Selective Activation of Gao by D2L Dopamine Receptors in NS20Y Neuroblastoma Cells

 
The Journal of Neuroscience, November 1, 1998, 18(21):8692–8699

Selective Activation of Gao by D2L Dopamine Receptors in NS20Y
Neuroblastoma Cells

Val J. Watts,1 Brenda L. Wiens,1 Medhane G. Cumbay,1 Minh N. Vu,1 Rachael L. Neve,2 and Kim A. Neve1
1Medical Research Service, Veterans Affairs Medical Center, and Department of Behavioral Neuroscience, Oregon Health
Sciences University, Portland, Oregon 97201, and 2Department of Genetics, Harvard Medical School, and McLean
Hospital, Belmont, Massachusetts 02178

D2L dopamine receptor activation results in rapid inhibition and                  blotting and inhibition of GTPgS-stimulated adenylate cyclase,
delayed heterologous sensitization of adenylate cyclase in sev-                   respectively. To assess the specificity of D2L-Ga coupling, cells
eral host cell types. The D2L dopamine receptor was stably                        were infected with herpes simplex virus (HSV) recombinants
transfected into NS20Y neuroblastoma cells to examine inhibi-                     expressing individual PTX-resistant G-protein a subunits and
tion and sensitization in a neuronal cell environment and to                      treated with PTX, and quinpirole-induced responses were mea-
identify the particular G-proteins involved. Acute activation of                  sured. Infection of NS20Y-D2L cells with HSV-Gao* rescued
D2L receptors with the selective D2 agonist quinpirole inhibited                  both inhibition and sensitization in PTX-treated cells, whereas
forskolin-stimulated cAMP accumulation, whereas prolonged                         infection with HSV-Gai1*, HSV-Gai2*, or HSV-Gai3* failed to
incubation (2 hr) with quinpirole resulted in heterologous sen-                   rescue either response. In summary, the current study provides
sitization (more than twofold) of forskolin-stimulated cAMP ac-                   strong evidence that the D2L dopamine receptor couples to Gao
cumulation in NS20Y-D2L cells. To unambiguously identify the                      in neuronal cells, and that this coupling is responsible for both
pertussis toxin (PTX)-sensitive G-proteins responsible for inhi-                  the acute and subacute effects of D2 receptor activation on
bition and sensitization, we used viral-mediated gene delivery                    adenylate cyclase activity.
to assess the ability of genetically engineered PTX-resistant
G-proteins (Gai1*, Gai2*, Gai3*, and Gao*) to rescue both re-                       Key words: dopamine D2L receptors; Gai/o ; NS20Y neuro-
sponses after PTX treatment. The expression and function of                       blastoma; adenylate cyclase; pertussis toxin; heterologous
individual recombinant G-proteins was confirmed with Western                      sensitization

Alterations in D2-like dopamine receptors and their signaling                     cyclase to subsequent stimulation (Sharma et al., 1975; Bates et
pathways are thought to be involved in the etiology and treatment                 al., 1991; Ammer and Schulz, 1996; Watts and Neve, 1996; Watts
of many neuropsychiatric disorders, including schizophrenia, de-                  et al., 1998). Heterologous sensitization has been proposed to be
pression, Parkinson’s disease, and drug abuse. Hence, identifying                 one mechanism by which a cell adapts to prolonged inhibition of
the signaling pathways evoked after D2-like dopamine receptor                     cAMP synthesis and may be a cellular model of drug tolerance
activation may help us to understand the biochemical changes                      and dependence (Sharma et al., 1975; Ammer and Schulz, 1996;
that occur in clinical settings. Such studies in native neuronal                  Nestler and Aghajanian, 1997). Although both D2-mediated inhi-
tissues are made difficult by the molecular diversity of D2-like                  bition and heterologous sensitization of forskolin-stimulated
dopamine receptors (D2S , D2L , D3 , and D4 ), the number of                      cAMP accumulation are blocked by PTX, no studies have di-
pertussis toxin (P TX)-sensitive G-proteins through which they                    rectly examined and compared the G-protein specificity for these
can couple (Gai1 , Gai2 , Gai3 , Gaoa , and Gaob ), and the many                  signaling events. Moreover, heterologous sensitization does not
signal pathways modulated by D2-like receptor activation (Huff,                   appear to be a direct result of decreased intracellular cAMP
1997; Watts and Neve, 1997).                                                      levels or reduced PKA activity (Watts and Neve, 1996; Watts et
   One of the characteristic features of D2-like dopamine recep-                  al., 1998), raising the possibility that different PTX-sensitive
tors and other Gai /o-coupled receptors is P TX-sensitive inhibi-                 G-proteins mediate inhibition and heterologous sensitization of
tion of cAM P accumulation. Also, persistent stimulation of Gai /                 adenylate cyclase.
o-coupled receptors such as the D2-like dopamine receptors and
                                                                                     Among the approaches that have been used to identify the
the m opioid receptor results in the sensitization of adenylate                   G-proteins that mediate D2 dopamine receptor signaling are
                                                                                  antisense oligonucleotide treatments (Liu et al., 1994), the use of
Received June 15, 1998; revised Aug. 17, 1998; accepted Aug. 19, 1998.            Ga subunit-specific antibodies (Lledo et al., 1992; Izenwasser and
  This work was supported by Grant MH45372 from the U.S. Public Health Service,
the Veterans Affairs Merit Review and Research Career Scientist Programs, and a
                                                                                  Côté, 1995), and rescue with PTX-insensitive Ga subunits
Young Investigator Award from the National Alliance for Research on Schizophre-   (Senogles, 1994; O’Hara et al., 1996). In the latter approach,
nia and Depression. We thank Dr. Amy Eshleman for careful reading of this         expression of individual PTX-resistant a subunits is used to
manuscript.
  Correspondence should be addressed to Dr. Kim A. Neve, Medical Research         determine the specificity of receptor-G-protein signaling after
Service (R&D-30), Veterans Affairs Medical Center, 3710 SW U.S. Veterans          elimination of endogenous receptor-G-protein coupling by treat-
Hospital Road, Portland, OR 97201.
  Dr. Watts’ present address: Department of Medicinal Chemistry and Molecular
                                                                                  ment with PTX (Taussig et al., 1992).
Pharmacology, Purdue University, West Lafayette, IN 47907.                           We used a defective herpes simplex virus vector (HSV) for
Copyright © 1998 Society for Neuroscience 0270-6474/98/188692-08$05.00/0          acute expression of PTX-insensitive mutants to compare the
Watts et al. • G-Protein Specificity for D2L Receptors in NS20Y Cells                             J. Neurosci., November 1, 1998, 18(21):8692–8699 8693

D2L:Ga protein specificity for inhibition and heterologous sensi-            solution, containing 15 mM H EPES, 1 mM isobutylmethylxanthine, 2%
tization of cAM P accumulation in a neuronal-like environment.               CBS, and 0.02% ascorbic acid). For inhibition experiments, cells were
                                                                             preincubated with 300 ml of assay buffer for 10 min and placed on ice. D2
The D2L dopamine receptor stably expressed in NS20Y cells                    agonists in the absence or presence of antagonists were added to wells
(NS20Y-D2L ) exhibited the appropriate pharmacological and                   before the addition of 10 mM forskolin. For sensitization experiments,
functional properties of endogenous D2 dopamine receptors.                   cells were preincubated for 2 hr in the presence of drugs at 37°C in a
Viral-mediated gene delivery of recombinant Ga subunits pro-                 humidified incubator with 10% C O2 and then washed three times for 3– 4
vided rapid (18 hr) expression of f unctional proteins in NS20Y-             min each with 300 ml of assay buffer. Forskolin (10 mM) was then added
                                                                             in the presence of spiperone (1 mM) to preclude acute effects of D2
D2L cells. We now report that stimulation of D2L receptors in                dopamine receptor activation by residual agonist (Watts and Neve,
NS20Y-D2L cells preferentially activates Gao for both the inhi-              1996). cAM P accumulation for inhibition and sensitization experiments
bition and sensitization of forskolin-stimulated cAMP                        was performed for 15 min at 37°C, the assay buffer was decanted, and the
accumulation.                                                                cells were placed on ice and lysed with 3% trichloroacetic acid. The
                                                                             24-well plates were then centrif uged at 1000 3 g for 15 min and stored at
MATERIALS AND METHODS                                                        4°C for at least 1 hr before quantification of cAM P. For f unctional
                                                                             studies using HSV recombinants, confluent NS20Y-D2L cells were in-
Materials. [ 3H]cAM P was purchased from N EN Life Science Products
                                                                             fected with viral preparations (approximately one infectious unit /cell) in
(Boston, M A), and [ 3H]spiperone (104 C i /mmol) was purchased from
                                                                             1 ml of growth medium for 18 hr, the medium was removed and replaced
Amersham Life Sciences (Arlington Heights, IL). Quinpirole, (1)-
                                                                             with medium containing P TX, as indicated in the figure legends, and
butaclamol, and forskolin were purchased from Research Biochemicals
                                                                             then sensitization or inhibition experiments were completed as described
International (Natick, M A). Dopamine (3-hydroxytyramine), pertussis
                                                                             above.
toxin, growth media, and most other reagents were purchased from
                                                                                Adenylate c yclase assay in NS20Y-D2L cell membranes. Confluent cell
Sigma (St. L ouis, MO). Fetal bovine serum (FBS) and iron-
                                                                             monolayers in six-well tissue culture plates were infected with viral
supplemented calf bovine serum (CBS) were purchased from HyC lone
                                                                             preparations (approximately one infectious unit /cell) for 18 hr in fresh
(L ogan, UT). Antisera to Gai1 and Gai2 were purchased from Signal
Transduction (San Diego, CA). Antisera to Gai3 and Gao were gener-           growth medium. C ells were harvested by lysis with ice-cold hypotonic
ously provided by Dr. David Manning (University of Pennsylvania,             buffer (2 mM Na 1-H EPES, pH 7.4, 2 mM EDTA, 1 mM DTT, and 0.3 mM
Philadelphia, PA). cDNAs encoding the pertussis toxin-resistant              PMSF), and the cell lysates were scraped from the plate, homogenized
G-proteins were generously provided by Dr. Ronald Taussig (University        with a Brinkmann Polytron homogenizer at setting 6 for 5 sec, and spun
of Michigan, Ann Arbor, M I).                                                at 30,000 3 g for 20 min. The resulting crude membrane fraction was
   Production and maintenance of cell lines. Transfection of NS20Y cells     resuspended (;1 mg /ml) in storage buffer (15 mM Na 1-H EPES, pH 7.4,
with the D2L vector was performed by calcium phosphate precipitation as      2 mM EDTA, 1 mM DTT, and 0.3 mM PMSF) and frozen at 270°C until
described previously (Cox et al., 1992). The plasmids pcDNA1-D2L (20         assayed. Adenylate cyclase assays were performed as described previ-
mg) and pBabe Puro (2 mg) were mixed with 0.5 ml of 0.25 M C aC l2 , and     ously with modifications (Watts et al., 1995a). Frozen membranes were
0.5 ml of 23 BBS [50 mM N,N-bis-(2-hydroxyethyl)-2-amino-                    thawed and added (10 –20 mg of protein /tube) to duplicate assay tubes
ethanesulfonic acid, 280 mM NaC l, 1.5 mM NaHPO4] was added. The             containing the reaction mixture (15 mM Na 1-H EPES, pH 7.4, 20 mM
mixture was incubated for 25 min and added dropwise to exponentially         phosphocreatine, 1 mM MgC l2 , 2 mM ATP, 1 mM isobutylmethylxan-
growing NS20Y cells in a 10 cm tissue culture plate. Transfectants were      thine, 5 U of creatine phosphokinase) and forskolin (30 mM) in the
isolated and screened by [ 3H]spiperone binding as described previously      absence or presence of GTPgS (1 mM), in a final volume of 100 ml.
(Cox et al., 1995). NS20Y-D2L cells were maintained in DM EM supple-         Incubations were performed for 15 min at 30°C and terminated by the
mented with 5% FBS and 5% CBS, penicillin – streptomycin, and puro-          addition of 3% trichloroacetic acid. T ubes were vortexed and centrif uged
mycin (2 mg /ml). C ells were grown in a humidified incubator at 37°C in     for 10 min at 15,000 3 g. cAM P in the supernatant was quantified as
the presence of 10% C O2.                                                    described below.
   Generation and pack ag ing of HSV vectors. The construction of P TX-         Quantification of cA MP. cAM P was quantified using a competitive
resistant mutant Ga (Ga*) cDNAs in which a serine replaced a cysteine        binding assay adapted with minor modifications from Nordstedt and
four residues from the C terminus has been described previously (Taussig     Fredholm (1990). Duplicate samples of the cell lysate (5–10 ml) were
et al., 1992; O’Hara et al., 1996). Mutant cDNAs were cloned into            added to reaction tubes containing cAM P assay buffer (100 mM Tris-
pHSV PrPUC using standard molecular techniques, and replication-             HC l, pH 7.4, 100 mM NaC l, 5 mM EDTA). [ 3H]cAM P (1 nM final
defective HSV vectors expressing mutant Ga subunits were prepared as         concentration) was added to each assay, followed by cAM P-binding
described (Neve et al., 1997). The titer of the helper virus component of    protein (;100 mg of crude adrenal extract in 500 ml of cAM P buffer).
each stock was 1–1.2 3 10 3 plaque-forming units/ml on 2–2 cells. HSV-       The reaction tubes were incubated on ice for 2 hr and harvested by
LacZ was prepared simultaneously with the individual HSV-Ga subunit          filtration as described for radioligand binding assays. cAM P concentra-
vectors with a titer of 2 3 10 5 infectious units/ml. E xpression of the     tions in each sample were estimated in duplicate from a standard curve
HSV-Ga* subunit vectors was confirmed by Western blotting (see               ranging from 0.1 to 100 pmol cAM P/assay.
Results).                                                                       Membrane preparation for immunodetection. For the standard mem-
   Radioligand binding assays. Confluent cells in 10 cm plates were har-     brane preparation, cells were infected with viruses and harvested as
vested by lysis with ice-cold hypotonic buffer (1 mM Na 1-H EPES, pH         described for membrane adenylate cyclase assays. The resulting mem-
7.4, 2 mM EDTA). After swelling for 10 –15 min, the cells were scraped       brane pellet was resuspended in 80 ml of H EPES buffer (15 mM Na 1-
from the plate and spun at 24,000 3 g for 20 min. The resulting crude        H EPES, pH 7.5, 1.0 mM DTT, and 0.3 mM PMSF), and protein concen-
membrane fraction was resuspended in Tris-buffered saline (50 mM             tration was determined using a BCA protein assay (Pierce, Rockford,
Tris-HC l, pH 7.4, with 155 mM NaC l) with a Brinkmann Polytron              IL). Proteins were equalized by dilution in SDS-PAGE sample buffer and
homogenizer (Westbury, N Y) at setting 6 for 10 sec and used for             frozen at 270°C until use. For the enriched preparation, membranes
radioligand binding assays. The binding of [ 3H]spiperone was assessed as    were prepared as described previously (Watts et al., 1998). C ells were
described previously (Starr et al., 1995; Watts and Neve, 1996). Aliquots    scraped with a rubber policeman in H EPES buffer with 0.25 M sucrose.
of the membrane preparation (5–15 mg of protein) were added to dupli-        The cells were collected by centrif ugation at 1,000 3 g for 10 min,
cate assay tubes containing the following: Tris-buffered saline, 0.001%      homogenized with a Teflon-glass homogenizer (eight strokes), and then
bovine serum albumin, radioligand, and appropriate drugs. (1)-               centrif uged at 600 3 g to remove the nuclei. The supernatant was
Butaclamol (2 mM) was used to define nonspecific binding. Incubations        decanted, and the resulting nuclear pellet was resuspended (two to three
were performed at 37°C for 45 min, in a volume of 1.0 ml, and terminated     strokes) and centrif uged at 1000 3 g for 10 min. The supernatants were
by filtration using a 96-well Tomtec cell harvester (Orange, C T). Filters   pooled and centrif uged at 48,000 3 g for 10 min. The membrane pellet
were allowed to dry, and 50 ml of BetaPlate scintillation fluid was added    was resuspended in H EPES buffer and centrif uged at 48,000 3 g for 10
to each sample. Radioactivity on the filters was determined using a          min. The final membrane pellet was resuspended in 400 ml of H EPES
BetaPlate scintillation counter (L K B-Wallac, Gaithersburg, MD).            buffer and frozen at 270°C until use. Protein was determined as de-
   cA MP accumulation assays. C ells were seeded at a density of 250,000 –   scribed above for the standard membrane preparation.
300,000 cells/ well in 24-well tissue culture clusters. E xperiments used       Immunodetection. Protein was subjected to SDS-PAGE through a 10%
confluent cells and were completed in assay buffer (Earle’s balanced salt    polyacrylamide gel and transferred to poly vinylidene difluoride (PV DF)
8694 J. Neurosci., November 1, 1998, 18(21):8692–8699                                   Watts et al. • G-Protein Specificity for D2L Receptors in NS20Y Cells

Figure 1. Specificity of D2L receptor-mediated inhibition of cAM P ac-
cumulation. cAMP accumulation was stimulated by forskolin (10 mM) in          Figure 2. Potency of quinpirole for inhibition and sensitization of
NS20Y-D2L cells in the absence or presence of quinpirole (1 mM) or            forskolin-stimulated cAM P accumulation. Each point is the average of
dopamine (1 mM) for 15 min. As indicated in the figure, some experiments      duplicate determinations, expressed as a percentage of forskolin-
were performed in the presence of 1 mM spiperone (1 Spip) or 1 mM             stimulated activity in the absence of quinpirole (F, Inhibition) or after 2
SCH23390 (1 SCH ). Data shown are the mean 6 SE of three to four              hr pretreatment with 10 mM quinpirole (E, Sensitization). Dose–response
independent experiments, each assayed in duplicate. *p , 0.01 compared        curves for quinpirole inhibition of cAM P accumulation were determined
with vehicle-treated cells (Dunnett’s post-repeated measures ANOVA).          in NS20Y-D2L cells stimulated with 10 mM forskolin and increasing
                                                                              concentrations of quinpirole for 15 min. For sensitization, NS20Y-D2L
membranes (Costar Corning, C ambridge, M A). Membrane sheets were             cells were treated with increasing concentrations of quinpirole for 2 hr
blocked overnight with 5% nonfat dry milk, washed with Tris-buffered          and washed, and cAM P accumulation was stimulated with 10 mM forsko-
saline, and incubated with specific G-protein a subunit antibodies for 3      lin. The experiments shown are representative of three independent
hr. The PV DF membranes were washed, and immunodetection was                  experiments. In the inhibition curve shown, the maximal inhibition was
accomplished using the ECF Western blotting kit (Amersham Life                88% and the IC50 value was 3.3 nM. Forskolin stimulation in the absence
Sciences, Buckinghamshire, England) according to the manufacturer’s           of quinpirole was 135 pmol / well. The EC50 value for sensitization was 18
instructions. Membranes were incubated with secondary antibody                nM; forskolin-stimulated cAM P accumulation was 130 pmol/well in
(fluoroscein-linked anti-rabbit Ig or fluoroscein-linked anti-mouse Ig) for   vehicle-treated cells and 320 pmol / well in cells treated with 10 mM
1 hr, washed, and then incubated with tertiary antibody (anti-fluoroscein-    quinpirole.
alkaline phosphatase conjugate) for 1 hr. Membranes were again washed,
exposed to ECF substrate for 7 min, dried at room temperature for 20          receptors (Monsma et al., 1989), the selective D1 antagonist
min, and then scanned using a Storm Imaging System (Molecular Dy-
namics, Sunny vale, CA).                                                      SCH23390 did not alter quinpirole- or dopamine-induced inhibi-
   Data anal ysis. Saturation isotherms for the binding of [ 3H]spiperone,    tion of cAMP accumulation in NS20Y-D2L cells (Fig. 1). Dose–
competition binding studies, and dose –response curves for stimulation        response curves for inhibition of cAMP accumulation by quinpi-
and inhibition of cAM P accumulation were analyzed by nonlinear re-           role revealed an IC50 value of 4.1 6 1.4 nM and a maximal
gression using the program GraphPAD Prism (San Diego, CA). Statis-
                                                                              inhibition of 88 6 5% (Fig. 2).
tical comparisons were made using ANOVA followed by Dunnett’s post
hoc t test comparing vehicle or control with treated groups, except where     Sensitization of forskolin-stimulated cAMP
indicated.                                                                    accumulation in NS20Y-D2L cells
RESULTS                                                                       Although acute activation of D2L receptors in NS20Y cells inhib-
Pharmacological characterization of D2L dopamine                              its forskolin-stimulated cAMP accumulation (Fig. 1), 2 hr treat-
receptors in NS20Y neuroblastoma cells                                        ment of NS20Y-D2L cells with dopamine or quinpirole enhanced
                                                                              subsequent forskolin-stimulated cAMP accumulation (Fig. 3).
The density of binding sites in membranes prepared from NS20Y
                                                                              This D2 agonist-induced heterologous sensitization was pre-
cells expressing the D2L receptor (NS20Y-D2L ), determined by
                                                                              vented by coincubation with spiperone or pretreatment with PTX
saturation analysis of [ 3H]spiperone binding, was 690 6 50
                                                                              (Fig. 3) but was not blocked by coincubation with the D1 antag-
fmol/mg of protein (n 5 7) with a KD for [ 3H]spiperone of 68 6
                                                                              onist SCH23390 (data not shown). The EC50 value for quinpirole-
7 pM. Competition binding studies completed in the absence of
                                                                              induced sensitization of forskolin-stimulated cAMP accumula-
GTP revealed shallow Hill slopes (nH ) for both dopamine (0.57 6
                                                                              tion in NS20Y-D2L cells was 26 6 4 nM, with a maximal increase
0.03, n 5 5) and quinpirole (0.72 6 0.02, n 5 5) when competing
                                                                              in cAMP accumulation that was 134 6 4% (n 5 3) greater than
for [ 3H]spiperone-labeled sites, consistent with an agonist profile.
                                                                              vehicle-treated cells (Fig. 2).
The Hill slope for the binding of sulpiride was 0.97 6 0.07 (n 5
3), consistent with an antagonist profile. The apparent affinity              Expression and function of PTX-resistant G-protein a
constants for each drug were 6.9 6 0.7 mM (dopamine), 6.7 6 0.8               subunits (Gai1*, Gai2*, Gai3*, and Gao*) using the HSV
mM (quinpirole), and 64 6 2 nM (sulpiride).                                   vector
   We examined the ability of D2 agonists to inhibit forskolin-               Before investigating the coupling of the D2L receptor to recom-
stimulated cAM P accumulation in NS20Y-D2L cells. Dopamine                    binant PTX-resistant Ga subunits (Ga*), we characterized the
and quinpirole markedly inhibited forskolin-stimulated cAMP                   expression and function of Ga* subunits under the conditions to
accumulation, and the inhibition by both agonists was completely              be used for D2L dopamine receptor functional assays. NS20Y-D2L
prevented by the D2 antagonist spiperone (Fig. 1). Although                   cells were infected with HSV recombinants expressing individual
NS20Y cells endogenously express low levels of D1-like dopamine               Ga* subunits (HSV-Ga*), and expression was examined by West-
Watts et al. • G-Protein Specificity for D2L Receptors in NS20Y Cells                            J. Neurosci., November 1, 1998, 18(21):8692–8699 8695

Figure 3. D2 agonist-induced heterologous sensitization. NS20Y-D2L
cells were treated with vehicle, quinpirole (1 mM), or dopamine (1 mM) for
2 hr at 37°C. Where indicated, some agonist treatments were completed in
the presence of 1 mM spiperone (1 Spip) or after overnight treatment with
50 ng/ml pertussis toxin (1 PT X ). C ells were extensively washed, and
cAMP accumulation was stimulated with forskolin (10 mM) for 15 min.
Data shown are the mean 6 SE of four to six independent experiments,
each assayed in duplicate. *p , 0.01 compared with vehicle-treated cells
(Dunnett’s post-repeated measures ANOVA).

ern blotting of membrane homogenates. Infection for 18 hr pro-               Figure 4. E xpression of HSV-Gai1*, -Gai2*, -Gai3*, and -Gao* in
duced robust expression of each of the recombinant Ga* subunits              NS20Y-D2L cell membranes. Lane 1 of each gel was loaded with 50 mg of
(Fig. 4). We also examined the f unction and confirmed the PTX               control NS20Y-D2L cell membranes from an enriched membrane prepa-
                                                                             ration (EP) as described in Materials and Methods. Lanes 2 and 3 were
resistance of each Ga* subunit. After infection with HSV-Ga*                 loaded with 10 mg of cell membranes from a standard membrane prepa-
viruses, cells were treated with P TX, and the effect of each                ration (StdP) of control and HSV-Gai1*, -Gai2*-, -Gai3*-, or -Gao*-
HSV-Ga* subunit on GTPgS-stimulated cAM P accumulation                       infected cells (18 hr), and Western analysis was completed using corre-
was measured. In control and HSV-LacZ-infected cells, the ad-                sponding a subunit specific antibodies. The data shown are from a single
                                                                             experiment representative of three independent experiments.
dition of GTPgS (1 mM) significantly potentiated forskolin-
stimulated cAM P accumulation by ;100% (Table 1). In contrast,
infection with the inhibitory Ga subunits HSV-Gai1*, -Gai2*,                 and uninfected NS20Y- D2L cells, and 73 6 2% in cells infected
-Gai3*, or -Gao* prevented GTPgS potentiation of forskolin-                  with HSV-LacZ but not treated with PTX.
stimulated cAM P accumulation (Table 1).
                                                                             G-protein a subunit specificity for heterologous
G-protein a subunit specificity for inhibition of                            sensitization in NS20Y-D2L cells
forskolin-stimulated cAMP accumulation in                                    D2L receptor-mediated heterologous sensitization of forskolin-
NS20Y-D2L cells                                                              stimulated cAMP accumulation in NS20Y-D2L cells is blocked by
D2 receptor-mediated inhibition of forskolin-stimulated cAMP                 PTX treatment (Fig. 3). NS20Y-D2L cells were infected with
accumulation in several cell lines is blocked by previous PTX                HSV-Ga* recombinants and incubated with quinpirole to induce
treatment, implicating P TX-sensitive Ga subunits (Neve et al.,              heterologous sensitization of forskolin-stimulated cAMP accu-
1989; Watts and Neve, 1996). To identif y the Ga subunit(s)                  mulation. In the absence of PTX, treatment with quinpirole (1
involved in D2L receptor-mediated inhibition of cAM P accumu-                and 10 mM) for 2 hr potentiated forskolin-stimulated cAMP
lation in NS20Y-D2L cells, we infected cells with HSV recombi-               accumulation in control NS20Y-D2L cells and in NS20Y-D2L cells
nants expressing P TX-resistant Ga subunits Gai1*, Gai2*, Gai3*,             infected with HSV-LacZ, or any of the PTX-resistant mutants
or Gao*. Under these conditions, treatment with P TX eliminates              (Fig. 6) (data for 1 mM not shown). Specifically, cAMP accumu-
coupling of D2L receptors to endogenous P TX-sensitive Ga sub-               lation was enhanced by 132 6 19% in control cells, 90 6 8% in
units, but not D2L receptor coupling to heterologous PTX-                    HSV-LacZ cells, and 91 6 22% (Gao*), 67 6 11% (Gai2*), 52 6
resistant Ga subunits. In the absence of P TX treatment, quinpi-             13% (Gai3*), and 38 6 8% (Gai1*) in HSV-Ga*-infected cells.
role inhibited forskolin-stimulated cAM P accumulation in                    Although quinpirole-induced sensitization was significant in each
NS20Y-D2L cells infected with HSV-LacZ, -Gai1*, -Gai2*,                      condition, the magnitude of sensitization was reduced in cells
-Gai3*, or -Gao*, whereas P TX pretreatment completely blocked               infected with HSV-Gai1*, -Gai2*, and -Gai3* ( p , 0.05, com-
quinpirole-induced inhibition of forskolin-stimulated cAMP ac-               pared with control cells; Dunnett’s post-repeated measures
cumulation in NS20Y-D2L cells and LacZ-infected NS20Y-D2L                    ANOVA). In some experiments, cells were initially treated with
cells (Fig. 5). P TX pretreatment also prevented quinpirole inhi-            PTX (500 ng/ml) for 2 hr, followed by a 2 hr incubation with
bition of cAM P accumulation in cells infected with Gai1*, Gai2*,            quinpirole in the presence of PTX (250 ng/ml). Consistent
or Gai3*. In contrast, quinpirole inhibited cAM P accumulation               with the data presented in Figure 3, PTX pretreatment com-
by 57 6 6% in P TX-treated cells that had been infected with                 pletely blocked quinpirole-induced sensitization of forskolin-
HSV-Gao*, compared with inhibition of 77 6 3% in untreated                   stimulated cAMP accumulation in control and HSV-LacZ-
8696 J. Neurosci., November 1, 1998, 18(21):8692–8699                                              Watts et al. • G-Protein Specificity for D2L Receptors in NS20Y Cells

Table 1. Effect of HSV-Ga* subunits on GTPgS-stimulated cyclic AMP accumulation in membranes from PTX-treated NS20Y-D2L cells

                                      cAM P accumulation (pmolzmg21zmin21)
                                                                           Forskolin                             1 GTPgS                               Stimulationa
Condition                             Basal                                (30 mM)                               (1 mM)                                (% of forskolin)
Control                                8.8 6 3.7                           178 6 39                              355 6 50*                             239 6 60
HSV-Gao                                6.8 6 2.1                           186 6 45                              246 6 71                              134 6 15
HSV-Gai1                              11.6 6 2.5                           272 6 70                              259 6 36                              112 6 19
HSV-Gai2                               9.0 6 1.5                           292 6 96                              261 6 63                              118 6 32
HSV-Gai3                              10.0 6 2.7                           191 6 43                              197 6 48                              101 6 3
HSV-LacZ                              12.7 6 2.3                           175 6 43                              313 6 53*                             200 6 27

NS20Y-D2L cells were infected with individual PTX-resistant Ga subunits (Ga*) for 18 hr and treated with PTX (500 ng/ml) for 4 hr. The medium was removed, and
membranes were prepared for adenylate cyclase assays as described in Materials and Methods. cAMP accumulation was stimulated with forskolin (30 mM) in the absence and
presence of GTPgS (1 mM). Data shown for basal, forskolin, and forskolin 1 GTPgS are expressed as pmolzmg21zmin21 and are the mean 6 SE of five independent
experiments, assayed in duplicate.
a
  Stimulation was calculated as the percentage of forskolin-stimulated cyclic AMP accumulation in the presence of GTPgS divided by cAMP accumulation with forskolin alone.
*p , 0.01 compared with forskolin alone (Student’s t test).

Figure 5. Ga subunit specificity for inhibition of cAM P accumulation in NS20Y-D2L cells. Quinpirole-induced inhibition of forskolin-stimulated cAMP
accumulation was examined in cells infected with P TX-resistant HSV-Ga subunits. NS20Y-D2L cells were untreated (Control ), infected with HSV-LacZ,
or infected with individual P TX-resistant Ga subunits for 18 hr. cAM P accumulation was stimulated with forskolin (10 mM) in the presence or absence
of quinpirole (10 mM) for 15 min. Where indicated, cAM P accumulation was assessed after 6 hr treatment with 100 ng /ml pertussis toxin (PTX ). Data
shown are the mean 6 SE of four or more independent experiments, assayed in duplicate. **p , 0.01, *p , 0.05 compared with forskolin alone (Student’s
t test).

infected NS20Y-D2L cells (Fig. 6). Rescue experiments with                             tion proteins makes it possible to study defined sets of signaling
PTX-resistant Ga subunits revealed that quinpirole treatment                           molecules in various cultured cells. In the current study, we
produced heterologous sensitization in cells infected with Gao*                        transfected the D2L dopamine receptor into NS20Y neuroblas-
(57 6 9%, n 5 5) after P TX treatment. In contrast, infection with                     toma cells, which have many characteristics of striatal cholinergic
Gai1*, Gai2*, or Gai3* failed to rescue sensitization in PTX-                          cells (Amano et al., 1972).
treated NS20Y-D2L cells. Similar results were seen in sensitiza-                          Initial characterization of NS20Y-D2L cells demonstrated that
tion experiments with 1 mM quinpirole (data not shown).                                acute activation of D2L receptors inhibited forskolin-stimulated
                                                                                       cAMP accumulation. Furthermore, subacute (2 hr) activation of
DISCUSSION                                                                             D2L receptors in NS20Y-D2L cells resulted in heterologous sen-
The complexity of the mechanisms by which a neurotransmitter,                          sitization of adenylate cyclase, a phenomenon we previously
such as dopamine, modulates intracellular processes is increas-                        characterized in the non-neuronal C6 glioma and HEK293 cell
ingly evident because of our expanding knowledge of interactions                       lines (Cox et al., 1995; Watts and Neve, 1996). D2L receptor-
among receptor subtypes, G-proteins, effectors, and other regu-                        mediated inhibition and heterologous sensitization of forskolin-
latory proteins. The molecular cloning of many signal transduc-                        stimulated cAMP accumulation in NS20Y-D2L cells are PTX-
Watts et al. • G-Protein Specificity for D2L Receptors in NS20Y Cells                                  J. Neurosci., November 1, 1998, 18(21):8692–8699 8697

Figure 6. Ga subunit specificity for heterologous sensitization in NS20Y-D2L cells. Quinpirole-induced sensitization of forskolin-stimulated cAMP
accumulation was examined in cells infected with P TX-resistant HSV-Ga subunits. NS20Y-D2L cells were untreated (Control ), infected with HSV-LacZ,
or infected with individual P TX-resistant Ga subunits for 18 hr. NS20Y-D2L cells were incubated in the presence or absence of quinpirole (10 mM) for
2 hr and extensively washed, and cAM P accumulation was stimulated with forskolin (10 mM) for 15 min. Some experiments were completed after
pertussis toxin (PTX ) treatment. C ells were initially treated with P TX (500 ng /ml) for 2 hr, followed by a 2 hr incubation with quinpirole in the presence
of PTX (250 ng/ml). Data shown are the mean 6 SE of five to seven independent experiments, assayed in duplicate. **p , 0.01 compared with matched
vehicle-treated cells (Student’s t test).

sensitive; thus, these cells provide a neuronal-like model system                cumulation in NS20Y-D2L cells. Expression of mutant Gai1*,
with which to examine the f unctional coupling specificity for D2L               Gai2*, and Gai3* subunits did not rescue inhibition or sensitiza-
receptor– Gai /o signaling events. This was accomplished by viral                tion in PTX-treated cells. Moreover, in the absence of PTX
(HSV)-mediated gene delivery of cDNAs encoding PTX-                              treatment, expression of each of the mutant Gai subunits ap-
resistant G-protein a subunits. The use of mutant P TX-resistant                 peared to reduce the magnitude of sensitization compared with
G-proteins is a powerf ul technique that has been used success-                  control NS20Y-D2L cells. This effect may be attributable to some
fully to study G-protein coupling to C a 21 currents (Taussig et al.,            degree of constitutive inhibition of adenylate cyclase by recombi-
1992) and D2 receptor-G-protein coupling to inhibition of cAMP                   nant Gai subunits. Alternatively, the reduction in D2L receptor-
accumulation in other cell types (Senogles, 1994; O’Hara et al.,                 mediated sensitization caused by expression of the Gai* subunits
1996). In the current study, HSV-mediated expression of each of                  could be attributable to sequestration of bg subunits, because bg
the Ga* subunits produced f unctional expression of the subunits                 sequestration by expression of Gat or the C terminus of
in NS20Y-D2L cells (Table 1). The expression of each Ga*                         b-adrenergic receptor kinase can prevent heterologous sensitiza-
subunit was robust (Fig. 4), and because all studies were per-                   tion (Avidor-Reiss et al., 1996; Thomas and Hoffman, 1996).
formed in one NS20Y-D2L cell line, confounds attributable to                        Although the exact mechanism responsible for heterologous
clonal variation in the expression level of D2 receptors and                     sensitization remains to be elucidated, we propose that persistent
endogenous components of the signaling pathways, such as                         activation of Gao-linked receptors leads to enhanced Gas-
G-proteins and adenylate cyclase, are likely to be minimal (Mul-                 adenylate cyclase coupling, possibly through a bg subunit-
laney et al., 1995; Watts et al., 1995b; Kenakin, 1997). Further-                dependent event (Avidor-Reiss et al., 1996; Thomas and Hoff-
more, the short duration of expression of the P TX-resistant Ga                  man, 1996). This hypothesis is based in part on observations that
subunits is likely to diminish adaptive changes in cellular signal-              heterologous sensitization of adenylate cyclase is associated with
ing and growth processes that may occur as a result of chronic                   an increase in the number of [ 3H]forskolin-labeled sites (Jones
expression of exogenous G-protein a subunits (Gordeladze                         and Bylund, 1990) and decreased palmitoylated Gas (Ammer and
et al., 1997).                                                                   Schulz, 1997), which could lead to increased adenylate cyclase
   Heterologous sensitization induced by Gai /o-coupled receptors                activity. Increased Gas–adenylate cyclase interactions have also
is blocked by P TX, but no studies have examined the G-protein                   been proposed as a mechanism for sensitization of adenylate
specificity for this neuroadaptive mechanism, which could be                     cyclase by antidepressant treatment (Chen and Rasenick, 1995).
mediated by a G-protein distinct from that mediating inhibition                  Our own data demonstrating that activation of D2 receptors
of adenylate cyclase (Watts and Neve, 1996; Watts et al., 1998).                 sensitizes multiple forms of Gas-activated adenylate cyclase add
However, using P TX-insensitive G-proteins, we found that selec-                 further support to this hypothesis (Watts and Neve, 1996).
tive activation of Gao* by D2L receptors mediated both inhibition                   We recently described mechanistic differences between short-
and heterologous sensitization of forskolin-stimulated cAMP ac-                  and long-term sensitization by D4 dopamine receptors in
8698 J. Neurosci., November 1, 1998, 18(21):8692–8699                             Watts et al. • G-Protein Specificity for D2L Receptors in NS20Y Cells

HEK293 cells (Watts et al., 1998). L ong-term (18 hr) agonist           which do not express endogenous Gao (B. L. Wiens, V. J. Watts,
treatment of H EK-D4 cells results in a greater magnitude of            K. A. Neve, unpublished observations).
sensitization in both intact cells and cell membranes compared             In the current study we have analyzed the selective activation
with short-term agonist treatment (2 hr). L ong-term sensitiza-         of PTX-sensitive G-proteins by D2L dopamine receptors in a
tion, but not short-term sensitization, is accompanied by de-           neuronal-like environment. We have demonstrated the utility of
creased immunoreactivity of Gai in membranes. A reduction of            the HSV expression vector for examination of recombinant sig-
Gai is likely to enhance adenylate cyclase activity and suggests        naling proteins. Overnight infection with PTX-resistant Ga sub-
that, in addition to increased Gas-adenylate cyclase interactions,      units resulted in marked increases in protein expression as as-
long-term sensitization involves other mechanisms. In light of the      sessed by Western blotting. The function and PTX resistance of
present results, it would be particularly interesting to examine        individual G-protein a subunits was confirmed by examining the
Gao levels in membranes after short- and long-term agonist              ability of Gai1*, Gai2*, Gai3*, or Gao* to inhibit GTPgS-
exposure in NS20Y-D2L cells. Moreover, the current observation          stimulated cAMP accumulation after PTX treatment. Under
that the selective stimulation of Gao was responsible for both the      these conditions, only Gao* demonstrated functional coupling to
acute (inhibition) and subacute (sensitization) effects of D2L          D2L dopamine receptors expressed in NS20Y cells. D2L receptor
receptor activation in NS20Y-D2L cells provides an impetus to           coupling to Gao* was confirmed for two PTX-sensitive signaling
examine the Ga subunit specificity involved in long-term heter-         events, one in which the acute response of D2L receptor activation
ologous sensitization.                                                  is measured and a second that requires more prolonged agonist
   Selective coupling of D2 receptors to Gao has been shown             occupation of the receptor. These are novel findings in neuronal-
using other approaches in both primary cultures of pituitary cells      like cells and are particularly striking when one considers that two
and pituitary-derived cell lines. Specifically, antiserum to Gao ,      separate signaling events resulting in opposing changes in ade-
but not Gai1/2 or Gai3 , blocks D2 receptor coupling to Ca 21           nylate cyclase activity are both mediated by the same class of Ga
channels in rat anterior pituitary cells (L ledo et al., 1992). Anti-   subunits. Heterologous sensitization of adenylate cyclase may be
sense depletion of Gao in pituitary GH4C1 cells revealed that           one neuroadaptive mechanism by which a single protein contrib-
activation of Gao is largely responsible for D2-mediated inhibi-        utes to maintenance of cellular homeostasis during a chronic
tion of C a 21 entry (Liu et al., 1994). In contrast, studies of cAMP   inhibitory signal.
metabolism suggested that selective activation of Gai subunits is
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