyruvzte (1 raM), and testosterone (10 riM). Lymphoblastoid IBW4 ceils growing in suspension and adherent stably transfec~ed fibroblasts (PC4 and PC10) were cultured in DMEM supplemented with 10% FCS, 20 mM Hopes, 20 mM NaI-ICOa, penicillin (100 U/ml) and streptomycin (50 ~g/ml). All cell lines were grown in 5% CO2 at 37~C. Transfected IBW4 and PC cells were treated with dexamethasone 24 h before harvesting, because the expression vector pMAMn¢o contains the g[ucocorticold-inducible MMTV-LTR prom3t~r and dexamethasone can be used to increase receptor espre~ion.

75-125 ~g of proteiln was incubated with [3H]rauwolscine (0.06~8 nM) in a final volume of 0.5 ml a," ~ for 30 rain. Non-specific binding was determined by including 10 pM phentolamine in parallel assays. Competition studies were performed using [3H~rauwolseine concentrations close to its K d at each receptor subtype and 8-16 concentrations of the competitors, prazusin, oxTmctazoline, ( - )-noradrenaline and ( - )-noradrenaline iv; the pre~nCe of ~ GTIP-aoalogue, Gpp(NH)~. A Gpp(NH)p concentration of 10 ~tM was selected for this study, based on pilot e~q~erimentscarried out with human platelet membranes (K. Luomala, unpublished observations). Bound radioactivity was separated by filtration on Whatman G F / B filter strips, using a Brandcl cell harvester 100 fmol/mg protein). Thus, results are presented for only three cell lines (SllS-a 2C4, $115-a2-C10 and IBW4-a2-CA). TransfectJon with these pMAMneo-based constructs did not affect the morphology or the growth rates of the cells, except for the a~uired resistaDce to the neomycin analogue G418. Saturation isotherms of [3I-l]rauwolscine binding and LIGAND-derivcd K d (receptor affinity) and B,m (receptor density) values were determined in three separate experiments for each cell line. Representative plots are shown in Fig. 2. The r~,epter ~xpression levels in these stably transfected cell lines (passages 2-23) were 590-900 fmo]/mg protein (Table 11. This corresponds to receptor densities of approx. 2- 10s per cell. The proportions of specific binding (defined by 10 p.M phentolamine) in $115 cells expressing az-CA and ~ 2 - C 1 0 receptors were 75 and 71%, and 95% in [BW4a2.C4 cells, when the concemration o f [3H]rauwoiscine was equal to each receptor's K d value. For comparison, some results are also shown for previously de.~..xibed fibroblastic PC4 and PCI0 cell lines [11]. All binding studies with PC cells were carried out in sodium/potassium-phosphate buffer instead of our standard conditions, because [3H]ranwoiscine binding to PC cell homngenates was erratic in TME buffer.


3000 _

~ ~ i ~ i ~






~~am .


2 4 6 ~HlRauwolscine (nM)



12000 o , . ~




.,o u©











dpm 18000 15000




' "


2 4 6 [SH]Rauwolscine [nM)




LIS I,IO l~s

• &

12000 9000

Fig, 2. Saturation isotherms of [:ZH]raewolscine binding (dpm) to homogonates of zransfected all5 (A, at-C-4; B, a2-ClO) and IDW4 ~ l b ( C nZl~)" 75--]~ ~g protein was incubated with the indicated concentrations of [aH]muwolsci;l©. tO ~M phentolamimewas used (o doienninc non-specific 1~nding, Tolal []Hjmu,~ni~ir,: bind]~]s ~s indicated by ~rctes. non-specific bindJsi[( by ~uaTcs and specific binding (total - non.specific) by triangles. Insets: Rosenthal p]ols of [~]-i~auwolscine binding. Data are representative of thTce separate experJrm=nl,with each point ped'ormed in [r;p]icate.


3000 0


2 4 ; ISHlRauwolscine (riM)




Characterls~ies of [-~H[rauwohci.e binding tO homgRenates of transfueled S)lS, IBW4 and PC cell lines

Jnhibiti~ of [~H]ramt~t~lm, B t ~ to arz-AR subl~l~s by lwa~os~, o.~.y~.~etrzol'm¢and { - ).na~drenallne in Jhe absence and presence n[ tO ~ M G o M N H ~ p

Specificity o[ binding was determined with l0 ~M phrnlolamine. Homogen~tes of S|1:~ and IBW4 c¢|ls were assayed in TME barfer and PC cells in s~lium/pot=ssium-phospbate buffer, as described under Materials and Methods. laW4 cells were trcuted with 5 .aM a~td PC cells with I p.M dexamethasone befo:e harvesling, Values ~eprexe.I means±SJ~, from three separate eXperimenls. B r ~ , ~©c~ptor density; K,~, IX~'~ptot affinity; nil, Hi~ coeft"tcienz(slope).



K~ tnM)


Sl lS~z~-C4

681 -J:55




831 4-13 58± 7 593+20 ]47± 17

0.8c~±0.02 tg,8-t0.08 0.7.8±0,0"~ 0.95-~0.07 6.40+0~52 0.99±0.0l I.t~ £ I).13 0.98 4- G.01

PC4 S|15-a2-Cl0 FClO

Saturation studies with [3H]rauwolscin¢ and S115 homogcnates produced similar results in both buffers (data not shown). Preliminary experiments wieh different dexamethasone concent,ations (0.3-10 p.M) indicated that 5 pM dexamethasone treatment of IBW4a2-CA cells resulted in optimal, 3-4-fold increases in receptor density compared to untreated cells. Receptor expression could not be increased further in $115 cells by dexamethasone. No endogenous expression of a2AR could be detected by [3H]rauwols¢ine binding in either of these cell lines before transfection ~r after transfeetion with another pMAMneo-based expression construct containing axons 4 and 5 of the haman 8rowth hormone 8rue (data not shown).

lnhihimr ~etazoline Prazosin


180 ±J.| qs 4-13 t I 1.4 4-0.60

0.94 12,6 "1:0-05± 3 3 LZ] ~,36~ ±O.Ol ±1415 H 3.8 ±2,8 0.88 + 0.t9 L 12[12 ±3"~ 1.00 1079 ±0.06 ±405

1.12 ±0.02 1,17 ±0.1D

105 ±]O 83 ±11 H 6.2 ~ 1.3

0.5 t + 0.O7


0-83 ±0J]6 1.00 -I-0.0~

+ 0.f16 L 8_'.N} :1:1% 862

±0.12 ±73

0.92 ±O.OI

The pharmacologkal s~,btype characteristics of the cell lines were investigated with two subt31~-selective iigands, prazosin and oxymetazotia¢, that competed with |3H]muwolscil~g with the expected order of potency at the two =2-AR subtypes (Fig. 3 and Table !!). The prazosin/ocjm~tazolin¢ Kfrafios, o.rten used to characterize a2-AR subtypes, were 0.5 for az-C4 and 260 for a2-Cl0 receptors in S115 cells and 0.8 for nz-C4 in IBW4 cells. The displacement curves were





40 20 '

40 t 20


JR.W4-,mZ-C4 K~ {aM) n H




S| 15~z-CI0 Kt (aM) n .

L715 4-18] N A + GI~(NH)p 948



S115-¢~-C4 K~ (aM) n H

Specific binding (Z|

Specific binding (g) 120 100

The eoaeenlratiou of the dlu$ that inhl~eited specific bindinll by 50% (ICa)) was used to eakutal© K I */.nines mhql tim ChenlPPrumff equalion. Values represcnl rr~ans± S.E. | ~ thrnn separate expcr~. taunts. H, hish affinity binding sil~ L, low affiniW binding site.



ConGentration (M|

o-s- to'-'



10-'o lo-' 10-" lo"


Concentration (M)

RS. 3. lnhibhlon of l~Hkanwobcm~ biflcLillqJby prazmln (sqttar~s)~.nd o,vxT~=czc,.~,_JineI,~!.esP. ~ , ~ deaole ee2-CAand open s",jmbu~.s ¢%-Cl0 retepton. Homogeaates of transfected $115 (At and IBW4 (B) cells were incubated with [3HJnuwol~ae at ¢eacentraliom nero- its K e and 8-12 cct:cenlratio~s of the ¢ompatito~, Data points ate aleam of three separate ex~dments pelf0fmed in lrildicat:, S l a d a r d errors were in tbe range of 0.4-'/%, and are not ~ for clarity.


steep with both compounds (see TaMe lI for Hill coefficients: n,), and the binding data was unambiguously modeled by a one-site fit in all cases.

Specific binding I~) 120 -

The natural agonist ( - ) - n o r a d r e n a l i n e was =~]so

hotrod with high affinity. Displacement curves with


(-)-noradrenatine were shallow for both receptor sub~

types in S115 cells (Hill coefficients 0.88 and 0.5I for o~z-C4 and ~2-C10) (Fig. 4 and Table II). Computer analysis with LIGAND indicated that the data could bc statistically significantly more accurately modeled with a two-site fit than with a one-site fit. The two-site analysis yielded K~ values of approx. !.4 nM and 715 nM for the high and low affinity forms of the az-C4 receptors. The proportion of high-affinity sites was 27-46%. At ~2-C10 receptors in Sl15 cells, the corresponding proportion of high-affinity sites was 44-56%, and the K~ values were approx. 3.8 nM and 1202 nM. This tendency to biphasic compelition of (-)-noradrenaline for ['~H]rauwolscine binding was abolished in the presence of i0 ,aM Gpp(NH)p. The displacement curves were shifted significantly to the right, and the slopes were clearly steeper (Hill coefficients I.O0 and 0.68 for otz-CA and -2-C10) compared to the

egperimems with (-)-noradrenaline in the absence of Gpp(NH)p. Similar results were obtained also with IBW4-~2-C4 cells (Fig. 4 and Table II). The coupling of the 0r2-AR subtypes expressed by S115 ceils to inhibition of adenylate cyclase activity was investigated using stimulation with 40 /~M forskolin. Forskolin-stimulated cAMP formation in SlI5 cell~ was effectively inhibited by 100 p.M.(- )-noradrenaline (by 72 and 70% for a2-C4 and az-C10, respectively); 100 pM cionidine inhibited cAMP formation by 51 and 41% in the same cells (Fig. 5). The expression of these two ¢2-AR subtype genes, az-C_A and a2-Cl0, was also detected on the transcriptional level using a sensitive RNA solution hybridization/RNase protection assay (Fig. 6). RNA isolated from stably transfected PC fibroblasts was used as positive control material, Total cellular RNA from untransfected SII5 and IBW4 cells was also tested, and no background levels of hybridization were detected. Neither was there any detectable cross-hybridization between the different subtypes. The effect o f dexamethasone induction in IBW4 cells expressing the a2-C4 receptor subtype was also evident as elevated amounts of specific RNA in the RNase protection assay (Fig. 6).


60 40 ~.0 0

10-1o 10-I












Specific binding (~) 120 100 80

60 40 2O 0


10-Io 10-o 10-e 10-1' 10-| 10-'5 10-4 IO-S Concentration (M) Specdm bindirJg IS) 120 100 80 80 40

FiB. 4. Inhibition of [3H]rattwolsein= binding Io homogenalcs of transrected S115 ceiis (A, ~2-C4; B, ¢*~-CiO} at|d ]BW4 ¢¢;l~ (C, =z,.C4) by (-)-ttoradTcnaline in Ihe presence(~uar¢i) ~nd absence (ci[cl=s) of l0 v.M GplgNH)p. Dala lminls are mea~s of three separateexperimentsperformed in triplieate. Standarderrc*s were in the rangeof 0.3-'/%, and are not shown for clarity.

Zo 0 10-to 10-e





C o n c e n t r a t i o n (M)



175 Convor$iofl '1o PHI,cAk,CP (Z]







i BA








Fig. 5~ Inl:ibition of forskolin-.stima]atnd{FSK, 40 #.M) ade.qylat¢ ~¢lase ac~iviP/by 100 gM (-)-noradrenaliR¢ (NA) and 10ft ,aM clonidine ~CLO) in S115 cell's Iransf¢cted tO eapress the ~ A R s,blypes az-C4 (A) and ot~-CI0(B). The bars represent means+ S.E. of sis individual e~periments.BA. basal ( ,. non-stimulated)cAMP formalion.


Cloning of th¢ huma.q geiies encoding distinct a2-AR subtyi~s now ~llows the separate J'ecombinam e x ~ e s sion of each individual subtype, which is a significant advantage compared to receptor material from natural tissue sources. Animal tissues represent ¢omp|ox mixtures of cells expressing different receptor subtypes, which hampers the examination and characterization of individual receptor subtypes. Reueaubinant expression systems may now be used ~¢ produce 8enetically defined receptor material for stud!es on.receptor stracture, function, and regelatidn, as well as to test new, 9otentially subtype-selective pharmacological agents [12]. In this communication we relmrt the transfection, selection and preliminary characterization of two dif[c~¢nt mammalian ceil lines, adherent SI15 mottse mammary tuw~our cells and human IBW4 lympheb~astold c©lgs, growing in suspension, e'~jpressing separately the human ag-AR s~:bt'ypes, az-C4 and a2-Cl0. The capacity of oxymetazoline and prazcsin to differentiate the a2-AR subtypes ~2-C4 &--t a~-C10 was verified in the new cell lines, which shoal- thus be suitable for use



Fig. 6. Reprmenmtive aumradiolpam o~ hybridization of s a ~ l l c ¢,RNA probes to Io~ ¢~llalar RNA hem aon-nanslg-cte."eand t-mnsiL-cted¢uItur¢~¢¢ils: Rlqas¢mmccrRmassay.

in binding assays exploring the subtype-selectlvity of a2-adrenergie agonists and antagonists. The calculated prazosin/oxymetazoline Ktoratios were 0.5 and 260 for the c~2-C4 and a t - e l 0 re~ptors expressed in $115 cells, and 0.8 for the a2-CA receptors expressed in IBW4 cells. These values are dose to the corresponding ratios of 0.5-0.7 and 160-170 reported in COS-7 cells 16.T]. and r a t i ~ of 0.3 and 380 in stable PCd and PC10 fib~oblast cell line~ [11'. ;~: our S11~ and ]BW4 evils, the ~.f~nities of ~auwol~ine and (-)-noradrenaline w¢~ somewhat higher to aa-CA than to a2-Cl0 receptors, as also previously reported f~r these a2.AR subtypes in COS-7 cells and PC cells [6,11]. It thus appea~ the.t_the relative affinity of ligands to receptor ~btypes would be t e n , r e e d across a range of mam.-naben host cells used for het¢l ologous expression, and would be a property determined by the genetic code of each retorter subtype. Ncveribeless, ~,s stated by BuckIcy et at. [12], it is ~till poss~te that differences in the environment or po~t.trauslational modifications of the receptor may influence its binding characteristics. The sensitivity of agonist binding to the GTP-an~ logue Gpp(NH)p suggested that the receptors i~ S11~ and IBW4 cells are efficiently coupled to intracellular signal transduction mechanisms [21,22]. Coupiing to one of the possible second messenger mechanisms of a2-AR [10,11,23], inhibition of adenylate c~clase, was also verified by direct measurement of forskolin-stimulated adenylate ~ydase activity in recombinant Sl15 cells. (-)-Noradrenaline, a full agonist, was able to inhibit cAMP formation by approx. 70%, whereas clonidine, a partial agonist, appeared to be.-l~ss effective (41-51% inhibition). No clear difference~, could be seen in this preliminat3, experiment between ~he a2-AR subtypes. Nevertheless, the higher propotio~ of a2-Cl0 receptors with high affinity for the agoni~t (-)-noradrenaline compared to ¢2-C_,4,may suggest more erEdent coupling of a2-Cl0 to G-proteins in c~115 cells. Because of this coupling to G-prote)ns, the present cell lines should be suitable for studies on receptor function as well as for binding a.~suys, Oxymetazoline, which in pharmacological tests may function as an ¢2-agonist or antagonist depending on the investigated tissue and receptor subtype, had steep, antagonist-type competition profiles at both receptor subtyl~S in our blndin8 studies; its functional properties remain to be investigated in the adenylate ~yda~ assay. The a~-AR cx~ressinu levels in our Sl15 and IBW4 clones were clearly hi~j~er than in natural tissue sources. In other published mammalian cell lines stably expressing a~-AR subtypes, the density of binding sites has typically been in the same range, between 0.3 and 1.4 pmol/mg membrane protein [11,22,24]. Higher expression, 18-35 pmol/mg membrane protein, was reported in C127 mouse mammary tumour cells transfected with a mammalian expression vector containing the mouse

metaliothionein promoter mMT-] [25]. Although the different expression levels may in part be explained by differences in assay procedures, it is also evident that the expression vector pBMT3X [25], which allows the selection of transfected clones in the presence of Cd 2+, may produce more efficient overexpression of mammalian membrane-hound receptors than the vector we used, pMAMneo. The dexam~thasonc cffe~ in IBW4 celts was relatively weak, and the MMTV-LTR prometer of pMAMneo does not appear to be very potent as an inducer of receptor gone expression. On the other hand, the present expression levels are clearly no~ deleterious to the growth and division of S[15 ceils. The MMTV-LTR promoter was probably maximally activated by the testosterone present in the culture medium, and dexamethasone failed to further increase receptor production, $115 cells require testosterone to maintain their fibroblastic morphology and high growth rate [13,26). We are ct:rrently investigating the use of alternative methcglr, to culture S115 cells (roller flasks and suspensio.q culture) in order to increase the yield of recombi,,, :at receptors compared to amounts produ¢~.~l h~ co~tventional adherent cultures. It should be noted, however, that the levels of heteroiogous receptor expressiott so far attained in mammalian cells may not be sufficient for experiments aimed to receptor purification, crystallization, and physical structure analysis. A recent report described the recombinant expression of human ~2-AR in yeast, where expression levels of more than 100 pmol/mg protein were achie~:d [27]. Thin, while stably tgansfueled mammalian cell lines, such as our recombinant SI15 and IW~'4 clones, appear suitable for phurmacological experiments and should be useful in the development of new, subtype-selectee drug molecules, alternative production strategies may be required for the large-scale production of receptor proteins for struc. lural analysb.

Admowl~gements This study was funded by 8rants from the Technology Development Centre of Finland and the Academy of Finland. The authors age grateful to Dr. RJ. Lefkowitz of the Howard Hughes Medical Institute, Duke University, for permission to use the a~-AR cDNAs and ftbroblast cell lines and for helpful discussions. We also thank Drs. P. Hlirk6nen and S. Jalkanen for gifts of cell lines, Dr. S, Lepp~ for help with culture and transfection of Sl15 cells, Dr. U. Pesonen for help with construction of expression vectors, Dr. R. Lammintau~ta for strategic advice, and Drs. S, Cure,hip and J.W. Lomasney for valuable ~ m m e m s on the manuscript.

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Stable expression of recombinant human alpha 2-adrenoceptor subtypes in two mammalian cell lines: characterization with [3H]rauwolscine binding, inhibition of adenylate cyclase and RNase protection assay.

Cloning of the genes encoding distinct subtypes of human alpha 2-adrenergic receptors (alpha 2-AR) allows the separate recombinant expression of each ...
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