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Regulation and molecular characterization of dopamine D2 receptors in a prolactin-secreting 7315a anterior pituitary tumor
European Journal of Pharmacology - Molecular PharraacologF Section, 188 (1990) 329-334
329
Elsevier EJPMOL 90080
Regulation and molecular characterization of dopamine Dz receptors in a prolactin-secreting 7315a anterior pituitary tumor J o w Y. Lew 1, H a l i n a Z a w a d z k a 1, David F e i g e n b l u m 1, Di T a n g 1 David Filer 2, Peter Benedetto 2 and Menek Goldstein Department of Psychiatry, I Neurochemistry Research Laboratories and 2 Millhauser Laboratories, New York University Medical Center, 560 First Avenae, New York, N Y 10016, U.S.A.
Received17 August 1989. revised MS received31 January 1990. accepted 13 February 1990
The regulation and molecular properties of the dopamine (DA) D: receptors were compared in the prolactin-secreting 7315a anterior pituitary tumor with those in the striatum of rats. Chronic treatment with haloperidol increases the maximal binding for [3H]spiroperidol in tumor and striatum, but the percent increase is much higher in tumor than in striatum. Photoaffinity labelling of D A D 2 receptors with N-(p-azido-m-[ 1~'5I]iodophenethyl)spiperone ([125I]N3oNAPS) yielded a major specifically labeled peptide with the M r of 32-34 kDa in tumor, and two specifically labeled pepfides with M r of 32-34 and 92-94 kDa in striatum. The analysis of D A D z receptor mRNA show~, that the size is similar in tumor and striatum. The DA I)2 receptor mRNA in tumor is very low and chronic treatment with halopeddol produces a considerable increase of the specific mRNA. It is postulated that the reported defect in regulation of prolactin release by DA agonists might be due to posttranslational changes in the tumor DA D2 receptor. Dopamine D2 receptors; Striatum (rat); Prolactin-secreting 7315a anterior pituitary tumor
!. Introduction Dopamine (DA) D 2 receptors are present in the rat prolactin-secreting 7315a anterior pituitary tumor (Cronin et al., 1981). The order of binding potency for various DA agonists and antagonists in the tumor correlates with that of the anterior pituitary gland. The DA receptors in the tumor were found to be linked to inhibition of adenylate cyclase activity and were characterized as a uniform population of DA D: receptors (Lin et al., 1987). However, DA does not mediate prolactin
Correspondence to: Menek Goidstein, Ph.D,, New York University Medical Center, Nearochemistry Research Laboratories, 560 First Avenue, Rm. H-544, New York, NY 10016, U.S.A.
release in tumor cells as it does in normal anterior pituitary glands (Cronin et al., 1981). This abnormality might result from a defect in the effector system which couples inhibition of adenylate cyclase and inhibition of prolaetin secretion (Cronin et al., 1981), or it might result from alterations in the molecular structure of the tumor D A D 2 receptor. In order to investigate potential mechanisms responsible for this defect, we compared the regulation and molecular properties of D A D z receptors in rat 7315a pituitary tumor with those in stfiatum. Specifically we compared the effects of chronic treatment with a neuroleptic on the increase in binding densities of DA D 2 receptors, electrophoretic properties of ligand binding subunits, and we investigated levels of DA D 2 receptor m R N A in tumors of untreated and haloperidol-treated animals.
0922-4106/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
330 2. Materials and method~ A group of female Buffalo rats (150-200 g; Simonsen, Gilroy, CA) were inoculated s,c. with a mince of the 7315a pituitary tumor (Cronin et al., 1981). The rats bearing this tumor were kindly given to us by Dr. R.M. MaeLeod (University of Virginia, Charlottesville, VA). The tumor was allowed to grow for 3-4 weeks. The animals were killed and tumor tissue and striata were immediately dissected. The necrotic regions of tumor were discarded and the remaining tumor tissue was homogenized in 20 mM HEPES buffer, pH 7.5, containing 250 mM NaCI and 5 mM EDTA. Rat striata were homogenized in 0.05 M Tris/HC1 buffer pH 7.7. The membranes from tumor and from striatum were prepared as previously described (Lew and Goldstein, 1984).
Z 1. Binding assay [~H]Spiropeddol (24-28 Ci/mmol; New England Nuclear) binding assay was carried out in triplicate in the presence of 20 nM ketanserin. The reaction mixture containing tumor membranes was incubated for 45 rain at 37 ° C and that containing Striata membranes for 15 min at 37"C. The bindhag reaction was terminated by filtration through G F / B filters and the samples were washed three times with 5 ml of 0.05 Tris/HCl buffer pH 7.7, containing 0.2 M NaC1. Specific binding was defined as the difference between the amount of radioactivity bound in presence and absence of 1 /LM (+)-butaclamol. Saturation isotherms were transformed using the method of Seatehard (1949). The K D and Bin,x were determined from the regression analysis of linear Scatchard plots by the least-squares method. The data were analyzed by an EBDA radioligand computer program.
2.2. C~ronic treatment with haloperidol One or two days after inoculation of the tumors, rats were treated daffy with haloperidol (1 mg/kg; s.c.). The animals were treated for 21 days and sacrificed 48 h after the last administration of haloperidoL
2.3. Photoaffinity labeling with N-(p-azido-m[t2Sl]iodophenethyOspiperone ([I:5I]Ns-NAPS) The rat striata and tumor membranes were prepared as described above, with the exception t'aat the homogenization buffer contained 20 mM EDTA and 5 #g/ml of the following protease inhibitors: aprotinin, leupeptin, pepstatin, chymostatin, soybean trypsin inhibitor, benzamidine and 1 mM phenyl-methylsulfonyl fluoride (PMSF). The membranes (2-3 mg/ml) were preincubat~ in a volume of 5 ml in the dark for 15 rain, and subsequently in the presence of 100-200 pM []z51]N3-NAPS and ia presence or absence of 10 -s M (+)- or (-)-buta¢lamol for 60 rain a* 25 ° C. Photolysis was performed by transferring the incubation mixture to 35 mm Petri dishes (kept on an ice bath) which were held 12 cm from the high pressure mercury lamp. The samples ,,',ere irradiated for 90 s and the labeled membranes were pelleted by centrifugation, washed and resuspended in sodium dodecyl sulfate-polyacrylamide gel dectrophoresis (SDS-PAGE) buffer (50 mM T r i s / H C l pH 6.8, 10~ glycerol, 5% flmercaptoethanol and 0.008% bromophenol blue). The samples were kept for 60-90 min at room temperature and aliquots of 50-100 /~1 were applied to each lane on SDS-PAGE gel containing 6-15~ acrylamide gradient in the resolving gel and 3% acrylan~de in the stocking gel. Following electrophoresis the gels were dried and an autoradiogram was developed (XAR Kodak film with a lightening-plus intensifying screen) at - 70 ° C.
2.4. b~orthern blot analysis The total RNA was extracted by the guadinium thiocianiate method (Chomczynski and Sacchi, 1987). The isolated RNA appeared to be intact, judged from the bands of rRNA (28S and 18S) after ethidium bromide-stained agarose gel dectrophoresis. The mRNA was isolated from total RNA by oligo dT column chromatography. The denatured mRNA samples were loaded onto 1.5~g agarose. The horizontal electrophoresis was run 4 h at 100 V at room temperature in 1 × MOPS (Maniatis et al., 1982). The RNA was then transferred to a Gene Screen membrane in 25 mM
331 phosphate buffer pH 6.5, overnight. The membrane was washed, baked at 80-100°C for 2 h under reduced pressure, and incubated first for 4 h at 420C in a prehybridization solution containing 50~ formamide, 2 × Denhardt's solution, 5 × saline sodium citrate buffer (SSC), 1% SDS, salmon sperm D N A (200/~g/rnl) and yeast tRNA 100 pg/ml). Subsequently m R N A was hybridized with random primed a-32p-labeled (4 x 106 d p m / m l ) D A D 2 receptor e D N A of 495 bp (Pst I fragment; Bunzow et al., 1988) in a prehybridization solution containing half the amount of salmon sperm D N A at 4 2 ° C for 18-22 h. The membrane was washed to the stringency of 0.2 x SSC and exposed to Kodak XO mat-AK film at - 20 ° C in presence of the intensifying screen. The radioactive hybrid was quantitated by computerized densitometric scanning (Hoefer Scientific !~stP~ments, GiS300) of the autoradiograrns.
3. Results
3.1. Effects of chronic treatment with haloperidol on [3H]spiroperidol binding Since chronic treatment with D A D 2 antagonists increases the binding of [3H]spiroperidol to striatal DA receptors, we compared the effects of chronic treatment with haloperidol on binding characteristics of [gH]spiroperidol to striatal and tumor DA receptors. The results presented in table 1 show that chronic treatment with haloperidol has no significant effect on the K D for [3H]spirop?ridol, but increases the maximal binding (Bronx) for [3H]spiroperidol in both striatum and tumor. The Brnaxvalues were increased in the striatum by
approximately 20~,, while in the tumor by approximately 270%.
3,2. Photoaffinity labeling of the DA D: receptors Since the ligand binding subunit of the D 2 subtype of the DA receptor in the striatum was identified by photoaffinity labeling with [125I]N3NAPS (Amlaiky and Caron, 1985), we used this photosensitive probe for identification of the binding subunit of the DA receptor in the tumor. In membrane preparations obtained from the tumor, [r'SI]N:NAPS labeled, in a photo-dependent manner, a peptide of M~ 32-34 kDa. The labeling of peptides with M r of approximately 92 kDa was also observed (fig. 1), but its intensity was weak and not visible in the underexposed radiographs, The labeling Gf :he 32-34 kDa peptide was blocked specifically by the D z antagonist (+)-butaclamol (fig. 1), while the inactive enantiomer ( - ) , butaclamol was ineffective in preventing the labeling (data not shown). In membrane preparations obtained from rat striatum [12-~I]N:NAPS labeled a peptide of M~ 92-9a kDa and a second peptide of M~ 32-34 kDa, The labeling of both peptides was blocked by (+)-butactamol (fig. 1), while (-)-butaclamol was ineffective in preventing the labeling of both peptides (data not shown). The labeling of a third peptide of approximately 140 kDa was also observed, but its intensity was weak and not visible in the underexposed radiographs.
3.3. Analysis of relative levels of D A D e receptor mRaVA In this study we used the 495-bp probe (from amino acids 246-411) which codes for the putative
TABLE l The effect of chronic treatment with haloperidol on the binding characteristics of [~H]spiroperidol to rat tumor 7315a .and striatal membranes. The values are the means_+S.E.M, from three experiments. The K B and Bm~, values were obtained from Scatchard analysis of [3H]spiroperidol (0.1-1.4 nM) binding to the striatal or tumor receptors. The values for eae~ eoneentrauon of [3H]spiroperidol (14 different concentrations) w~,red~termined in triplicate, Treatment None Haloperidol
KD (riM) Striatum 0.23+_0.02 0.202:0.02
Tumor 0.27_+0.03 0.202:0.03
Bm~ (fmol/mg protein) Striatum 1 185.0+_75.0 ~ 1 460.0~ 85.0
a The Bm~ is significantlydifferent from that obtained from haloperidol-treatedanimals.
Tumor 94.6_+ 6.5 ~ 371.02:24.0
332
A
2). This band migrated to a position of approximately 2.5 kb which is similar to the size of m R N A for D A D 2 receptors previously reported (Bunzow et al., 1988). In order to determine whether m R N A levels in the tumor were altered by chronic treatment with haloperidol, we compared the relative levels in untreated with those in neuroleptic-treated animals. The amount of DA D 2 receptor m R N A as estimated by densitometric scanning was approximately 2.5 times higher in haloperidol-treated tumors than in untreated tumors (fig. 2).
B
"140,----,~
1 I'
/-
"7"-
2
3
,7""
..¢ o/ooO.o o
2.~
Fig. 1. SDS-PAGEpattern of the labeling of rat striatal and 7315a tumor membraneswith[12sIIN3.NAPS.Membraneswere incubated in the presence or absence (control) of 10-s M butaclamol. The patterns show are typical of four to six experiments.(A) Striatal membranes;(B) 7315a tumor membranes.
sixth and seventh membrane spanning domains as well as for most of the variable cytoplasmic loop between the fifth and sixth transmembrane domains of the D A D z receptor. The variable cytoplasmic loop (from amino acids 228-338) shows no homology to other known G protein-linked receptors and seems to be specific for the D A D 2 receptor (Bunzow et al., 1988). Under the stringency conditions used, a single m R N A band which hybridizes to the D A D 2 receptor probe (60% of the probe codes for the variable cytoplasmic loop) was obtained from striatum and tumor tissue (fig.
Fig. 2. Northern blot analysis of D A D 2 recepto:"mRNA isolated from rat striatum and 7315a tumor. (The conditions are as described in Materials and methods). (Lanes) 1, rat striatum (not treated with halopefidol);2, rat 7315a tumor (not treated with haloperidol); 3, rat 7315a tumor (treated with baloperidol); all lanes mRNA2/~g. The Northern blot analysis is typicalof three experiments.
333
4. Discussion
The findings that chronic treatment with haloperidol increases the density of the DA receptor binding sites (Br~) and the levels of DA Dz receptor mRNA in the tumor to a much greater extent than in the striatum (M. Goldstein, unpubfished data) raises the question as to whether the phenomenon reflects regulation of the receptor or a change in the growth of the tumor. Although the weights and sizes of the tumors do not appear to differ in the neuroleptic-treated animals from the untreated controls, the possibility that the properties of the tumor were altered by the treatment cannot be excluded. It is noteworthy that some transmitter-receptors have the potential to act as regulators in mitogenesis, and it is conceivable that the D A D 2 receptor, like the 5-HTlc receptor, functions as a protooncogene when expressed in a nonneuronal environment (Julius et al., 1989). The regulation of the D A D z receptor in the tumor by chronic treatment with neuroleptics might reflect the bl~kade of the response to DA derived from the systemic circulation. Since the density of D A D 2 receptors in the tumor is very low in untreated rats, the blockade of the D A D 2 receptor by treatment with neuroleptics should be very efficient and should elicit a relatively high increase in the D A D 2 receptor mRNA and DA D2 receptor density. A similar phenomena was observed for the a2-adrenoceptors in the salivary gland following reserpine treatment (Bylund and Martinez, 1980). Based on the present data it cannot be ascertained whether the increase in DA D2 receptor mKNA is due to an increase in the rate of transcription of the gene or to a decrease in the rate of degradation of D A D 2 receptor mRNA. Although the molecular size of D A D 2 receptor mRNA in the tumor is similar to that in the striatum, the possibility is not excluded that the gene in the tumor might have been altered by mutation or that it represents a subtype of the D A D 2 receptor. Indeed, the probe used in this study will nat differentiate between the recently described two forms of the D A D 2 receptor (Snyder et al., 1989), and the broad single mRNA band might represent the two forms. The possible labeling of another G
protein-linked receptor mRNA by the probe is unlikely since under the stringent conditions only a single band was obtained, and chronic treatment with haloperidoI predominantly alters the D A D z receptors. Furthermore, the increase in D A D 2 receptor mRNA levels parallels the increase in the maximal [3H]spiroperidol binding sites. The difference in the M r of the D A D 2 receptor binding subunit between tumor and ~hat of striaturn might be due to posttranslational changes. The labeled peptide with an M r of 32-34 kDa isolated from tumor was also isolated as a second minor labeled peptide in striatum (Amlaiky and Carom 1986; fig. 1). It was suggested that this peptide probably represents a proteolytic product of the M r 94- and 140-kDa-labeled peptides (Amlaiky and Caron, 1986) and the 32-34 kDa peptide in the tumor might also be a proteolytic product of the larger peptides. It is noteworthy that the tumor receptor ~s not glycosylated to the same extent or differently than the striatal receptor (Lew and Goldstein, 1988). Thus, the solubilized striatal D A D 2 receptor is almost completely adsorbed on wheat-germ agglutinia agarose colurrms, while only a small proportion of the tumor receptor is adsorbed on this lectin. It is therefore conceivable that the low M r binding subunit of the tumor might represent a different glyeosylated or de~ycosylated form of the receptor. This idea is further supported by recent findings that deglycosylation of the striatal DA D2 receptor reduces the M r of the labeled DA D~ receptor subunits and that the 32-34-kDa suburdt is insensitive to neuroaminidase digestion (Jarvie et al., 1988). Based on these results we postulate that the reported defect ha regulation of prolactin release by DA agonists in tumor might not necessarily be due to a defect at a postreceptor site in the tumor cell but might be due to posttransiational changes of a specific DA 13, receptor subtype.
Acknowledgements These studies were supported in pozt by granls from the NIMH (MH 432.30 and MH 02717) and NINCDS (0~801). We also wish to thank Dr. Oliver Civelli of The Oregon Health Sciences University, Portland, for the cDNA receptor probe.
334
References Amlaiky, N. and M.G. Caron, 1985, Photoaffinity labeling of the D2-dopamine receptor using a novel high affinity radioiodinated probe, J. Biol. Chem. 260, 1983. Amlaiky, N. and M.G. Caron, 1986, Identification of the D2-dopamine receptor binding subnnit in several mammalian tissues and species by photoaffinity labeling, J. Neurochem. 47, 1986. Bunzow, J.R., H.H.M. Van To]. D.K. Grandy, P. Albert, J. Salon, M. Christie, C.A. Machida, K.A. Neve and O. Civdli, 1988, Cloning and expression of a rat D 2 dopamine receptor eDNA, Natu~ 336, "/83. Byluad, D.B. and Martinez, J.R, 1980~ ~2-Adrenergic receptors appear in rat salivary glands after reserpine treatment, Nature 285~ 229. Chomczynski, P. and N. Sacchi, 1987, Single-step method of RNA isolation by acid-guanidium thiocyanate phenol-chloroform extraction, Anal. Biochem. 162, 156. Cronin, M.J., C.A. Valdenegro, S.N. Perkins and ILM. MacLeod, 198"1, The 7315a pituitary tumor is refractory to dopaminergic inhibition of prolactin release bet contains dopamine receptors, Endocrinol. 109, 2160.
Jar'vie, K.R., H.B. Niznik and P. Seeman, 1988, Dopamin¢ D 2 receptor binding subunits of M, =140000 and 94000 in brain: Deglycosylafion yields a common unit of M r = 44 0~0, Mol. Pharmacol. 34, 91. Julius, D., TJ. Livclli, T.M. Jcssell and R. Axcl, 1989, Ectopic expression of the serotonin l c receptor and the triggering of malignant transformation. Science 24,4, 105% Lew, J.Y. and M. Goldstein, 1984, Solubilization and characterization of striatal dopamine receptors, J. Neorochem. 42, 1298. Lew, LY. and M. Goldstein, 1988, Solubitization and purification of D2 dopamine (DA) receptors from a rat anterior pituitary tumor, Soc. Neurosci. Abstr. 165, 11. Lin, C., P. McGonigl¢ and P. Molinoff, 1987, Characterization of D-2 doparaine receptors in a tumor of the rat anterior pituitary gland, J. PliarraacoL Exp. Ther. 242, 950. Maniatis, T , E.F. Fri~.sch and 3. Sarabrook, 1982, Molecular Cloning, a Laboratory Manual (Cold Spring Harbor Laboratories), Cold Spring Harbor Laboratories, NY). Scatchard, G., 1949, The attraction of proteins for small molecules, Ann. N.Y. Aead. Sci. 51,660. Snydcr, L.A., L. Brosius, J.L. Roberts and S.C. Sealfon, 1989, Polymerase chain reaction cloning of D 2 receptor subtypes, Soc. Neurosci. 1"/0, 17.
329
Elsevier EJPMOL 90080
Regulation and molecular characterization of dopamine Dz receptors in a prolactin-secreting 7315a anterior pituitary tumor J o w Y. Lew 1, H a l i n a Z a w a d z k a 1, David F e i g e n b l u m 1, Di T a n g 1 David Filer 2, Peter Benedetto 2 and Menek Goldstein Department of Psychiatry, I Neurochemistry Research Laboratories and 2 Millhauser Laboratories, New York University Medical Center, 560 First Avenae, New York, N Y 10016, U.S.A.
Received17 August 1989. revised MS received31 January 1990. accepted 13 February 1990
The regulation and molecular properties of the dopamine (DA) D: receptors were compared in the prolactin-secreting 7315a anterior pituitary tumor with those in the striatum of rats. Chronic treatment with haloperidol increases the maximal binding for [3H]spiroperidol in tumor and striatum, but the percent increase is much higher in tumor than in striatum. Photoaffinity labelling of D A D 2 receptors with N-(p-azido-m-[ 1~'5I]iodophenethyl)spiperone ([125I]N3oNAPS) yielded a major specifically labeled peptide with the M r of 32-34 kDa in tumor, and two specifically labeled pepfides with M r of 32-34 and 92-94 kDa in striatum. The analysis of D A D z receptor mRNA show~, that the size is similar in tumor and striatum. The DA I)2 receptor mRNA in tumor is very low and chronic treatment with halopeddol produces a considerable increase of the specific mRNA. It is postulated that the reported defect in regulation of prolactin release by DA agonists might be due to posttranslational changes in the tumor DA D2 receptor. Dopamine D2 receptors; Striatum (rat); Prolactin-secreting 7315a anterior pituitary tumor
!. Introduction Dopamine (DA) D 2 receptors are present in the rat prolactin-secreting 7315a anterior pituitary tumor (Cronin et al., 1981). The order of binding potency for various DA agonists and antagonists in the tumor correlates with that of the anterior pituitary gland. The DA receptors in the tumor were found to be linked to inhibition of adenylate cyclase activity and were characterized as a uniform population of DA D: receptors (Lin et al., 1987). However, DA does not mediate prolactin
Correspondence to: Menek Goidstein, Ph.D,, New York University Medical Center, Nearochemistry Research Laboratories, 560 First Avenue, Rm. H-544, New York, NY 10016, U.S.A.
release in tumor cells as it does in normal anterior pituitary glands (Cronin et al., 1981). This abnormality might result from a defect in the effector system which couples inhibition of adenylate cyclase and inhibition of prolaetin secretion (Cronin et al., 1981), or it might result from alterations in the molecular structure of the tumor D A D 2 receptor. In order to investigate potential mechanisms responsible for this defect, we compared the regulation and molecular properties of D A D z receptors in rat 7315a pituitary tumor with those in stfiatum. Specifically we compared the effects of chronic treatment with a neuroleptic on the increase in binding densities of DA D 2 receptors, electrophoretic properties of ligand binding subunits, and we investigated levels of DA D 2 receptor m R N A in tumors of untreated and haloperidol-treated animals.
0922-4106/90/$03.50 © 1990 ElsevierSciencePublishers B.V. (BiomedicalDivision)
330 2. Materials and method~ A group of female Buffalo rats (150-200 g; Simonsen, Gilroy, CA) were inoculated s,c. with a mince of the 7315a pituitary tumor (Cronin et al., 1981). The rats bearing this tumor were kindly given to us by Dr. R.M. MaeLeod (University of Virginia, Charlottesville, VA). The tumor was allowed to grow for 3-4 weeks. The animals were killed and tumor tissue and striata were immediately dissected. The necrotic regions of tumor were discarded and the remaining tumor tissue was homogenized in 20 mM HEPES buffer, pH 7.5, containing 250 mM NaCI and 5 mM EDTA. Rat striata were homogenized in 0.05 M Tris/HC1 buffer pH 7.7. The membranes from tumor and from striatum were prepared as previously described (Lew and Goldstein, 1984).
Z 1. Binding assay [~H]Spiropeddol (24-28 Ci/mmol; New England Nuclear) binding assay was carried out in triplicate in the presence of 20 nM ketanserin. The reaction mixture containing tumor membranes was incubated for 45 rain at 37 ° C and that containing Striata membranes for 15 min at 37"C. The bindhag reaction was terminated by filtration through G F / B filters and the samples were washed three times with 5 ml of 0.05 Tris/HCl buffer pH 7.7, containing 0.2 M NaC1. Specific binding was defined as the difference between the amount of radioactivity bound in presence and absence of 1 /LM (+)-butaclamol. Saturation isotherms were transformed using the method of Seatehard (1949). The K D and Bin,x were determined from the regression analysis of linear Scatchard plots by the least-squares method. The data were analyzed by an EBDA radioligand computer program.
2.2. C~ronic treatment with haloperidol One or two days after inoculation of the tumors, rats were treated daffy with haloperidol (1 mg/kg; s.c.). The animals were treated for 21 days and sacrificed 48 h after the last administration of haloperidoL
2.3. Photoaffinity labeling with N-(p-azido-m[t2Sl]iodophenethyOspiperone ([I:5I]Ns-NAPS) The rat striata and tumor membranes were prepared as described above, with the exception t'aat the homogenization buffer contained 20 mM EDTA and 5 #g/ml of the following protease inhibitors: aprotinin, leupeptin, pepstatin, chymostatin, soybean trypsin inhibitor, benzamidine and 1 mM phenyl-methylsulfonyl fluoride (PMSF). The membranes (2-3 mg/ml) were preincubat~ in a volume of 5 ml in the dark for 15 rain, and subsequently in the presence of 100-200 pM []z51]N3-NAPS and ia presence or absence of 10 -s M (+)- or (-)-buta¢lamol for 60 rain a* 25 ° C. Photolysis was performed by transferring the incubation mixture to 35 mm Petri dishes (kept on an ice bath) which were held 12 cm from the high pressure mercury lamp. The samples ,,',ere irradiated for 90 s and the labeled membranes were pelleted by centrifugation, washed and resuspended in sodium dodecyl sulfate-polyacrylamide gel dectrophoresis (SDS-PAGE) buffer (50 mM T r i s / H C l pH 6.8, 10~ glycerol, 5% flmercaptoethanol and 0.008% bromophenol blue). The samples were kept for 60-90 min at room temperature and aliquots of 50-100 /~1 were applied to each lane on SDS-PAGE gel containing 6-15~ acrylamide gradient in the resolving gel and 3% acrylan~de in the stocking gel. Following electrophoresis the gels were dried and an autoradiogram was developed (XAR Kodak film with a lightening-plus intensifying screen) at - 70 ° C.
2.4. b~orthern blot analysis The total RNA was extracted by the guadinium thiocianiate method (Chomczynski and Sacchi, 1987). The isolated RNA appeared to be intact, judged from the bands of rRNA (28S and 18S) after ethidium bromide-stained agarose gel dectrophoresis. The mRNA was isolated from total RNA by oligo dT column chromatography. The denatured mRNA samples were loaded onto 1.5~g agarose. The horizontal electrophoresis was run 4 h at 100 V at room temperature in 1 × MOPS (Maniatis et al., 1982). The RNA was then transferred to a Gene Screen membrane in 25 mM
331 phosphate buffer pH 6.5, overnight. The membrane was washed, baked at 80-100°C for 2 h under reduced pressure, and incubated first for 4 h at 420C in a prehybridization solution containing 50~ formamide, 2 × Denhardt's solution, 5 × saline sodium citrate buffer (SSC), 1% SDS, salmon sperm D N A (200/~g/rnl) and yeast tRNA 100 pg/ml). Subsequently m R N A was hybridized with random primed a-32p-labeled (4 x 106 d p m / m l ) D A D 2 receptor e D N A of 495 bp (Pst I fragment; Bunzow et al., 1988) in a prehybridization solution containing half the amount of salmon sperm D N A at 4 2 ° C for 18-22 h. The membrane was washed to the stringency of 0.2 x SSC and exposed to Kodak XO mat-AK film at - 20 ° C in presence of the intensifying screen. The radioactive hybrid was quantitated by computerized densitometric scanning (Hoefer Scientific !~stP~ments, GiS300) of the autoradiograrns.
3. Results
3.1. Effects of chronic treatment with haloperidol on [3H]spiroperidol binding Since chronic treatment with D A D 2 antagonists increases the binding of [3H]spiroperidol to striatal DA receptors, we compared the effects of chronic treatment with haloperidol on binding characteristics of [gH]spiroperidol to striatal and tumor DA receptors. The results presented in table 1 show that chronic treatment with haloperidol has no significant effect on the K D for [3H]spirop?ridol, but increases the maximal binding (Bronx) for [3H]spiroperidol in both striatum and tumor. The Brnaxvalues were increased in the striatum by
approximately 20~,, while in the tumor by approximately 270%.
3,2. Photoaffinity labeling of the DA D: receptors Since the ligand binding subunit of the D 2 subtype of the DA receptor in the striatum was identified by photoaffinity labeling with [125I]N3NAPS (Amlaiky and Caron, 1985), we used this photosensitive probe for identification of the binding subunit of the DA receptor in the tumor. In membrane preparations obtained from the tumor, [r'SI]N:NAPS labeled, in a photo-dependent manner, a peptide of M~ 32-34 kDa. The labeling of peptides with M r of approximately 92 kDa was also observed (fig. 1), but its intensity was weak and not visible in the underexposed radiographs, The labeling Gf :he 32-34 kDa peptide was blocked specifically by the D z antagonist (+)-butaclamol (fig. 1), while the inactive enantiomer ( - ) , butaclamol was ineffective in preventing the labeling (data not shown). In membrane preparations obtained from rat striatum [12-~I]N:NAPS labeled a peptide of M~ 92-9a kDa and a second peptide of M~ 32-34 kDa, The labeling of both peptides was blocked by (+)-butactamol (fig. 1), while (-)-butaclamol was ineffective in preventing the labeling of both peptides (data not shown). The labeling of a third peptide of approximately 140 kDa was also observed, but its intensity was weak and not visible in the underexposed radiographs.
3.3. Analysis of relative levels of D A D e receptor mRaVA In this study we used the 495-bp probe (from amino acids 246-411) which codes for the putative
TABLE l The effect of chronic treatment with haloperidol on the binding characteristics of [~H]spiroperidol to rat tumor 7315a .and striatal membranes. The values are the means_+S.E.M, from three experiments. The K B and Bm~, values were obtained from Scatchard analysis of [3H]spiroperidol (0.1-1.4 nM) binding to the striatal or tumor receptors. The values for eae~ eoneentrauon of [3H]spiroperidol (14 different concentrations) w~,red~termined in triplicate, Treatment None Haloperidol
KD (riM) Striatum 0.23+_0.02 0.202:0.02
Tumor 0.27_+0.03 0.202:0.03
Bm~ (fmol/mg protein) Striatum 1 185.0+_75.0 ~ 1 460.0~ 85.0
a The Bm~ is significantlydifferent from that obtained from haloperidol-treatedanimals.
Tumor 94.6_+ 6.5 ~ 371.02:24.0
332
A
2). This band migrated to a position of approximately 2.5 kb which is similar to the size of m R N A for D A D 2 receptors previously reported (Bunzow et al., 1988). In order to determine whether m R N A levels in the tumor were altered by chronic treatment with haloperidol, we compared the relative levels in untreated with those in neuroleptic-treated animals. The amount of DA D 2 receptor m R N A as estimated by densitometric scanning was approximately 2.5 times higher in haloperidol-treated tumors than in untreated tumors (fig. 2).
B
"140,----,~
1 I'
/-
"7"-
2
3
,7""
..¢ o/ooO.o o
2.~
Fig. 1. SDS-PAGEpattern of the labeling of rat striatal and 7315a tumor membraneswith[12sIIN3.NAPS.Membraneswere incubated in the presence or absence (control) of 10-s M butaclamol. The patterns show are typical of four to six experiments.(A) Striatal membranes;(B) 7315a tumor membranes.
sixth and seventh membrane spanning domains as well as for most of the variable cytoplasmic loop between the fifth and sixth transmembrane domains of the D A D z receptor. The variable cytoplasmic loop (from amino acids 228-338) shows no homology to other known G protein-linked receptors and seems to be specific for the D A D 2 receptor (Bunzow et al., 1988). Under the stringency conditions used, a single m R N A band which hybridizes to the D A D 2 receptor probe (60% of the probe codes for the variable cytoplasmic loop) was obtained from striatum and tumor tissue (fig.
Fig. 2. Northern blot analysis of D A D 2 recepto:"mRNA isolated from rat striatum and 7315a tumor. (The conditions are as described in Materials and methods). (Lanes) 1, rat striatum (not treated with halopefidol);2, rat 7315a tumor (not treated with haloperidol); 3, rat 7315a tumor (treated with baloperidol); all lanes mRNA2/~g. The Northern blot analysis is typicalof three experiments.
333
4. Discussion
The findings that chronic treatment with haloperidol increases the density of the DA receptor binding sites (Br~) and the levels of DA Dz receptor mRNA in the tumor to a much greater extent than in the striatum (M. Goldstein, unpubfished data) raises the question as to whether the phenomenon reflects regulation of the receptor or a change in the growth of the tumor. Although the weights and sizes of the tumors do not appear to differ in the neuroleptic-treated animals from the untreated controls, the possibility that the properties of the tumor were altered by the treatment cannot be excluded. It is noteworthy that some transmitter-receptors have the potential to act as regulators in mitogenesis, and it is conceivable that the D A D 2 receptor, like the 5-HTlc receptor, functions as a protooncogene when expressed in a nonneuronal environment (Julius et al., 1989). The regulation of the D A D z receptor in the tumor by chronic treatment with neuroleptics might reflect the bl~kade of the response to DA derived from the systemic circulation. Since the density of D A D 2 receptors in the tumor is very low in untreated rats, the blockade of the D A D 2 receptor by treatment with neuroleptics should be very efficient and should elicit a relatively high increase in the D A D 2 receptor mRNA and DA D2 receptor density. A similar phenomena was observed for the a2-adrenoceptors in the salivary gland following reserpine treatment (Bylund and Martinez, 1980). Based on the present data it cannot be ascertained whether the increase in DA D2 receptor mKNA is due to an increase in the rate of transcription of the gene or to a decrease in the rate of degradation of D A D 2 receptor mRNA. Although the molecular size of D A D 2 receptor mRNA in the tumor is similar to that in the striatum, the possibility is not excluded that the gene in the tumor might have been altered by mutation or that it represents a subtype of the D A D 2 receptor. Indeed, the probe used in this study will nat differentiate between the recently described two forms of the D A D 2 receptor (Snyder et al., 1989), and the broad single mRNA band might represent the two forms. The possible labeling of another G
protein-linked receptor mRNA by the probe is unlikely since under the stringent conditions only a single band was obtained, and chronic treatment with haloperidoI predominantly alters the D A D z receptors. Furthermore, the increase in D A D 2 receptor mRNA levels parallels the increase in the maximal [3H]spiroperidol binding sites. The difference in the M r of the D A D 2 receptor binding subunit between tumor and ~hat of striaturn might be due to posttranslational changes. The labeled peptide with an M r of 32-34 kDa isolated from tumor was also isolated as a second minor labeled peptide in striatum (Amlaiky and Carom 1986; fig. 1). It was suggested that this peptide probably represents a proteolytic product of the M r 94- and 140-kDa-labeled peptides (Amlaiky and Caron, 1986) and the 32-34 kDa peptide in the tumor might also be a proteolytic product of the larger peptides. It is noteworthy that the tumor receptor ~s not glycosylated to the same extent or differently than the striatal receptor (Lew and Goldstein, 1988). Thus, the solubilized striatal D A D 2 receptor is almost completely adsorbed on wheat-germ agglutinia agarose colurrms, while only a small proportion of the tumor receptor is adsorbed on this lectin. It is therefore conceivable that the low M r binding subunit of the tumor might represent a different glyeosylated or de~ycosylated form of the receptor. This idea is further supported by recent findings that deglycosylation of the striatal DA D2 receptor reduces the M r of the labeled DA D~ receptor subunits and that the 32-34-kDa suburdt is insensitive to neuroaminidase digestion (Jarvie et al., 1988). Based on these results we postulate that the reported defect ha regulation of prolactin release by DA agonists in tumor might not necessarily be due to a defect at a postreceptor site in the tumor cell but might be due to posttransiational changes of a specific DA 13, receptor subtype.
Acknowledgements These studies were supported in pozt by granls from the NIMH (MH 432.30 and MH 02717) and NINCDS (0~801). We also wish to thank Dr. Oliver Civelli of The Oregon Health Sciences University, Portland, for the cDNA receptor probe.
334
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