Journal of NeurochemistryLippincott—Raven Publishers, Philadelphia© 1998 International Society for Neurochemistry

Continuous Activation of Pituitary Adenylate Cyclase-Activating Polypeptide Receptors Elicits Antipodal Effects onCyclic AMP and Inositol Phospholipid Signaling Pathways inCATH.a Cells: Role of Protein Synthesis and Protein Kinases

*André Muller, Bernadette Lutz-Bucher, Pascal Kienlen-Campard,

Bernard Koch, and Jean-Philippe Loeffler

IPCB, Laboratoire de Neurophysiologie et de Neurobiologie des Systèmes Endocrines, UMR CNRS 7519,and * Clinique de la Douleur, Hospices Civils de Strasbourg, Strasbourg, France

Abstract: Continuous exposure of cells to agonists de-velops a process that determines the extent to which thecells eventually respond to further stimuli. Here we usedCATH.a cells (a catecholaminergic neuron-like cell line),which express pituitary adenylate cyclase-activatingpolypeptide (PACAP) receptors linked to both adenylylcyclase and phospholipase C-ß pathways, to investigatethe influence of prolonged hormonal treatment on dualsignaling and gene transcription. Prolonged incubation ofcells with PACAP failed to down-regulate the density andaffinity of membrane binding sites and caused oppositechanges in messenger systems: PACAP-stimulated cy-clic AMP accumulation was attenuated in a time- anddose-dependent fashion (t112 = 6.7 h and IC50 = 0.1 nM),whereas phosphoinositide turnover was overstimulated.Both effects were insensitive to pertussis toxin, whereasthe drop in cyclic AMP concentration was also un-changed in the presence of 3-isobutyl-1 -methylxanthine,indicating that neither G-like proteins nor cyclic nucleo-tide phosphodiesterases play a critical role in these pro-cesses. Blockade of protein synthesis with cyclohexi-mide, as well as inhibition by H89 of cyclic AMP-depen-dent protein kinase (but not by bisindolylmaleimide ofprotein kinase C) antagonized the influences exerted byPACAP on adenylyl cyclase activity and inositol phos-phate formation. Transcription of the chimeric GAL4-CREB construct, transiently transfected into CATH.acells, was stimulated by PACAP, and this effect was po-tentiated as a result of chronic PACAP treatment. Theresults of the present investigation provide new insightinto the possible differential regulation and cross-talksof transduction signals of receptors linked to multiplexsignaling. They demonstrate that prolonged exposure ofCATH.a cells to PACAP results in the desensitization ofthe cyclic AMP pathway and superinduction of the inositolphosphate signal, through protein neosynthesis andcyclic AMP-dependent protein kinase activation. At thesame time, they show that desensitization of cyclic AMPsignaling not only fails to hamper, but actually amplifiesPACAP-stimulated CREB-regulated transcription. KeyWords: CATH.a cells—Pituitary adenylate cyclase-

activating polypeptide— Cyclic AMP—Phosphoinositideturnover— Desensitization — CREB expression.J. Neurochem. 70, 1431—1440 (1998).

As a result of continued activation of hormone andneurotransmitter receptor systems, cells adapt them-selves by developing a gradual attenuation/desensitiza-tion (Hausdorff et al., 1990; Gershengom, 1994; Kar-oor et al., 1996) or, on the contrary, an amplification]up-regulation of their response to agonists (Penhoat etal., 1989; Thomas et al., 1992; Hettinger-Smith et al.,1996). Thus, the receptor system provides itself witha “memory“ that remembers the cell of its first en-counter with a ligand and, as a result, determines themagnitude of its subsequent response to new stimuli.Disruption of these processes, which may result inoverstimulation of cells, can have deleterious effects,and malfunctioning of signaling pathways have actu-

Received September 12, 1997; revised manuscript received No-vember 4, 1997; accepted November 14, 1997.

Address correspondence and reprint requests to Dr. B. Koch atIPCB, Laboratoire de Neurophysiologie et de Neurobiologie desSystèmes Endocrines, UMR CNRS 7519, 21, rue René Descartes,67084 Strasbourg Cedex, France.

Abbreviations used: AC, adenylyl cyclase; Br-cAMP, 8-bro-moadenosine 3‘,S‘-cyclic monophosphate; BSA, bovine serum albu-min; CaMK, Ca

2~/calmodulin-dependentprotein kinase; cAMP, cy-clic AMP; CATH.a cells, a catecholaminergic neuron-like cell line;CRE, cyclic AMP-response element; CREB, CRE-binding protein;Cx, cycloheximide; DMEMIF12, Dulbecco‘s modified Eagle‘s me-dium/Ham‘s mixture F-12; G protein, guanine nucleotide-bindingprotein; IBMX, 3-isobutyl- I -methylxanthine; InsP

1, inositol mono-phosphate; InsP2, inositol bisphosphate; InsP3, inositol trisphosphate;PACAP, pituitary adenylate cyclase-activating polypeptide; PDE,cyclic nucleotide phosphodiesterase; PI, phosphoinositide; PKA, cy-clic AMP-dependent protein kinase; PKC, protein kinase C; PLC,phospholipase C-ß; PMA, phorbol I 2-myristate 13-acetate; PTX,pertussis toxin; VIP, vasoactive intestinal peptide.

1431

1432 A. MULLER ET AL.

ally been linked to various states of illnesses (Spiegelet al., 1992; Levitzki, 1996).

In the case of guanine nucleotide-binding (G) pro-tein-coupled receptor systems, the desensitization pro-cess calls into play complex mechanisms. These mayinvolve various phenomena, such as trafficking of Gproteins in plasma membrane, second messenger-me-diated phosphorylations—through protein kinases—of various elements of the receptor—G protein—ef-fector system, modulation by accessory proteins, andactivation of cyclic nucleotide phosphodiesterases(PDEs) (Conti et al., 1991; Neer, 1995; Denhardt,1996; Karoor et al., 1996). Besides cyclic AMP(cAMP)-dependent protein kinase (PKA) and proteinkinase C (PKC), so-called G protein-coupled receptorkinases, in conjunction with proteins of the arrestinfamily, play a marked role in the process of uncouplingof the receptor with G proteins (Hausdorffet al., 1990;Wilson and Applebury, 1993; Premont et al., 1995;Karoor et al., 1996). Intracellular sequestration of re-ceptor also represents an important means to shut offcell response, although loss of membrane receptorsmay not always be a strict prerequisite in that process(Hausdorff et al., 1990; Olson et al., 1991; Chern etal., 1993; Olson and Schimmer, 1994).

Some types of G protein-coupled receptors appearto be linked to dual signaling, as ligand binding stimu-lates both adenylyl cyclase (AC) and phospholipid-phospholipase C-ß (PLC) activities. These include re-ceptors for thyrotropin-stimulating hormone, pituitaryadenylate cyclase-activating polypeptide (PACAP),and luteinizing hormone (Milligan, 1993; Spengler etal., 1993). Although transduction pathways are thoughtto flow through different species of G protein subunits(G. and Gq, as well as G47 released upon dissociationof the trimeric protein), the very mechanisms that drivethis divergence remain essentially unclear (Milligan,1993; Allgeier et al., 1994; Herrlich et al., 1996). In arecent study, evidence was provided that two distinctroutes, before G protein—receptor coupling, may con-vey the different messages from receptor to effectors(Gilchrist et al., 1996).

Using cathecholaminergic neuron-like CATH.acells, we have demonstrated previously the existencein these cells ofPACAP receptors coupled to multiplexsignaling, as activation of these receptors resulted ina coincident stimulation of bothAC and PLC pathways(Muller et al., 1996). More recent studies resulted inthe unexpected observation that chronic exposure toPACAP failed to down-regulate cell membrane bind-ing sites in these very cells, thus providing a uniquemodel system in which to study the desensitizationmechanism and to test the hypothesis of possible dif-ferential regulation and cross-talks of transduction cas-cades. Hence, the following series of experimentsaimed at examining whether the following occur inCATH.a cells: (a) long-term stimulation of PACAPreceptors causes a shutdown of both signaling routesor, alternatively, alters these routes in a distinct way;

(b) protein synthesis and/or protein kinases eventuallyplay a role in these processes; and (c) functionalchanges in cell signaling may impact on gene expres-sion and, especially, on the transcription of cAMP-response element (CRE)-binding protein (CREB) intransiently transfected cells.

EXPERIMENTAL PROCEDURES

MaterialsPACAP-38 and PACAP-27, as well as vasoactive intesti-

nal peptide (VIP), were purchased from Bachem (BachemBiochimie SARL, France). Pertussis toxin (PTX), 3-isobu-tyl- 1-methylxanthine (IBMX), forskolin, cycloheximide(Cx), phorbol 12-myristate 13-acetate (PMA), 8-bromoade-nosine 3‘,5‘-cyclic monophosphate (Br-cAMP), andDulbec-co‘s modified Eagle‘s medium/Ham‘s mixture F-12(DMEM/F12) were obtained from Sigma Chimie (SaintQuentin Fallavier, France). myo-[

3H]Inositol (102 Ci/mmol)was from Amersham France SA. Inhibitors of PKA (H-89)and of PKC (bisindolylmaleimide I) were obtained fromCalbiochem (France Biochem, Meudon, France), polyethy-lenimine (PET) from Aldrich Chemie, and Dowex AG1-X8resin (formate form; 100—200 mesh) from Bio-Rad (Labora-toiresBio-Rad SA, Paris, France). ‘251-PACAP-27 (2,200 Cilmmol) was purchased from NEN (Du Pont de NemoursFrance SA).

Culture of CATH.a cellsCATH.a cells were generously donated by D. M. Chika-

raishi (Boston, MA, U.S.A.). They were seeded in 24-wellcluster plates (Costar) for cAMP measurements and in 35X 10 mm dishes for inositol phosphate assay and cell trans-fection, at initial densities of 2 X io~and iO~cells, respec-tively. Cells were maintained in DMEMIF12 supplementedwith 10% fetal calf serum and antibiotics, in a humidifiedatmosphere of 5% CO

2 in air, for 3—4 days before experi-ments. All experiments were performed in serum-free culturemedium supplemented with 0.1% (wtlvol) bovine serum al-bumin (BSA).

Cell membrane preparation and receptorbinding assay

CATH.a cells, pretreated or not with PACAP (10 nM, 16h) in DMEM/F12 containing 0.1% BSA, were rinsed withphosphate-buffered saline and briefly incubated with 50 mMglycine in 100 mM NaC1 (pH 3.0) to dissociate receptor-bound peptide (Penhoatet al., 1989). Cells were then washedtwice with andharvested in ice-cold 50 mM Tris-HC1 buffer(pH7.4) supplemented with 5 mM MgCl2, 0.2 mg/mi bacitra-cm, 2 ~ig/ml phenylmethylsulfonyl fluoride, and 20 ‚ag/mlaprotinin (Tatsumo et al., 1991). They were homogenizedwith a Dounce homogenizer, and the homogenate was spunat 250 g for 10 min at 4°C to get rid of cell nuclei andunbroken cells. The resulting supernatant was centrifugedfurther at 50,000 g for 30 min at 4°Cto obtain a crudemembrane fraction. The latter was then resuspended in thesame Tris-HC1 buffer enriched with 0.2% BSA. Aliquots of100 /21 of membrane suspension (equivalent to 30—50 ~ ofprotein) were incubated at 18°Cfor 90 min in the presenceof 30—40 fmol of

1251-PACAP-27, alone or together with10_le_10_6 M unlabeled PACAP-38. The reaction wasstopped by the addition of 1.5 ml of ice-cold Tris-HC1 buffercontaining 0.5 mM EDTA and 0.1% BSA, and the mixture

J. Neurochem., Vol. 70, No. 4, 1998

DESENSITIZATION OF PACAP RECEPTORS 1433

was rapidly filtered through Whatman GF/C filters (pre-soaked for 2 days in 0.5% polyethylenimine). The filterswere washed twice with 4 ml of the same bufferand countedfor radioactivity in a Packard y-counter. Nonspecific bind-ing, obtained upon incubation of membrane suspension inthe presence of 1

1iM peptide, was ‚-~15% of total binding.

Measurement of cAMP productionCATH.a cells were incubated in the absence or presence

of various drugs in serum-free DMEM/F12 (containing 0.1%BSA) for varying time periods. They were then washed twicebefore being challenged with peptides for 15 mm, as indi-cated in the figure legends. After completion of the incuba-tion period, the reaction was arrested by the addition to thecell culture of 1 volume of ice-cold 0.2 M HC1. After afreeze—thaw cycle, cells were disrupted further by sonica-lion, and the suspensions were spun at 10,000 g for 15 min.The resulting supernatants were stored at —25°Cfor mea-surement of cAMP by radioimmunoassay (Lutz-Bucher etal., 1996).

Measurement of inositol phosphate accumulationAfter 2—3 days in culture, CATH.a cells were cultured

for 2 additional days in the presence of myo-[3H]inositol (4

/sCilml) in myo-inositol-free DMEMIF12 culture mediumsupplemented with 2% fetal calf serum. After being pre-treated with peptides or Br-cAMP, cells were washed withand incubated for 10 min in 10 mM LiC1 in HEPES buffer,composed of 150 mM NaC1, 5 mM KC1, 0.8 mM MgSO

4, 1mMCaCl2, 5 mMHEPES, 5.5 mM glucose, and 0.1% BSA,at pH 7.4. They were then exposed to PACAP for 20 minin the same medium. At the completion of the incubationperiod, cells were recovered in ice-cold 5% perchloric acidand homogenized. After centrifugation of the homogenateat 10,000 g for 15 mm, the supernatant was recovered andneutralized with 10 M KOH. The clear supernatant obtainedafter afinal centrifugation wasapplied to DowexAG- 1 mini-columns. Columns were washed with water to remove free[3H]inositol, and glycerophosphoinositol was washed out

with a mixture of 60 mM ammonium formate and 5 mMsodium tetraborate. Inositol monophosphate (InsP

1), inositolbisphosphate (InsP2), and inositol trisphosphate (InsP3) werethen eluted by means of a stepwise gradient of 0.1 M formicacid in 0.2, 0.4, and 0.7 M ammonium formate, respectively(Berridge et al., 1982).

Transfection studiesWe used a chimeric plasmid encoding the DNA binding

domain of the yeast protein GAL4 (l—147) fused to CREB,which was a kind gift of M. E. Greenberg (Boston, MA,U.S.A.). CATH.a cells were transfected by means of thepolyethylenimine method (Boussif et al., 1995). In brief,cells were cotransfected for 8 h with 1.5 ~g each of GAL4-CREB and reporter gene GAL4-luciferase, using 3 /21 of 10mM polyethylenimine//2g of DNA, in serum-free DMEM/F12. After this transfection period, cells were washed andpretreated, or not, with PACAP-38 for 16 h. The cells werethen washed again and left untreated for 8 h before beingreexposed to PACAP for 6—8 h. Luciferase activity wasmeasured as indicated by the manufacturer (BoehringerMannheim France).

Statistical analysisStatistical significance of data was assessed by means of

analysis of variance, followed by the Student—Newman—Keuls test for multiple comparisons, using Instat2 software

FIG. 1. Concentration of PACAP binding sites. A: Competitivedisplacement of

1251-PACAP-27 binding to membranes ofCATH.a cells by graded doses of unlabeled PACAP-38. Mem-brane fractions were isolated from naive (Ctr.) and PACAP-38(PAC)-pretreated cells (10 nM, 16 h). IC

50 values were calculatedto be 2.3 and 2.4 nM, respectively. Each point is the mean ofduplicates in a representative experiment. B: Concentrations ofspecific binding sites (B~,,)in membranes from control and PA-CAP-treated cells. Specific binding was the difference in bindingobserved in the absence (total) and presence (nonspecific) of 1~sMunlabeled PACAP. Each column represents the mean ±SEof three experiments.

(Graphpad). Binding data were analyzed with the aid ofGraphpad‘s Prism and Biosoft‘s Kell programs. Data seenin figures are representative of at least three independentexperiments, which yielded similar results.

RESULTS

Binding of PACAP to cell membranesAnalysis of binding data obtained by incubating

membrane fractions of CATH.a cells with a constantconcentration of ‘

251-PACAP, along with graded dosesof unlabeled peptide, clearly indicated that the densityof binding sites was not changed significantly in PA-CAP-pretreated cells compared with controls (154±11 and 175 ±18 fmol/mg of protein, respectively;Fig. 1), nor were the dissociation constants, whosevalues were calculated to be 0.35 ±0.11 and 0.30±0.07 nM, respectively. It thus appears that chronicexposure to PACAP is not associated with down-regu-lation of membrane receptor sites in CATH.a cells,thereby offering a suitable model system in which toinvestigate possible cross-talks between multiplextransduction signals.

Desensitization of the cAMP pathwayWhen CATH.a cells were pretreated with 2 nM

PACAP for various time periods, there was a delayedand gradual decrease (t

112 = 6.7 ±0.8 h) in theirability to accumulate cAMP in response to a subse-quent exposure to PACAP (Fig. 2). Dampening ofthe cAMP response did not occur rapidly in thesecells, as a decrease of —~30%was registered after 4

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1434 A. MULLER ET AL.

FIG. 2. Time and dose dependency of homologous desensitiza-tion of the cAMP response to PACAP. CATH.a cells were pre-treated, or not (zero time point), with 2 nM PACAP-38 for varioustime periods before being exposed to a test dose of 1 riM PA-CAP-38 for 15 min. Inset: Homologous desensitization is dose-dependent in cells stimulated for 16h, with IC50 = 0.1 nM. Eachpoint is the average ±SE of four determinations done in dupli-cate. Data represent net values: final cAMP concentration minusresidual activity of PACAP-pretreated cells.

h, whereas an inhibition of ~-.‘80%was reached at16 h (a time point that was used for all further experi-ments). Homologous desensitization elicited by PA-CAP was dose-dependent, with half-maximal inhibi-tion (IC50) being achieved with 0.1 ±0.03 nM (insetto Fig. 2). Chronically treated cells, incubated for16 h in fresh culture medium, showed no apparentsign of resensitization when exposed to PACAP(data not shown).

To test whether the fall in cAMP formation waspossibly due to the effect of inhibitory G1-like pro-teins or todownstream degradation by cyclic nucleo-tide PDEs, we carried out experiments using drugsknown to block these proteins. Preincubation of cellswith PTX (Fig. 3A), which blocks G~-likeproteins,produced the expected amplification of PACAP-stimulated cAMP formation in control cells, but didnot counter the desensitization process caused bylong-term treatment of cells with PACAP. Also, in-hibition by IBMX of PDEs (Fig. 3B) did not hamperthe reduction of PACAP-induced cAMP accumula-tion, precluding an effect due to mere cyclic nucleo-tide degradation.

Stimulation of PKC by PACAP, via activation of thePLC route, could also be involved in altered AC activity(Houslay, 1991). We tested that possibility and foundthat pharmacological stimulation by phorbol ester of PKCprofoundly depressed (by ‘=63% versus control; p<0.001) PACAP-stimulated cAMP formation. This ef-fect was not prevented by lBMX, precluding a possibleinfluence of PDEs in the process (Table 1).

FIG. 3. Effects of PTX and IBMX on PACAP-induced cAMP de-sensitization. CATH.a cells were cultured for 16h in the absence(Ctr.) and presence of PTX (50 ng/ml), either alone or with 2 nMPACAP-38. They were then reexposed to 1 nM PACAP for 15mm, either without (A) or with (B) 0.5 mM IBMX. 0-P and P-Prefer, respectively, to untreated and PACAP-pretreated cells thatwere then exposed to PACAP. Each column is the average±SE of four determinations done in duplicate. Data in this, aswell as in subsequent, figures represent net values: final cAMPconcentration minus baseline or residual activity of treated cells(1.7 ±0.3 and 5.1 ±0.9 pmol/well, respectively). **~.rr~p<0.001,compared with corresponding controls.

Pretreatment of cells with VIP, but not withforskolin, mimicked the effect of PACAP oncAMP formation

We next examined whether blunting of the cAMP re-sponse could be reproduced by VIP (which cross-reactswith PACAP receptors) or by forskolin (which bypassesthe very receptor—G protein complex and directly stimu-lates AC). Treatment with I /2M VIP did mimic the effectseen with 2 nMPACAP (Fig. 4A), as subsequentPACAP-stimulated cAMP formation was reduced by 45%, as com-pared with 57%, respectively (p < 0.01 and p < 0.001,versus control). This was clearly not the case with for-skolin, which not only failed to dampen second messengerformation under these conditions, but even caused an ele-vation of its concentration with a 10 1iM concentration of

TABLE 1. Effect of activation by PMA of PKC onPACAP-induced cAMP formation

Drug additioncAMP formation

(pmollwell)

None 73.7 ±2.1PMA 27.5 ±1.1°IBMX 123.7 ±3.9IBMX + PMA 35.3 ±1.7°

CATH.a cells were treated with 10 nM PMA, alone or with 0.5roM IBMX, for 10 mia before exposure to I nM PACAP-38 for 15min. Results are means ±SE of four determinations done in dupli-cate. Basal cAMP concentrations were 2.9 ±0.4 pmol/well.

“p < 0.001, versus respective controls.

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DESENSITIZATION OF PACAP RECEPTORS 1435

FIG. 4. Effect of cell treatmentwith VIP and forskolin on PACAP-induced cAMP accumulation.CATH.a cells were cultured for 16h in the absence (0) or presenceof 2 nM PACAP-38 (P), 1 pM VIP(V), or 1

1.zM or 10 pM forskolin(Fi and F10) before being restim-ulated with 1 nM PACAP (P). Eachcolumn is the average ± SE offour determinations done in dupli-cate. °°p< 0.01, ~ < 0.001,compared with control (0-P).

the drug. This strongly argues in favor of regulatory ele-ments upstream of AC being involved, namely, the recep-tor—G protein complex.

Effect of PACAP on stimulation ofphosphoinositide (PI) hydrolysis

Data in Fig. SA clearly show that PACAP-38 causedsignificant changes of Pl turnover in CATH.a cells, asevident from elevations in the concentrations of InsP1,InsP2, and InsP3. Because incubation was carried outin the presence of Lit, which blocks myo-inositol 1-

FIG. 5. Amplification by chronic treatment with PACAP of PA-CAP-induced inositol phosphate production. A: Cells, labeledwith myo-[

3H]inositol, were incubated for 20 min in the absence(Ctr.) or presence of either 10 riM PACAP-38 (PAC) or 1 pMVIP, in HEPES buffer containing 10 mM LiCI. Inositol phosphatespecies (InsP

1, lnsP2, and InsP3) were separated on Dowex mini-columns using a gradient of ammonium formate in 0.1 formicacid. *p < 0.05, °°p< 0.01, °°°p< 0.001, compared with Ctr.B: Cells were cultured for 16 h in control medium (0) and inmedia to which were added PACAP-38 (P; 0.1 —100 nM), VIP (1pM), and Br-cAMP (1 mM) and further tested for their ability toaccumulate lnsP1 in response to 10 nM PACAP (P) during a 20-min incubation period. Each column is the mean ± SE of fourdeterminations. *p < 0.05, °°p< 0.01, between columns con-nected with a horizontal line and compared with group O-P forcells treated with VIP or cAMP, respectively.

phosphatase (Berridge et al., 1982), the most dramaticeffect of PACAP (an augmentation of about seventimes the basal level) was seen with lnsP1. The latterspecies was thus selected for measurement in all subse-quent experiments. The parent hormone VIP producedno significant effect, providing evidence for the pres-ence in these cells of type I PACAP receptors.

In marked contrast to the desensitization seen forcAMP, long-term stimulation of PACAP receptors notonly failed to desensitize the PLC-PI pathway but, incontrast, caused a striking amplification of InsP1 for-mation as triggered by subsequent stimulation withPACAP (Fig. SB). The effect was concentration-de-pendent (EC50 = 0.11 ± 0.05 nM), with a significantaugmentation of 49% being apparent with 0.1 nMPACAP (p < 0.01 compared with naive cells) and anincrease of >200% with 100 nM PACAP. Similarly,VIP (1 /2M) and Br-cAMP (1 mM), when added for16 h to the culture medium of CATH.a cells, alsocaused significant (p < 0.01) elevations of InsP1 accu-mulation in response to a test dose of 10 nM PACAP.

Protein synthesis is a requisite for PACAP-induced alterations of transduction signals

Because the mechanism of cell desensitizationcaused by prolonged stimulation of G protein-coupledreceptors involves a variety of so-called accessory pro-teins (Gershengorn, 1994; Neer, 1995; Berman et al.,1996), we investigated the effect of inhibition by Cxof protein synthesis. The efficacy of inhibition wasassessed in a preliminary experiment, which showedthat Cx (10 /2g/ml) blocked the incorporation of ~‘

4C]-leucine into cell proteins by 90 ± 3% (n = S).

When added to culture medium along with PACAPduring long-term treatment, Cx appeared to reversecompletely the desensitization of the cAMP pathway(Fig. 6A): in the absence of the inhibitor, the cAMPresponse to a test dose of PACAP was reduced by 70%(p < 0.01 compared with naive cells), whereas in thepresence of the drug there was no significant changerelative to controls. The likelihood that this effect couldhave resulted from inhibition by Cx of PDE synthesiswas further excluded by the observation that closelysimilar results were yielded in the presence of IBMX(data not shown).

In a similar way, Cx also caused a complete oblitera-tion of PACAP-induced overproduction of lnsP

1 (Fig.6B). Accumulation of InsP~,which was heightened to2.4 times the control level in PACAP-pretreated versusnaive cells, remained unchanged in cells cocultured inthe presence of PACAP along with Cx. Cells culturedwith Cx alone showed no significant change in re-sponse to PACAP in terms of either cAMP or InsP1formations (data not shown).

Effect of inhibitors of PKA and PKCGiven the evidence that protein kinase-induced

phosphorylation of various substrates is involved inthe desensitization process (Böhm et al., 1997), wetested the effects of inhibitors of PKA (H89) and PKC

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1436 A. MULLER ET AL.

FIG. 6. Effect of inhibition of protein synthesis on PACAP-in-duced changes of transduction signals. Cells were incubated for16 h in the absence (0) or presence of 2 nM PACAP-38, eitheralone (P) or with 10 pg/mI Cx (Cx/P). They were then challengedwith 1 nM and 10 nM PACAP for measurement of cAMP (A) orlnsP1 (B) production, respectively. Each column is the mean± SE of three or four determinations. *~p< 0.01 °°°p< 0.001,versus PACAP-stimulated naive cells (0-P). Addition of Cx aloneto the medium of naive cells did not change their response toPACAP (data not shown). Each bar is the mean ± SE of fivedeterminations.

(bisindolylmaleimide). We found that H89 signifi-cantly reversed the drop of PACAP-induced cAMPformation seen in PACAP-pretreated cells, whichplummeted by 70% compared with controls (Fig. 7A;p < 0.001 and p < 0.5, respectively). Bisindolylmalei-mide, in contrast, appeared to cause only a modest andnonsignificant reversal of PACAP-elicited desensitiza-tion.

FIG. 7. Effects of H89 and bisindolylmaleimide (BIS) on AC andPLC pathways. Cells were cultured for 16 h in the absence (0)or presence of 2 nM PACAP, either alone (P) or together with10 pM H89 (H89/P) or 1 pM BIS (BIS/P). They were then stimu-lated with 1 cr10 nM PACAP (P) for measurements of cAMP (A)and lnsP1 (B), respectively. °°°p< 0.001, versus group O-P. ~p< 0.001, versus group P-P. Each bar is the mean ± SE of fourdeterminations.

It is interesting that the PKA inhibitor likewise coun-tered the overinduction of chronic PACAP treatmenton Pl hydrolysis. The concentration of InsP1 achievedin PACAP-treated cells and in response to a test doseof PACAP showed a threefold increase over controllevels. When cells were cultured with PACAP in thepresence of H89, overstimulation of PLC activity wasobliterated (Fig. 7B). In this case, the PKC inhibitoralso failed to affect hydrolysis of Pl in a significantmanner.

Effect of chronic PACAP on CREB-driventranscriptional activity

We next aimed at determining if persistent stimula-tion of PACAP receptors in CATH.a cells, which re-sults in altered transduction signaling, would eventu-ally be accompanied by downstream modifications ofgene expression. To this end, we measured transcrip-tion closely dependent on CREB by cotransfecting anexpression vector encoding a chimeric protein: CREBfused to the DNA/dimerization binding domains oftranscriptional activator GAL4, along with a GAL4-luciferase reporter gene.

As depicted in Fig. 8, PACAP-38 dose-dependentlyenhanced CREB-dependent transcription, with maxi-mal and half-maximal effects being achieved with 10nM and 0.7 nM, respectively. VIP had to be presentat 1,000 times the concentration of PACAP to stimulatea similar augmentation, whereas forskolin was less po-tent in this respect, with only about a twofold increasecompared with control cells (Fig. 9A and B). Becauseforskolin is known to exert both cAMP-dependentand -independent actions (Laurenza et al., 1989), wealso tested the direct effect of the cell-permeable Br-cAMP. The latter, at a concentration of 1 mM, in-creased CREB-regulated transcription about fourfold,from a resting level of 506 ±28 to 2,173 ±439arbitrary units (n = 4; p < 0.01).

We next addressed the question of whether alter-ations of transduction signaling in cells chronicallyexposed to either PACAP, VIP, or forskolin wouldimpact on gene transcription. As seen in Fig. 9C, pre-treatment of cells withPACAP and VIP enhanced sub-

FIG. 8. Activation of CREB-me-diated transcription by gradeddoses of PACAP. CATH.a cellswere cotransfected for 8 h withGAL4-CREB and reporter geneGAL4-luciferase, as describedin Experimental Procedures.They were then washed and leftin serum-free culture mediumfor 24 h before being restimu-lated with increasing concentra-tions of PACAP-38 for 8 h. Eachpoint is the mean ± SE of fourdeterminations. AU., arbitraryunits.

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DESENSITIZATION OF PACAP RECEPTORS 1437

FIG. 9. Effect of long-term exposure of cells to PACAP, VIP,and forskolin on PACAP-induced activation of CREB-mediatedtranscription. Transfected cells were pretreated, or not (0), with2 nM PACAP-38 (P), 1 pM VIP (V), or 5 pM forskolin (F) for 16h before being exposed, or not, to 1 nM PACAP orS pMforskolinfor 8 h. A: Naive (O) and pretreated cells (P, V, or F) were incu-bated in culture medium alone (0). °°°p< 0.001, versus group0-0. B: Naive (0) cells were stimulated with P, V, or F. *p <0.05,***p <0.001, versus respective controls in A. C: Cells pretreatedwith P, V, or F were reexposed to P and F. **p < 0.01, versusgroups 0-P and 0-V, respectively. Each point is the mean ± SEof four determinations. AU., arbitrary units.

sequent PACAP-induced CREB-regulated transcrip-tion. In sharp contrast, preexposure of cells to forskolinproduced no significant change in cells restimulatedwith either PACAP or forskolin (Fig. 9B and C).

DISCUSSION

One way to counter excessive stimulation by ago-fists is for the cell to engage in a desensitization pro-cess, which results in the dampening of its responseto further stimuli. However, although numerous studieshave been devoted to the molecular mechanisms in-volved in the shutoff of AC-dependent receptor sys-tems (Hausdorff et al., 1990; Böhm et al., 1997), com-paratively little information is available regarding re-ceptors linked to the PLC pathway, not to mentionthose linked to both signaling routes.

Here we investigated the long-term effect ofPACAPon CATH.a cells, which express type I receptors (bothshort and hop forms, as defined by Spengler et al.,1993) coupled to both AC and PLC (Muller et al.,1996). Continuous exposure of CATH.a cells to PA-CAP surprisingly failed to reduce membrane densityof PACAP binding sites and binding affinity. Althoughreceptor internalization provides an efficient means forthe cells to protect themselves against prolonged expo-sure to agonists, previous studies have already shownthat receptor sequestration/degradation may not alwaysbe a necessary requisite for receptor desensitization tooccur (Hausdorff et al., 1990; Olson et al., 1991; Chernet al., 1993; Olson and Schimmer, 1994), implying thatmechanisms downstream of agonist — receptor interac-tion also play a determinant role.

We show here for the first time that chronic stimula-tion of CATH.a cells with PACAP affected dual signal-ing in a differential way: PACAP-induced accumula-tion of cAMP appeared to be desensitized in the longrun, whereas Pl turnover, in contrast, was enhanced.Based on quite different experimental settings, previ-ous studies revealed similar functional relationshipsbetween AC and PLC activities. Thus, permanent over-expression of G~,.in a thyroid cell line was found tocause desensitization of PLC (Laglia et al., 1996),whereas pharmacological stimulation of the cAMP-PKA chain in hepatocytes and 3T3 fibroblasts en-hanced Pl hydrolysis in the presence of vasopressinand epidermal growth factor, respectively (Olashawand Pledger, 1988; Pittner and Fain, 1989).

Recent evidence suggests the existence of two sepa-rate “transmembrane conductors,“ which link the lu-teinizing hormone receptor to AC and PLC effectors(Gilchrist et al., 1996). Our findings are in line with thisnotion, in that PACAP-induced messenger cascadesactually evolved in an antipodal fashion. They alsofavor a model system based on interactive signalingroutes. Thus, G protein /3y subunits released on disso-ciation with either G

5,. or Gq,. subspecies involvedin the stimulation of AC and PLC, respectively—arelikely to act as additional stimulators of PLC (Iyengar,1993; Neer, 1995; Exton, 1997). Alternatively, PLCisoforms may also be activated through PKA-depen-dent phosphorylations (Liu and Simon, 1996), whereasPKA and other messenger-dependent protein kinasesare likely to phosphorylate any element of the recep-tor—G protein—PLC complex. Conversely, dephos-phorylation by phosphatases of phosphorylated sub-strates may antagonize the shutting down of signalsand, consequently, also represent an important regula-tory mechanism (Hunter, 1995). In contrast, stimula-tion of PKC elicited on activation of PLC may directlyaffect AC activity (Houslay, 1991). In fact, we actuallydemonstrate that pharmacological enhancement byphorbol ester of PKC activity markedly depressesPACAP-stimulated cAMP formation, suggesting acontribution of PKC in AC desensitization. However,the failure of bisindolylmaleimide, a potent PKC inhib-itor, to reverse the chronic effect of PACAP on cAMPproduction, does not favor such a mechanism of action.In this connection, it is of interest to point out thatCATH.a cells possess two isoforms of PACAP recep-tors: the short type and the hop type (Muller et al.,1997). The latter bears an insert in the third intracellu-lar domain, which encodes phosphorylation sites forPKC (Spengler et al., 1993). It is thus conceivable thatPACAP-stimulated PKC activity may eventually alterthe phosphorylation state of the receptor, precisely ata site—the third intracellular loop—that is thought tointeract with G proteins (Premont et al., 1995; Karooret al., 1996). -

The molecular mechanisms that govern the processof receptordesensitization— or up-regulation— are farfrom being fully understood. A number of intricate

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1438 A. MULLER ET AL.

factors are at play, including various G proteins andsecond messenger-activated protein kinases, G protein-coupled receptor kinases, accessory proteins, anddownstream cyclic nucleotide PDEs (Premont et al.,1995; Chuang et al., 1996; Karoor et al., 1996; Böhmet al., 1997). In the present study, we found that neitherPTX nor IBMX antagonized desensitization of thecAMP pathway in CATH.a cells, arguing against apossible involvement of G-like proteins or PDEs inthe reduction of cellular accumulation of cAMP. Incu-bation of cells in the presence of the PKA inhibitorH89 obliterated the long-term effect of PACAP onboth the decrease of cAMP production and the potenti-ation of Pl turnover. Under the same experimental con-ditions, the PKC inhibitor exerted only a modest andnonsignificant influence. Together with the observationthat VIP (which is solely coupled to AC) similarlyattenuated AC activity and amplified inositol phos-phate formation, our results point to the cAMP-PKAchainas playing a predominant role incells chronicallytreated with PACAP. However, in apparent oppositionto that view, pretreatment of cells with forskolin wasfound not to reduce PACAP-stimulated cAMP produc-tion. Because the site of action of the drug lies down-stream of the receptor—G protein complex, it is tempt-ing to speculate that receptor-dependent activation ofG protein subspecies plays a preeminent role in theprocess.

An important mechanism of action by which cellsmay adapt themselves to sustained exposure to agonistsis to alter the levels of G proteins, either by down- orup-regulation (Milligan, 1993). Indeed, a decrease ofG5,. and Gq,. levels has actually been demonstrated tooccur in neuroblastoma—glioma hybrid cells (NG1O8-15) and Chinese hamster ovary cells, as a result oflong-term treatment with prostaglandin and carbachol,respectively. Importantly, however, for these effects tobecome apparent, receptors had to be expressed at highlevels and thus interacted with a large amount of Gproteins. This probably was not the case for FRTL-5thyroid cells, in which overstimulation of the cAMP-PKA route caused desensitization of PLC activity inthe absence of measurable down-regulation of Gq,.(Laglia et al., 1996). In the present study, we showedPACAP-elicited desensitization of cAMP productionto be independent of G-like proteins, but the questionsof whether there is a reduction of G5,. or an incrementof Gq,.-like proteins (in relation to the overstimulationof PLC activity) remain to be clarified.

Evidence has been provided recently that variousaccessory proteins may serve as regulators of G proteinsignaling proteins or as binders of specific mRNAsof G protein-coupled receptors (Berman et al., 1996;Denhardt, 1996; Karoor et al., 1996). In an attempt todetermine whether protein synthesis may be involvedin some way in the alteration by chronic PACAP ofdual transmembrane signaling, we blocked protein syn-thesis in CATH.a cells by means of Cx. It is notewor-thy that, in the presence of the drug, both desensitiza-

tion of the cAMP pathway and overstimulation of thePLC pathway, as triggered by persistent exposure ofcells to PACAP, were obliterated. Blockade of proteinsynthesis by Cx, according to the regimen used here,appeared to have no deleterious effect on cell function,at least as far as PACAP-stimulated productions ofcAMP and InsP1 are concerned. Taken together, ourdata may thus be interpreted to indicate that the antipo-dal changes that prolonged treatment with PACAP ex-erts on dual signaling require newly synthesized pro-tein(s), which may possibly represent substrate(s) forPKA-mediated phosphorylation. Previous studies, like-wise based on the use of Cx, have demonstrated a needfor de novo protein synthesis in the desensitization ofthe mitogen-activated protein kinase signal (Grahamet al., 1996) and the induction by cAMP-PKA of /3-arrestin 1 mRNA expression (Chuang et al., 1996).

Finally, the fact that CATH.a cells fail to down-regulate receptors upon chronic exposure to PACAPalso offers the possibility of investigating whether dif-ferential alteration of signaling may impact on genetranscription. Because PACAP activates cAMP andhence affects cAMP-responsive genes, we examinedthe CRE-dependent transcription pathway. Expressionof CRE is under the control of a number of factors,including CREB, CREB-binding protein, CRE modu-lator, and other CRE binders (Meyer and Habener,1993), whereas CREB is regulated by PKA and otherprotein kinases such as Ca

2~/calmodulin-dependentprotein kinases (CaMKs) and ras-dependent Sen33 pro-tein kinase (Sheng et al., 1991; Sun et al., 1994;Hunter, 1995). To analyze CREB-driven transcription,we used cells transfected with vectors in which CREBis fused to the heterologous GAL4-DNA binding do-main, which targets the GAL4-luciferase reporter con-struct (Sheng et al., 1991). Using this experimentalapproach, we found PACAP to stimulate CREB tran-scriptional activity in a dose-dependent manner, withan EC

50 concentration close to the dissociation constantof the receptor. High concentrations of VIP, as wellas forskolin, likewise stimulated CREB-driven tran-scription. As to the regulation of the transcription ofCREB itself, previous studies found this factor to beunder the negative control of the cAMP cascade, be-cause stimulation of CATH.a cells with forskolin re-sulted in a decrease of both CREB protein and mRNAlevels (Widnell et al., 1994, 1996).

The state of phosphorylation of Ser‘33 on CREB,

which directly determines the degree of transcrip-tional inducibility, results mainly from the antago-nistic influences of protein kinases and phospha-tases, as well as from the presence/activity of nu-clear PKA inhibitor PKI (Hunter, 1995). WhereasPKA and CaMKs of types II and IV activate CREB,CaMKII is also likely to block CREB activation byphosphorylating Ser142 (Sun et al., 1994). Here wefound long-term treatment of cells with PACAP orVIP to potentiate CREB transcriptional activity inresponse to a subsequent exposure to PACAP. This

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DESENSITIZATION OF PACAP RECEPTORS 1439

was clearly not the case with cells pretreated withforskolin, suggesting that activation of elementsupstream of PKA, namely the receptor—G proteincomplex, exert a preeminent influence. Amplifica-tion of CREB-dniven transcription in PACAP-pre-treated cells could be explained in several, thoughnot exclusive, ways. It may result from augmentedactivation of CaMKs, consecutive to PACAP/VIP-induced overstimulation of the PLC cascade, whichultimately culminates in the elevation of ~Ca2~I

1.The likelihood also exists that, in PACAP chroni-cally treated cells, the activity of phosphatases and/or of nuclear PKA inhibitor PKI may be down-regulated, thereby allowing for increased phosphor-ylation of Ser‘

33 on CREB. Alternatively, assumingCaMKII to be present in CATH.a cells and phos-phorylating Ser142, dephosphorylation by phospha-tases of this inhibitory site would end up in CREBinduction as well.

Acknowledgment: We are grateful to D. M. Chikaraishiand M. E. Greenberg for their generous gifts of CATH.acells and GAL4-CREB construct, respectively. We alsothank Mrs. L. Lepersonnic and F. Herzog for their excellenttechnical assistance. This work was supported in part by theAssociation pour la Recherche sur le Cancer (grant ARC no.6089 to J.-P.L.).

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