differential coupling of serotonin 5-ht1a and 5-ht1b receptors to activation of erk2 and inhibition...

Differential Coupling of Serotonin 5-HT1A and 5-HT1B

Receptors to Activation of ERK2 and Inhibition of AdenylylCyclase in Transfected CHO Cells

Jorge Mendez, Tapan M. Kadia, Ravi K. Somayazula, Khaled I. El-Badawi, andDaniel S. Cowen

Department of Psychiatry, University of Medicine and Dentistry of New Jersey–Robert Wood Johnson Medical School,Piscataway, New Jersey, U.S.A.

Abstract: Although the subtypes of serotonin 5-HT1 re-ceptors have distinct structure and pharmacology, it hasnot been clear if they also exhibit differences in couplingto cellular signals. We have sought to compare directlythe coupling of 5-HT1A and 5-HT1B receptors to adenylylcyclase and to the mitogen-activated protein kinaseERK2 (extracellular signal-regulated kinase-2). We foundthat 5-HT1B receptors couple better to activation of ERK2and inhibition of adenylyl cyclase than do 5-HT1A recep-tors. 5-HT stimulated a maximal fourfold increase inERK2 activity in nontransfected cells that express endog-enous 5-HT1B receptors at a very low density and amaximal 13-fold increase in transfected cells expressing230 fmol of 5-HT1B receptor/mg of membrane protein. Incontrast, activation of 5-HT1A receptors stimulated only a2.8-fold maximal activation of ERK2 in transfected cellsexpressing receptors at 300 fmol/mg of membrane pro-tein but did stimulate a 12-fold increase in activity in cellsexpressing receptors at 3,000 fmol/mg of membrane pro-tein. Similarly, 5-HT1A, but not 5-HT1B, receptors werefound to cause significant inhibition of forskolin-stimu-lated cyclic AMP accumulation only when expressed athigh densities. These findings demonstrate that althoughboth 5-HT1A and 5-HT1B receptors have been shown tocouple to G proteins of the Gi class, they exhibit differ-ences in coupling to ERK2 and adenylyl cyclase. KeyWords: Extracellular signal-regulated kinase-2—Mito-gen-activated protein kinase—Cyclic AMP—5-HT1A re-ceptors—5-HT1B receptors—Serotonin.J. Neurochem. 73, 162–168 (1999).

At least 16 types of mammalian receptors for seroto-nin [5-hydroxytryptamine (5-HT)] have been reported(for reviews, see Hoyer et al., 1994; Scalzitti and Hen-sler, 1996). All are G protein-coupled receptors with theexception of 5-HT3 receptors, which are ligand-gated ionchannels. Those receptors that couple to G proteins of theGi class and inhibit adenylyl cyclase have been classifiedas 5-HT1 receptors. They have been further subtyped as5-HT1A, 5-HT1B, 5-HT1D, 5-HT1E, and 5-HT1F recep-tors. Although these subtypes of 5-HT1 receptors have

clear distinctions in pharmacology and structure, differ-ences in coupling to cellular signals have not been pre-viously demonstrated. In contrast, comparison of resultsacross studies suggests that the receptors may be func-tionally equivalent, bringing into question the signifi-cance of expression of multiple subtypes in brain.

In fact, there has been a tendency to lump together allreceptors that couple to G proteins of the Gi class, withthe assumption that they all activate identical cellularsignals. Clearly, various studies have demonstrated thatunder the appropriate cellular conditions some of thesereceptors can exhibit identical function. For example, inaddition to coupling negatively to adenylyl cyclase,many Gi-coupled receptors have been reported to acti-vate the mitogen-activated protein (MAP) kinases ERK1and ERK2 (extracellular signal-regulated kinase-1 and-2, respectively) (Meloche et al., 1992; Koch et al., 1994;Flordellis et al., 1995; Luttrell et al., 1995; Cowen et al.,1996; Garnovskaya et al., 1996; Pullarkat et al., 1998).However, some of these studies have used stable trans-fected cell lines that overexpress receptor at densities notobserved in cells expressing endogenous receptor. Otherstudies have used cells transiently transfected withcDNA for the receptors. Such cells do not express re-ceptor at a homogeneous density. In contrast, some cellsexpress receptors at extremely high density, whereasother cells express no receptors. Studies using cells over-

Received November 23, 1998; revised manuscript received February19, 1999; accepted March 6, 1999.

Address correspondence and reprint requests to Dr. D. S. Cowen atDepartment of Psychiatry, University of Medicine and Dentistry ofNew Jersey–Robert Wood Johnson Medical School, 125 PatersonStreet, New Brunswick, NJ 08901, U.S.A.

Abbreviations used:cAMP, cyclic AMP; CHO, Chinese hamsterovary; ERK, extracellular signal-regulated kinase; 5-HT, 5-hydroxy-tryptamine (serotonin); IBMX, 3-isobutyl-1-methylxanthine; MAP, mi-togen-activated protein;p-MPPI, 4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylbenzamide hydrochloride; 8-OH-DPAT, (6)-8-hydroxy-N,N-dipropyl-2-aminotetralin hydrobromide;RIA, radioimmunoassay.

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Journal of NeurochemistryLippincott Williams & Wilkins, Inc., Philadelphia© 1999 International Society for Neurochemistry

expressing receptor can correctly demonstrate that a par-ticular receptor can couple to a specific signal whenreceptors are expressed at the high density. However,such studies may miss the fact that the receptor wouldnot couple at lower (more physiological) densities. Whenreceptor function has been studied under conditionswhere receptors are expressed at the same densities,differences have been found. For example, using stabletransfected cell lines expressing receptors in the pico-mole per milligram of membrane protein range, Flordel-lis et al. (1995) demonstrated thata2B- and a2D-adren-ergic receptors but nota2C-adrenergic receptors coupleeffectively to MAP kinase.

The MAP kinases ERK1 and ERK2 are serine/threo-nine protein kinases. They have been shown to phosphor-ylate several transcription factors, including c-Jun,p62TCF/Elk-1, c-Fos, and c-Myc and also appear to reg-ulate translation of mRNA (for review, see Denton andTavare, 1995). Touhara et al. (1995) recently described amodel for Gi-coupled receptor stimulation of MAP ki-nase requiring G proteinbg subunits, an unidentifiedtyrosine kinase, Shc, phosphatidylinositol 3-kinase,Grb2/SOS, and Ras. Other components of the pathwayare thought to include Raf and MAP kinase kinase(MEK/MKK). The pathway for activation of MAP ki-nase is therefore much different from that for inhibitionof adenylyl cyclase, which is directly mediated by Gi,through a and bg subunits (for review, see Tang andHurley, 1998).

In the present study we sought to identify differencesin the coupling of 5-HT1A and 5-HT1B receptors tocellular signals. Both receptors have been shown in sep-arate studies to inhibit the activity of adenylyl cyclase(Fargin et al., 1989; Unsworth and Molinoff, 1992; Berget al., 1994, 1996; Harrington et al., 1994; Clarke et al.,1996; Giles et al., 1996) and in some cases to evokesmall increases in intracellular Ca21 level (Fargin et al.,1989; Harrington et al., 1994; Dickenson and Hill, 1995;Giles et al., 1996). However, attempts to make compar-isons of receptor function across separate studies arecomplicated by the use of different cell types and of cellsexpressing receptors at different densities. In the presentstudies we used the same parent cell line [Chinese ham-ster ovary (CHO) cells] for all studies and selected stabletransfected cell lines that express receptors at specificdensities. We chose to study coupling to inhibition ofadenylyl cyclase and activation of ERK2 because verydifferent pathways regulate these signals. In earlier stud-ies we previously demonstrated that 5-HT1B receptors,even when expressed in nontransfected CHO cells at lowdensity (a density below the sensitivity of our bindingassay), effectively couple to activation of ERK2 (Pullar-kat et al., 1998). In separate studies we and others (Co-wen et al., 1996; Garnovskaya et al., 1996) have alsoshown in transfected CHO cells that activation of5-HT1A receptors expressed at high density (.1pmol/mg of membrane protein) similarly stimulatesERK2. However, in the present studies we have directlycompared the efficacy of each receptor subtype to acti-

vate ERK2 and to inhibit adenylyl cyclase in cells ex-pressing receptors at the same densities. It is significantthat we found that coupling by 5-HT1A receptors, but not5-HT1B receptors, requires expression of receptor at ahigh density. This suggests that the coupling of 5-HT1Areceptors to MAP kinase and to adenylyl cyclase in CNSneurons would be significantly more sensitive to changesin density of receptor expression.

MATERIALS AND METHODS

Materials(6)-8-Hydroxy-N,N-dipropyl-2-aminotetralin hydrobro-

mide (8-OH-DPAT) and 4-iodo-N-[2-[4-(methoxyphenyl)-1-piperazinyl]ethyl]-N-2-pyridinylbenzamide hydrochloride( p-MPPI) were purchased from Research Biochemicals Inter-national (Natick, MA, U.S.A.). Pertussis toxin, forskolin, and3-isobutyl-1-methylxanthine (IBMX) were purchased fromCalbiochem (San Diego, CA, U.S.A.). Protein A-Sepharoseand 5-HT were purchased from Sigma (St. Louis, MO, U.S.A.).

Cell cultureCHO-K1 cells were obtained from the American Type Cul-

ture Collection (Rockville, MD, U.S.A.). A stable transfectedCHO-K1 cell line expressing 5-HT1B receptors at a density of230 fmol/mg of membrane protein was obtained by transfectingCHO-K1 cells with cDNA for the mouse 5-HT1B receptor andselecting for resistance to hygromycin B (200mg/ml) (Pullarkatet al., 1998). Two stable clones expressing 5-HT1A receptors ata density of 300 and 3,000 fmol/mg of membrane protein wereobtained by transfecting CHO-K1 cells with cDNA for thehuman 5-HT1A receptor (Fargin et al., 1988; Cowen et al.,1996) and selecting for resistance to geneticin (400mg/ml). Thedensity of 5-HT1B receptors was measured as described byUnsworth and Molinoff (1992) using the radioligand(6)-[125I]iodocyanopindolol obtained from DuPont NEN (Bos-ton, MA, U.S.A.). The density of 5-HT1A receptors was mea-sured as previously described (Cowen et al., 1997) using theradioligand [125I]p-MPPI obtained from DuPont NEN. Trans-fected cells were maintained in medium containing Ham’s F-12nutrient mixture withL-glutamine, 10% dialyzed fetal bovineserum (dialyzed in membranes with 1,000-Da cutoffs against a100-fold greater volume of 150 mM NaCl to remove endoge-nous 5-HT), 1% penicillin-streptomycin, and either 400mg/mlgeneticin (for cells expressing 5-HT1A receptors) or 200mg/mlhygromycin B (for cells expressing 5-HT1B receptors) at 37°C(95% air, 5% CO2).

Assay of MAP kinase activityMAP kinase activity was measured following immunopre-

cipitation with rabbit polyclonal IgG recognizing ERK2 (C-14)obtained from Santa Cruz Biotechnology (Santa Cruz, CA,U.S.A.), essentially as previously described (Flordellis et al.,1995; Cowen et al., 1996). The day before use, cells werewashed with phosphate-buffered saline and cultured overnightunder serum- and geneticin/hygromycin B-free conditions.Cells were stimulated with the specified concentrations ofagonists for 5 min and lysed. ERK2 was immunoprecipitatedfrom the cytosol, and activity was measured by incorporation of32P into a nine-amino-acid MAP kinase substrate peptide cor-responding to amino acids 95–98 of myelin basic protein (Up-state Biotechnology, Lake Placid, NY, U.S.A.). Phosphocellu-lose filters were spotted with the phosphorylated substrate,washed extensively in 0.75% phosphoric acid and acetone, andcounted for radioactivity by scintillation spectrometry.

J. Neurochem., Vol. 73, No. 1, 1999

1635-HT1A AND 5-HT1B RECEPTOR COUPLING TO ERK2 AND cAMP

Assay of adenylyl cyclase activityCells were cultured in 12-well plates until confluent. The day

before use, cells were washed with phosphate-buffered salineand cultured overnight under serum- and geneticin/hygromycinB-free conditions. The appropriate agonists were added to eachwell, immediately followed by 1mM forskolin and 100mMIBMX. The plates were incubated for 5 min at 37°C, and thereaction was stopped by aspiration and addition of 0.1Mhydrochloric acid. The cells were scraped from the plates andlysed with a 26-gauge needle. The lysate was neutralized, andcyclic AMP (cAMP) was quantified, after acetylation, by ra-dioimmunoassay (RIA). The RIA protocol, anti-cAMP, andcAMP standard were obtained from Calbiochem. Adenosine39,59-cyclic phosphoric acid 29-O-succinyl[125I]iodotyrosinemethyl ester was from DuPont NEN.

RESULTS

Differential activation of the MAP kinase ERK2 by5-HT1A and 5-HT1B receptors

Studies were designed to compare directly the magni-tude of activation of ERK2 elicited by 5-HT1A and5-HT1B receptors. All studies were done in transfectedand nontransfected CHO cells. This avoided potentialcellular differences that would have complicated inter-pretation of results had different cell lines expressingendogenous receptors been used. 5-HT1B receptors werestudied in nontransfected cells that express endogenousreceptor at a very low density and in a stable transfectedcell line. The transfected cell line (referred to in thisarticle as CHO-1B-230) expresses receptor with aKD forthe radioligand (6)-[125I]iodocyanopindolol of 50 pM, ata density of 230 fmol/mg of membrane protein. Thedensity of endogenous 5-HT1B receptors expressed innontransfected CHO cells is below the sensitivity of ourbinding assays—30 fmol/mg of membrane protein.5-HT1A receptors were studied in two stable transfectedcell lines expressing receptor at densities of 300 and3,000 fmol/mg of membrane protein, referred to asCHO-1A-300 and CHO-1A-3000, respectively. TheKDfor the radioligand [125I]p-MPPI is ;0.2 nM in bothCHO-1A-300 and CHO-1A-3000 cell lines.

As we have previously reported (Pullarkat et al.,1998), treatment of nontransfected CHO-K1 cells with5-HT causes stimulation of ERK2 activity through en-dogenous 5-HT1B receptors. 5-HT stimulated a maximalfourfold increase in activity with an EC50 of 45 nM (Fig.1A). It is significant that in CHO-1B-230 cells 5-HTstimulated a larger, 13-fold, increase in activity with anEC50 of 10 nM (Fig. 1A).

In contrast to the activation of ERK2 by 5-HT1Breceptors, which occurred even when the density ofreceptors was very low, the activation by 5-HT1A recep-tors required expression of receptor at a high density.Treatment of CHO-1A-300 cells with 8-OH-DPAT, anagonist selective for 5-HT1A receptors, stimulated only amaximal 2.8-fold increase in ERK2 activity with an EC50of 60 nM (Fig. 1B). Therefore, 5-HT1A receptors ex-pressed at a density of 300 fmol/mg of membrane proteinstimulated only a 2.8-fold increase in ERK2 activity, in

contrast to the 13-fold increase stimulated by 5-HT1Breceptors expressed at 230 fmol/mg of membrane proteinin CHO-1B-230 cells. The basal activity of ERK2 wassimilar in both cell types. Stimulation of ERK2 to amagnitude comparable to that seen by 5-HT1B receptorsin CHO-1B-230 cells occurred only in CHO-1A-3000cells. 8-OH-DPAT stimulated, in those cells, a maximal12-fold increase in ERK2 activity with an EC50 of 10 nM(Fig. 1B). It is significant that the stimulation of ERK2elicited by 8-OH-DPAT in CHO-1A-300 cells, althoughsmall, was selectively mediated by 5-HT1A receptors.8-OH-DPAT, at concentrations of,10 mM, did notstimulate ERK2 activity in nontransfected CHO cellsthat do not express 5-HT1A receptors (Fig. 1B). There-fore, 8-OH-DPAT, at the concentrations used in thesestudies, did not act nonselectively as an agonist at en-dogenous 5-HT1B receptors.

Because 8-OH-DPAT stimulated only a small activa-tion of ERK2 in CHO-1A-300 cells, it was important todemonstrate that this did not reflect selection of a mu-tated clonal cell line that had an impaired pathway foractivation of MAP kinase. This was not the case. ERK2could, in fact, be activated in CHO-1A-300 cells to anextent greater than that stimulated by 8-OH-DPAT. As

FIG. 1. Differential activation of ERK2 by 5-HT1A and 5-HT1Breceptors. A: Nontransfected (wild) CHO cells and CHO-1B-230cells were incubated for 5 min with the indicated concentrationsof 5-HT. B: Wild CHO, CHO-1A-300, and CHO-1A-3000 cellswere incubated for 5 min with the indicated concentrations of8-OH-DPAT. ERK2 was immunoprecipitated, and its activity wasmeasured as described in Materials and Methods. In A data aremean 6 SE (bars) values of three separate experiments, eachperformed in triplicate, with activity expressed as a percentageof unstimulated activity. *p , 0.05 versus wild CHO by two-sidedunpaired Student’s t test. In B data are mean 6 SE (bars) valuesof six separate experiments, each performed in triplicate. *p, 0.05 versus CHO-1A-300 by two-sided unpaired Student’s ttest.


164 J. MENDEZ ET AL.

shown in Fig. 2, treatment with 5-HT caused an eightfoldstimulation of ERK2, compared with the 2.8-fold activa-tion with 8-OH-DPAT. This larger stimulation was likelythe result of activation of both transfected 5-HT1A recep-tors and endogenous 5-HT1B receptors. However, analternative hypothesis for the poor activation by 8-OH-DPAT, relative to 5-HT, in CHO-1A-300 cells couldhave been that 8-OH-DPAT was not a full agonist for5-HT1A receptors. This proved also to be not the case.8-OH-DPAT stimulated a large, 13-fold, activation ofMAP kinase in CHO-1A-3000 cells that was similar inmagnitude to that stimulated by 5-HT (Fig. 2).

We further demonstrated that we were not looking ata partial agonist effect in studies in which 5-HT was usedas the agonist. We first showed that the antagonistp-MPPI was selective for 5-HT1A receptors and does notinhibit the activation of ERK2 stimulated in nontrans-fected cells through 5-HT1B receptors (Fig. 3A). It cantherefore be used to inhibit selectively the activation by5-HT of transfected 5-HT1A receptors without inhibitingthe activation by 5-HT of endogenous 5-HT1B receptors.As expected, 1mM p-MPPI almost completely inhibitedthe activation of ERK2 stimulated by 8-OH-DPAT inCHO-1A-300 cells (Fig. 3B). However, it only slightlyinhibited the activation by 5-HT, consistent with inhibi-tion of a small 5-HT1A (relative to 5-HT1B) receptor-mediated response. In contrast,p-MPPI almost com-pletely inhibited the activation of ERK2 by both 5-HTand 8-OH-DPAT in CHO-1A-3000 cells (Fig. 3C). InCHO-1A-3000 cells 5-HT1A receptors are expressed atsufficient density to mediate most of the activation ofERK2 by 5-HT, with the remaining small effect medi-ated by endogenous 5-HT1B receptors. Therefore, weconclude that although both human 5-HT1A and rodent5-HT1B receptors stimulate ERK2, 5-HT1A receptors ef-fectively do so only when expressed at high density. It issignificant that, although 5-HT1B receptors were found tocouple better to ERK2 than did 5-HT1A receptors, bothreceptors used pathways requiring Gi-type G proteins.

Pretreatment with pertussis toxin caused almost com-plete inhibition of 5-HT1A receptor- and 5-HT1B recep-tor-mediated activation of ERK2 (data not shown).

Differential inhibition of adenylyl cyclase activity by5-HT1A and 5-HT1B receptors

Endogenous 5-HT1B receptors expressed on nontrans-fected CHO cells have been previously reported to cou-ple negatively to adenylyl cyclase (Berg et al., 1994;Dickenson and Hill, 1995; Giles et al., 1996). We foundthat 5-HT caused a maximal 40% inhibition of forskolin-stimulated cAMP accumulation in these cells with an

FIG. 3. 8-OH-DPAT (DPAT) and 5-HT display similar efficacy forstimulating 5-HT1A receptor-mediated activation of ERK2. A:Nontransfected (wild) CHO cells were incubated for 5 min with 1mM 5-HT in the presence or absence of 1 mM p-MPPI (MPPI).CHO-1A 300 cells (B) and CHO-1A-3000 cells (C) were incu-bated for 5 min with 1 mM 5-HT or 1 mM DPAT in the presenceor absence of 1 mM MPPI. ERK2 was immunoprecipitated, andits activity was measured as described in Materials and Meth-ods. Data are mean 6 SE (bars) values of three separate exper-iments, each performed in triplicate, with activity expressed as apercentage of the activity stimulated by the individual agonist inthe absence of antagonist. ***p , 0.001 versus value in theabsence of MPPI by two-sided paired Student’s t test.

FIG. 2. Differential activation of ERK2 by 5-HT and 8-OH-DPAT(DPAT) in cells expressing transfected 5-HT1A receptors andendogenous 5-HT1B receptors. CHO-1A-300 and CHO-1A-3000cells were incubated for 5 min with either 1 mM 5-HT or 1 mMDPAT. ERK2 was immunoprecipitated, and its activity was mea-sured as described in Materials and Methods. Data are mean6 SE (bars) values of 10 separate CHO-1A-300 experiments andfour CHO-1A-3000 experiments, each performed in triplicate,with activity expressed as a percentage of unstimulated activity.*p , 0.05, ***p , 0.001 versus DPAT for each cell type bytwo-sided paired Student’s t test.



EC50 of 100 nM (Fig. 4A). It is significant that 5-HTstimulated a larger, 60% inhibition in CHO-1B-230 cellswith an EC50 of 80 nM (Fig. 4A).

In contrast, 5-HT1A receptors were found to coupleeffectively to inhibition of adenylyl cyclase only whenexpressed at a high density. Treatment of CHO-1A-300cells with 8-OH-DPAT caused a maximal 20% inhibitionof forskolin-stimulated cAMP accumulation with anEC50 of 200 nM (Fig. 4B). However, 8-OH-DPAT didinhibit forskolin-stimulated cAMP accumulation by 80%in CHO-1A-3000 cells with an EC50 of 3 nM (Fig. 4B).The accumulation of cAMP stimulated by forskolin wassimilar in all cell types.

DISCUSSION

Our findings demonstrate that in addition to havingdistinct structures and pharmacology, individual sub-types of 5-HT1 receptors also exhibit differences in cou-pling to cellular signals. 5-HT1B receptors were found tocouple better to both inhibition of adenylyl cyclase andto activation of ERK2 than do 5-HT1A receptors. Acti-vation of 5-HT1A receptors in CHO-1A-300 cells causedonly a 20% inhibition of forskolin-stimulated cAMPaccumulation and a 2.8-fold increase in ERK2 activity.In contrast, activation of 5-HT1B receptors in CHO-1B-

230 cells caused a 60% inhibition of forskolin-stimulatedcAMP accumulation and a 13-fold increase in MAPkinase activity. Even more striking was the 40% inhibi-tion of adenylyl cyclase activity and fourfold activationof ERK2 seen in nontransfected CHO cells. These cellsexpress endogenous 5-HT1B receptors at a density belowthat detectable by our binding assays and that of others(Giles et al., 1996). Because our binding assay is sensi-tive enough to measure receptor expression as low as 30fmol/mg of membrane protein, we can assume that thereceptors are expressed at a density below that and con-sequently at a density at least 10-fold lower than that atwhich 5-HT1A receptors are expressed on CHO-1A-300cells. It is significant that although we have not been ableto detect expression of 5-HT1B receptors in nontrans-fected CHO cells, we have previously demonstrated thatthe pharmacology for agonist-induced activation ofERK2 (Pullarkat et al., 1998) is consistent with theirexpression. Similarly, the pharmacology for inhibition ofadenylyl cyclase (Berg et al., 1994; Dickenson and Hill,1995; Giles et al., 1996) and activation of p70 S6 kinase(Pullarkat et al., 1998) in nontransfected CHO cells isconsistent with mediation by 5-HT1B receptors. There-fore, the more effective coupling by 5-HT1B receptors toactivation of ERK2 and inhibition of adenylyl cyclase,relative to 5-HT1A receptors, is striking.

It should be pointed out that our findings cannot beattributed to cellular differences in transfected cell linesresulting from clonal selection. Studies using a differenttransfected CHO cell line expressing human 5-HT1Areceptors at a density of 250 fmol/mg of membraneprotein confirmed our results from CHO-1A-300 cells,that agonists for 5-HT1A receptors cause a small activa-tion of ERK2 when receptors were expressed at a lowdensity (data not shown). In contrast, Garnovskaya et al.(1996), using a transfected CHO cell line expressing5-HT1A receptors at 1,000 fmol of receptor/mg of pro-tein, reported a fivefold increase in ERK2 activity. Thatmagnitude of stimulation fits well with our finding of a2.8-fold activation in cells expressing 300 fmol of recep-tor/mg of membrane protein and a 12-fold activation inour CHO-1A-3000 cells expressing 3,000 fmol of recep-tor/mg of membrane protein. The results of our studiesdemonstrate the importance of receptor density whencomparisons are made of receptor function.

Ideally, studies of receptor function in transfected celllines should use cells that express receptors at densitiessimilar to those found in vivo. 5-HT1A receptors havebeen reported to be expressed at;200 fmol/mg of mem-brane protein in rat hippocampus (Butkerait et al., 1995),and 5-HT1B receptors have been reported to be expressedat a similar density in rat cortex (Hoyer et al., 1985).Therefore, our CHO-1A-300 and CHO-1B-230 cells ap-pear to express receptors at a density similar to that foundin vivo. However, the densities reported in hippocampusand cortex represent an average of the cellular expressionin those regions. Because the density of receptors ex-pressed on individual cells in those regions is not homo-geneous, those neurons that do express receptors would

FIG. 4. Differential inhibition of forskolin-stimulated cAMP accu-mulation by 5-HT1A and 5-HT1B receptors. A: Nontransfected(wild) CHO cells and CHO-1B-230 cells were incubated for 5 minwith 1 mM forskolin, 100 mM IBMX, and the indicated concen-trations of 5-HT. B: CHO-1A-300 and CHO-1A-3000 cells wereincubated for 5 min with 1 mM forskolin, 100 mM IBMX, and theindicated concentrations of 8-OH-DPAT. Intracellular cAMP wasquantified by RIA, as described in Materials and Methods. Dataare mean 6 SE (bars) values of three separate experiments, eachperformed in triplicate, with the activity expressed as the per-centage of forskolin-induced increases in cAMP accumulation inthe absence of receptor agonists. **p , 0.01 versus CHO-1A-3000 by two-sided unpaired Student’s t test.



be expected to do so at densities of.200 fmol/mg ofmembrane protein. Therefore, our CHO-1A-300 andCHO-1B-230 cell lines express receptors at levels simi-lar to, or below, that expressed on neurons in hippocam-pus and cortex.

Our findings that receptor coupling to adenylyl cyclaseand MAP kinase required expression of 5-HT1A, but not5-HT1B, receptors at a high density suggest that thecellular effects elicited by 5-HT1A receptors in brain maybe more sensitive to changes in receptor expression. Infact, Yocca et al. (1992) used the irreversible antagonistN-ethoxycarbonyl-2-ethoxy-1,2-dihydroquinoline to ex-amine, in rat hippocampus, the relationship between5-HT1A receptor occupancy and inhibition of forskolin-stimulated cAMP accumulation. Their findings wereconsistent with a lack of receptor reserve. Also, 5-HT1Areceptors do not universally couple to adenylyl cyclase.Although the activation of postsynaptic 5-HT1A recep-tors in hippocampus causes inhibition of forskolin-stim-ulated cAMP accumulation, activation of somatoden-dritic 5-HT1A receptors in the dorsal raphe does not(Clarke et al., 1996). Although it is not known why5-HT1A receptors in the dorsal raphe do not couplenegatively to adenylyl cyclase, our findings suggest thatthe density of receptor expression relative to G proteinexpression may be insufficient.

In our studies of 5-HT1A and 5-HT1B receptors wefound that the magnitude of activation of ERK2 waspositively correlated with the magnitude of inhibition offorskolin-stimulated cAMP accumulation. However, inprevious studies we have demonstrated that inhibition ofadenylyl cyclase activity is not required for activation ofERK2 (Cowen et al., 1996). Pretreatment of CHO-1A-3000 cells with the permeable cAMP analogues dibu-tyryl cAMP or 8-bromo cAMP caused no inhibition of8-OH-DPAT-stimulated MAP kinase activity. We simi-larly found that pretreatment with forskolin or IBMX hadno effect. Therefore, activation of MAP kinase by5-HT1A receptors is not the result of decreases in cAMPconcentration. Instead, it more likely fits the model de-scribed for other Gi-coupled receptors (Touhara et al.,1995), requiring G proteinbg subunits, phosphatidylino-sitol 3-kinase, Grb2/SOS, and Ras. That there was acorrelation in the magnitude of change elicited in ERK2and adenylyl cyclase activity suggests that both signalsmay be regulated by the same G protein.

However, it is not currently known which pertussistoxin-sensitive G proteins are required for coupling of5-HT1A and 5-HT1B receptors to ERK2 and adenylylcyclase. It is also not known if the receptors use differentG proteins. Of the pertussis toxin-sensitive G proteins,CHO-K1 cells express primarily Gia2 and Gia3 (Gia2. Gia3), but not Gia1, and in some studies have beenfound to express small amounts of GoaA (Raymond et al.,1993; van Biesen et al., 1996). It is possible that both5-HT1A and 5-HT1B receptors couple to an identical Gprotein (or G proteins) but that 5-HT1B receptors couplemore effectively. Alternatively, 5-HT1A and 5-HT1B re-ceptors may preferentially use different G proteins. The

more effective coupling of 5-HT1B receptors to ERK2and adenylyl cyclase could then be a result of differencesin the efficacy of individual G proteins. The G proteinused by 5-HT1B receptors might be more effective incoupling to these signals than that used by 5-HT1B re-ceptors.

Our present study used rodent 5-HT1B receptors tocompare the coupling of 5-HT1A and 5-HT1B receptors tocellular signals. The endogenous expression of rodent5-HT1B receptors in CHO cells allowed studies of en-dogenous receptors expressed at a very low density, inaddition to transfected receptors expressed at a specifichigher density. Although there are some differences inreceptor pharmacology, the amino acid sequence of rat5-HT1B receptors is 94% identical to that of human5-HT1B receptors (Veldman and Bienkowski, 1992).Therefore, rat and murine (98% identical to rat) receptorsare often used as models for studying the function of5-HT1B human receptors. We would expect that human5-HT1B receptors would be similar to rodent receptors indisplaying functional differences compared with human5-HT1A receptors.

Our results help provide some insight into understand-ing the significance of expression of multiple subtypes of5-HT1 receptors in brain. Similar differences in functionappear also to be relevant to other receptor subtypesclassified within individual families. For example,Flordellis et al. (1995) used stable transfected cell linesexpressing similar densities of receptors to demonstratethat the subtypes ofa2-adrenergic receptors exhibit dif-ferences in coupling to second messengers.a2B- anda2D-adrenergic receptors, but nota2C-adrenergic recep-tors, were found to couple effectively to MAP kinase.Some differences have also been found in dopaminereceptors of the D2 family: D2 and D3 dopamine recep-tors have been reported to be better than D4 dopaminereceptors with respect to inhibiting dopamine release(Tang et al., 1994). In summary, there are accumulatingdata demonstrating that Gi-coupled receptors are notidentical but exhibit functional differences.

Acknowledgment: These studies were supported by anNARSAD Young Investigator Award to D.S.C., a grant fromthe Foundation of the University of Medicine and Dentistry ofNew Jersey, and grant GM55145-02 from the National Insti-tutes of Health. We thank Dr. Julie Hensler (University ofTexas Health Science Center, San Antonio) for subcloning the5-HT1A receptor construct into pcDNA3 and Dr. Robert Hamer(University of Medicine and Dentistry of New Jersey–RobertWood Johnson Medical School) for advice on statistical anal-yses.

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differential coupling of serotonin 5-ht1a and 5-ht1b receptors to activation of erk2 and inhibition...

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