parathyroid hormone-related peptide receptor of the human cerebellum and functional expression in human neuroblastoma SK-N-MC cells

ELSEVIER

MOLECULAR BRAIN RESEARCH Molecular Brain Research 36 (1996) 127-136

Research report

Structure of a parathyroid hormone/parathyroid hormone-related peptide receptor of the human cerebellum and functional expression in human neuroblastoma SK-N-MC cells M. Eggenberger ~, B. Fliihmann a, R. Muff a, M. Lauber h, W. Lichtensteiger b, W. Hunziker c, J.A. Fischer a, W. Born ~ ' " a Research Laboratory for Calcium Metabolism, Departments of Orthopedic Surgery and Medicine. Unicersity of Zurich, Forch~'tras~e 340, 8008 Zurich, Switzerland h Institute of Pharmacology, Unicersity of Zurich-lrchel, 8057 Zurich, Switzerland c F. Hoffmann-La Roche, Ltd., 4002 Basel, Switzerland Accepted 20 September 1995

Abstract Cloning and functional expression of a cDNA from the human cerebellum revealed a parathyroid hormone/parathyroid hormone-related peptide (PTH/PTHrP) receptor protein of 593 amino acids, identical in sequence to the PTH/PTHrP receptor of the human kidney and an osteoblast-like cell line (Schipani et al., Endocrinology, 132 (1993) 2157-2165). Expression of m RNA hybridizing with the cloned cDNA, indistinguishable in size on Northern blots from a 2.3 kb transcript in kidney and liver, was detected in eight brain areas. In situ hybridization histochemistry in rat brain tissue sections revealed predominant signals in the Purkinje cell layer of the cerebellum and in the mesencephalic nucleus of the trigeminal nerve. In human neuroblastoma (SK-N-MC) cells, stably transfected with the cloned eDNA, hPTH(I-84) and hPTH(1-34) displaced binding of 125 pM [t251][Tyr36}chPTHrP(1-361 to the PTH/PTHrP receptor with IC5, values of 4.0 + 0.6 nM and 2.00 + 0.08 nM, and stimulated cyclic AMP accumulation with ECso values of 0.19 + 0.06 nM and 0.09 + 0.01 nM. respectively. 16 out of 48 cells responded to 100 nM hPTH(I-341 with a 2-10-fold transient increase of cytosolic free calcium concentrations. In conclusion, a PTH/PTHrP receptor, identified in the human cerebellum, has the primary structure of the corresponding receptors of kidney and bone. Expression in human neuroblastoma SK-N-MC cells revealed functional properties indistinguishable from those of non-neuronal tissues. The widespread distribution of PTHrP and its receptor in brain implies biological functions remaining to be elucidated. Keywords." Parathyroid hormone/parathyroid hormone-related peptide receptor; Human brain; Cerebellum; SK-N-MC cell line: Nervous system

1. Introduction Parathyroid hormone (PTH), a polypeptide of 84 amino acid residues, is predominantly produced by the parathyroid glands. The secretion of PTH is inversely related to extracellular calcium levels. Predominant target organs are the kidney and bone. Evidence for the synthesis and local action of PTH has been reported in the central nervous system [8]. Immunoreactive PTH, and expression of PTH m R N A has been detected in the rat hypothalamus by Northern blot analysis, in situ hybridization and polymerase chain reaction (PCR) amplification of c D N A re-

• Corresponding author. Fax: 1411 (l) 386 16 52. 0169-328X/9~/$15.00 ~" 1996 Elsevier Science B.V. All rights reserved SSDI (~1 f~q-328X(~.~51I)0253 -7

verse transcribed from hypothalamic m R N A [5,211]. Local action of PTH identified in the hypothalamus was suggested, based on the observed stimulation of dopamine turnover by intracerebroventricularly (i.c.v.) administered PTH, and perifusion and incubation of hypothalamus tissue slices with micromolar concentrations of PTH [9,10]. PTH stimulated calcium transport into brain synaptosomes of the rat and raised cytosolic calcium concentrations [4,26]. Along these lines, PTH inhibited calcium currents in mouse neuroblastoma cells and the firing rate of neurons in the ventromedial nucleus of the hypothalamus, and i.c.v, administered PTH prevented urethane-induced hypocalcemia [18,21]. Parathyroid hormone-related peptide (PTHrP), associated with the syndrome of humoral hypercalcemia of

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M. Eggenberger et al. / Molecular Brain Research 36 (1996) 127-136

istry revealed widespread distribution of m R N A hybridizing with PTH/PTHrP receptor e D N A and antisense RNA [28,29]. Dose-dependent stimulation of cyclic AMP accumulation by PTH- and PTHrP(1-34) fragments have been observed in isolated rat cerebral microvessels and in murine brain cells [11,13,17]. Here we report the cloning and functional expression in a human neuroblastoma (SK-N-MC) cell line of c D N A encoding a PTH/PTHrP receptor of the human cerebellum. Tissue distribution of PTH/PTHrP receptor expression was assessed by Northern blot analysis of R N A from several human brain areas and by in situ hybridization histochemistry of adult rat brain tissue sections.

malignancy is also expressed in non-malignant tissues such as the brain [6,30]. In situ hybridization histochemistry revealed pronounced expression of PTHrP m R N A in neurons of the cerebral cortex, hippocampus and cerebellar cortex. Biological effects of PTHrP in the brain remain to bc investigated. Seven transmembrane domain receptors common to PTH and PTHrP and linked to heterotrimcric guanine nucleotide-binding proteins (G-protein) have been identified in human kidney and bone cell lines of man and rat [1,23,24]. lntracellular signaling occurs through activation of adenylyl cyclase and phospholipase C. In the rat brain, Northern blot analysis and in situ hybridization histochem-

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Fig. 1. Nucleotide and deduced amino acid sequence of a eDNA of the human cerebellum encoding a human PTH/P'l~rP-receptor. Solid lines indicate the putativetransmembranedomains I-VII. Arrows show the positions and the sense ( --*) and antisense ( ,- ) orientationof the oligonucleotides RT1 u~d for reveme tran~ription and of PC1, -2, -3, -5, -6 and -7 fi~r PCR amplificationand cloning of the 5'- and Y-end of the eDNA. Boxed restriction sites indicate oligonucleotide extensions introduced for subcloningof PCR products.The positions of single Bglll and Xhol sites used for reconstructionof the full length eDNA are also indicated. Capital letters in the nucleotide sequence stand for the coding sequence flanked by noncoding sequences shown in lower case letters. Numberingof the amino acid residues (left) and of the nuclcotides(right).

M. Eggenberger et a l . / Molecular Brain Re.~earch 36 (1996~ 127-136

2. Materials and methods

2.1. Cloning and sequencing of cDNA A cDNA fragment of 487 bp (nucleotide sequence 909 to 1395, Fig. 1), identical in sequence to the corresponding part of cloned cDNA encoding a human PTH/PTHrP receptor (Genbank accession numbers L04308 and X68596) [23,24], was amplified from 107 pfu of a human cerebellum Agtll cDNA library (Clontech, Palo Alto, CA) by polymerase chain reaction (PCR) with two pairs of nested degenerate primers. The two 5'-primers, 5'-AACTACITCCACAT[C]G C[A]AG[C]CTGTT-3' and 5'G[C]CAACTA[T]C[T]IICTGGA[C]TG[T]CTG-3' and the two 3'-primers, 5'-ACCTCT[A/G]C[T]C[G]T'I'GC[A]A [G]GAAA[G]CAGTA-Y and 5'-GCA[T]ACG[C]AA[C]A [C/G]AA[G]T[A]CCCTGGAA-3', were homologous to conserved nucleotide sequences encoding parts of the transmembrane domains II, 11I and VII of the PTH/PTHrP/calcitonin (Cq')/secretin receptor subfamily. Two rounds of PCR amplifications were done for 25 cycles (95°C-54°C-72°C, 1 min each) with initial denaturation at 96°C for 3 min and a final incubation at 72°C for 10 min. The 487 bp cDNA fragment was subsequently used for hybridization screening of 0.5 X 10 ~' independent ,~ phage plaques of a human cerebellum eDNA library as previously described [3]. Two independent double-positive plaques, AI and )t2, were purified, and the cDNA inserts were subcloned into the EcoRI site of the Biuescript( + )vector (Stratagene, La Jolla, CA) for sequencing with the T7 sequencing" kit (Pharmacia, Uppsala, Sweden). Sequence alignment with the previously identified cDNA in bone and kidney revealed two short interrupted regions of homology in the )~1 insert and sequence identity of both strands of the 1644 bp )~2 insert which, however, lacked 81 bp of 5'- and 54 bp of 3'-coding sequence. The subsequent strategy for cloning missing 5'- and 3'-flanking cDNA pieces of A2 by PCR amplification was based on the assumption that the two eDNA ends were similar or identical to the sequences identified in cDNA encoding PTH/PTHrP receptors in bonc and kidney. The missing 3'-end of the cDNA was amplified from the same human cerebellum )tgtl I cDNA library with two pairs of nestcd primers PCI, PC2, PC3 (Fig. 1), and PC4 with the sequence 5'-GTACAATGGA'Iqq'CCTFACGCG-3' of ,~gtl 1 next to the EcoRl cloning site. The first round of PCR amplification with the primers PC1 and PC4 was done for 30 cycles (95°-56°-72°C, 1 min each). Conditions in the second round with the primers PC2 and PC3 were the same except for the primer annealing temperature which was set at 65°C. Initial denaturation was carried out at 95°C for 3 min and final incubations were done at 72°C for 10 rain. The missing 5'-end of P T H / P T H r P receptor encoding cDNA was cloned from 10 /xg of human cerebcllum total RNA by reverse transcription combined with PCR. Reverse transcription was primed with the oligo-

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nucleotide RTI (Fig. 1), and the cDNA was amplified in two rounds of PCR with one forward primer PC5 and two reverse primers PC6 and PC7 (Fig. 1). Each round of PCR was done for 30 cycles (95°C, 15 s; 60°C, 1 min: 72°C, I min) followed by a final incubation for 10 min at 72°C. In the second round primer annealing was done at 56°C for 1 min. Candidate 5'- and 3'-end PCR-products werc selected by size after electrophoresis in 2% low melting agarosc. Primer-derived restriction sites at thc 5'- and 3'-ends dcsigned for subcloning of the PCR products and for rcconstruction of the full length cDNA were digested with the appropriate cnzymes and thc products subcloned into the Bluescript(+)-vector (Stratagene, La Jolla, CA), for scquencing of both strands. The eDNA encoding a full length human PTH/PTHrP-receptor was reconstructed from the cloned insert of It2 (nucleotidc 110 to nucleotidc 1753, Fig. 1), and the PCR-amplified 5'-flanking (nuclcotide 1 to the Bglll site, Fig. 1) and 3'-flanking (Xhol site to nucleotide 1877, Fig. 1) cDNA fragments. For PTH/PTHrP receptor expression in eukaryotic ccll lines, the reconstructed full length eDNA was cloned into the previously described cukaryotic exprcssion vector pN34~ [3]. The flanking sequences including thc reconstruction sites Xhol and Bglll were rcconfirmed bv DNA sequencing of both strands.

2.2. Northern blot analysis Northern blots of RNA from different human tissues including several brain regions were obtained from Clontech (Palo Alto, CA). The cDNA fragment of 487 bp that had been used for hybridization screening of the human cerebellum cDNA library was labeled with [ot-32P]dATP by the PRIME-ITT"-Iabeling system (Stratagene, La Jolla, CA), and hybridization was performed according to the manufacturers' protocol. The membranes were autoradiographed at - 70°C for 4 weeks.

2.3. In situ hybridization Tissue sections of adult rat brain were paraformaldehyde fixed as previously described [16]. Two 60-met oligonucleotides, 5'-TCCCAGCTGCCATI'GCGGTCACAGCGTCTGTAGG CATGGCCTIq'GTGA'I"FGAAGTCATAA-3' and 5'-CACGGTGCAG('AGGAAAATCTGTTCCTCTIq'GGTAAAGACATCGT('CGCATCCACCAGCG-3', complementary to nucleotide sequences encoding N- and C-terminal portions of the first extracellular domain of the rat PTH/PTHrP receptor (Genbank accession number M77184) [1] were 3'-end-labeled by tailing with [a-33p]dATP and terminal transferase (Boehringer, Mannheim, Germany) and purified over a Bio-Spin 30 column (Bio-Rad Laboratories, Glattbrugg, Switzerland). Tissue sections were hybridized with 811 /xl per 1 cm 2 area of hybridization buffer containing 6 x l0 t' c p m / m l of individual ~P-labeled oligonucleotides. Incubations wcrc

130

M. Eggenberger et al. / Molecular Brain Research 36 (1996) 127-136

carried out overnight in a humidified chamber at 45°C with the sections covered with Sealon Fuji film (Van den Brink Instrumenten, Veenendaal, The Netherlands). The hybridization solution consisted of 5 × SSC (0.75 M NaCI, 75 mM sodium citrate), 125 m g / m l dextran sulfate, 50% formamide, 1.25 x Denhardt's solution (2.5 m g / m l ficoll (type 400, Pharmacia, Uppsala, Sweden), 2.5 m g / m l polyvinylpyrrolidone, 2.5 m g / m l bovine serum albumin (fraction V, Sigma, St. Louis, MO), 125 mM dithiothreitol, 0.25 m g / m l herring sperm DNA, 0.25 m g / m l polyadenylic acid and 0.25 m g / m l E. coli transfer RNA. Subsequently, sections were sequentially washed twice with 1 × SSC, 2 mM dithiothreitol and twice with 0.5 × SSC, 2 mM dithiothreitol for 15 min at 55°C. The sections were then dehydrated through a series of ethanol washes and apposed to Hyperfilm 13-max (Amersham, Buckinghamshire, UK). Exposure time was 2 to 3 months, depending on the efficiency of the oligonucleotide labeling reaction. The extent of non-specific hybridization was estimated on adjacent sections under the same conditions in the presence of a 100-fold excess of both non-labeled oligonucleotides. 2.4. PTH / PTHrP receptor expression in COS-7 and SKN-MC cells COS-7 cells (ATCC CRL 1651) were cultured in Ham F-12 and Dulbecco's modified Eagle's minimum essential medium (DMEM) (1:1) supplemented with 10% fetal calf serum (FCS) and 2 mM glutamine. Human neuroblastoma (SK-N-MC) cells (ATCC HTB 10) were cultured in Ham F-12 medium containing 10% FCS and 2 mM glutamine. Both types of cells were subcultured weekly by trypsinization with 0.1% trypsin and 0.5 mM ethylenediaminetetraacetic acid in phosphate-buffered saline. For transient transfections, COS-7 cells were grown in 24-well plates to subconfluency of approx. 50%, washed twice with serumfree DMEM and incubated with 1.3 p,g/ml DNA of the PTH/PTHrP receptor expression construct and 4 /xl/ml transfectam (Promega Corporation, Madison, Wl) in the same medium for 4 h at 37°C. Subsequently, the cells were washed with corresponding culture medium (see above) and incubated at 37°C for 2 days before binding studies and cyclic AMP measurements were done. The same protocol was used for stable transfection of SK-N-MC cells in 25 cm 2 culture fasks with a mixture of 5 /xg/ml DNA of the PTH/PTHrP receptor expression construct, 0.5 ~ g / m l of the neomycin resistance plasmid pSV2neo (Clontech, Palo Alto, CA) and 15 /zl/ml transfcctam. Selection for neomycin resistance was started 2 days after transfection with 400 /xg/ml geneticin (G418; Life Technologies, Inc., Gaithersburg, MD). Cells surviving 4 weeks in selection medium after two subcultivation steps were considered to be stably transfected. Measurements of cellular cyclic AMP accumulation in transfected and non-transfected cells, the preparation of ~25I-labeled

[Tyr 36]chPTHrP(1-36)amide ([ 125I]chPTHrP(1-36)) for receptor binding studies and receptor autoradiography of single cells were performed as previously described [14]. [12~l]chPTHrP(1-36) binding and displacement by hPTH(1-84), hPTH(1-34), hPTH(3-34), chPTHrP(I-36) (donated by Ciba, Basel, Switzerland) and hPTHrP(I-34) (Bachem, Bubendorf, Switzerland) was carried out in DMEM/Ham F-12 (1:1) supplemented with I% bovine serum albumin (BSA) at 15°C for 3 h with SK-N-MC cells and for 2 h with COS-7 cells. Incubations were stopped by a single wash of the cells with ligand-free incubation medium and bound radioactivity was determined after cell lysis in 0.5 mi sodium dodecylsulfate (0.5%). The same peptides were used for the stimulation of cyclic AMP accumulation. 2.5. Measurement of cytosolic free calcium Stably transfected SK-N-MC cells were trypsinized and seeded at a density of 5000 cells/well into flexiperm chambers (Heraeus, Basel, Switzerland). Cells were kept overnight in culture medium without geneticin, loaded for 1 h at 37°C with 1 /zM fura-2/AM (Calbiochem, Lucerne, Switzerland) in a medium containing 136 mM NaCI, 5.4 mM KCI, 1 m g / m l glucose, 1 mM Na2HPO 4, 1 mM MgSO~, 1 mM CaCI 2, 10 mM Hepes, pH 7.4, supplemented with 0.1% BSA. The cells were washed once and then incubated in 100 /zl of loading medium without fura-2/AM. Test substances were added in 300 /xi of prewarmed medium. Cytosolic free calcium concentrations ([ Ca2+ ]i) were measured in single SK-N-MC cells at 37°C by dual excitation microfluorimetry on an inverted microscope (epifluorescence mode, objective Nikon F40, Nikon, Tokyo, Japan). Excitation light alternated at 0.5 Hz between 350 nm and 380 nm (SPEX Fluorimeter, Glen Creston, London, UK). Fluorescence was monitored through a rectangular diaphragm and an interference filter (500 nm) by photon counting. The intensity of the diaphragm alone was subtracted at each wavelength. [Ca 2" ], was calculated from the ratio R = F35o/F3s o according to the formula: [ C a 2 + ] , = K j × 13 X ( R - R m i , ) / ( R m a ~ - R ) where K d is 224 nM [7], and 13 is the fluorescence ratio of free and bound fura-2/AM at 380 nm. Calibration was carried out in separate experiments with cells loaded with fura-2/AM in the absence of extracellular calcium and presence of 0.5 mM ethyleneglycol tetraacetic acid for Rm,n (1.92 + 0.03). R,,,ax (24.1 +__1.1) and /3 (7.93 + 0.32) (n = 6) were obtained with 10 /xM ionomycin and 20 mM calcium. 2.6. Data analysis IC5o and ECso values were calculated by non-linear regression analysis using Fig.6.0 (Biosoft, Cambridge, UK).

M. Eggenberger et al. / Molecular Brain Research .t6 (1996) 127-136

131

rupted and flanked by sequences (not shown) without open reading frames. When compared to previously published parts including intron exon boundaries of the human P T H / P T H r P receptor gene [15], these new DNA sequences likely represent introns in a short fragment of eDNA derived from unspliced mRNA, or of genomic DNA ligated into AI. Northern blot analysis of mRNA of peripheral tissues, whole brain and different brain regions revealed a strong hybridization signal of mRNA, 2.3 kb in size, in the kidney (Fig. 2), confirming previously published observations [24]. Weak hybridization signals of the same size transcript were visible in liver, and in the brain in amygdala, caudate nucleus, corpus caliosum, hippocampus, hypothalamus, substantia nigra, subthalamic nucleus and thalamus. PTH/PTHrP receptor mRNA was undetectable in mRNA of lung, placenta, heart, skeletal muscle and pancreas and of whole brain (Fig. 2), and of the cerebellum (not shown). The distribution of PTH/PTHrP receptor mRNA in thc rat brain was investigated by means of in situ hybridization of frontal sections of different anterior to posterior regions. In a first series of experiments hybridization of the two oligonucleotides (see Materials and methods) to individual adjacent sections revealed a similar pattern of mRNA

Data are expressed as means + SEM. Significance of differences between means was calculated by analysis of variance.

3. Results

A cDNA of 1877 bp was reconstructed from three overlapping cDNA fragments that were cloned (for details see Materials and methods) by hybridization screening (nucleotides 110-1753, Fig. 1), and by PCR amplification from a human cerebellum Agtll cDNA library (Xhol site to nucleotide 1877, Fig. 1) and by combined reverse transcription and PCR amplification from human cerebellum total RNA (nucleotide 1 to the Bglll site, Fig. 1). The eDNA contains an open reading frame encoding a protein of 593 amino acids. The sequence is identical to previously published hPTH/PTHrP receptor cDNA cloned from cDNA libraries of human kidney and a human osteoblastlike osteosarcoma cell line [23,24]. A DNA insert, 1376 nucleotides in length, of a second A phage clone, A1, of the same human cerebellum cDNA library contained two segments with open reading frames homologous to the sequences 1063-1144 and 1145-1241 of the cDNA shown in Fig. 1. The segments are inter-

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Fig. 2. Northern blot analysis of 2 /.Lgof poly(A) + RNA from human peripheral tissues and selected brain areas. A, heart; B, whole brain; C, placenta; D, lung; E, liver; F, skeletal muscle; G, kidney; H, pancreas; I, amygdala; K, caudate nucleus; L, corpus eallosum; M, hippocampus; N. hypothalamus; O. substantia nigra; P, subthalamic nucleus; Q, thalamus.

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Fig. 4. Displacement of [12SlkhPTHrP(l-36) binding ( 1:3) and stimulation of cellular cyclic AMP accumulation by chPTHrP(1-36) ( A ) in COS-7 cells transiently transfected with the PTH/PTHrP receptor expression construct. Basal cyclic AMP levels were 1 pmol/well. Each point indicates the mean of triplicate determinations of a representative experiment, carried out three times.

distribution (not shown). In subsequent experiments the oligonucleotide probes were therefore pooled to increase the sensitivity of mRNA detection. As shown in Fig. 3, abundant hybridization was detected in the mesencephalic nucleus of the trigeminal nerve and in the Purkinje cell layer of the cerebellum. A weaker in situ hybridization signal was recognized in the reticulotegmental nucleus (not shown), the lateral reticular nucleus and in the granular cell layer of the cerebellum and no signal was seen in a section including the hippocampal area and the dentate gyrus. The extent of non-specific hybridization in the presence of a 100-fold excess of the unlabeled oligonucleotides was low (Fig. 3). Receptor binding properties and cyclic AMP accumulation of the PTH/PTHrP receptor encoded by the cloned cDNA were studied in COS-7 cells not expressing detectable endogenous PTH/PTHrP receptors. Upon transient transfection with the P T H / P T H r P receptor expression construct, specific binding of 50000 cpm (125 pM) [ t2s I]chPTHrP(1-36) reached equilibrium at 15°C after 2 h (not shown). Total binding ranged from 9000 to 18000 cpm/well in individual transfection experiments. Binding in the presence of 1 /,tM chPTHrP(1-36), considered to be non-specific, was 300-700 cpm/weil. In a representative experiment shown in Fig. 4, half-maximal inhibition (ICs,) of specific [i 25I]chPTHrP(1-36) binding and half-maximal stimulation (E('s,) of cyclic AMP accumulation occurred with 7 and 0.09 nM chPTHrP(1-36), respectively. In non-transfected COS-7 cells specific binding was below

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Fig. 5. Displacement of [~2~I]chPTHrP(I-36)binding (top) and stimulation of cellular cyclic AMP accumulation (bonom) in stably transfected SK-N-MC cells with the PTH/PTHrP receptor expression construct. Displacement of [12SlkhPTHrP(1-36) binding by hPTH(I-34) (,k), hPTtt(I-84) (41,), hPTHrP(I-34) (A), chPTHrP(I-36) (1:3) and hPTH(3-34) ( O ) was performed at 15°C for 3 h and stimulation of cyclic AMP accumulation at 37°C for 15 min. Basal cyclic AMP levels were 2 pmol/well. In both panels the values indicate the mean of triplicate determinations of a representative experiment, carried out three times.

0.5% of added ligand, and up to 1 /xM chPTHrP(1-36) did not raise cyclic AMP accumulation above basal levels of 1 to 2 pmol/well. Additional properties of the human PTH/PTHrP receptor were examined in human neuroblastoma SK-N-MC cells, in non-transfected SK-N-MC cells, specific binding of 125 pM [125I]chPTHrP(1-36) was below 0.5% of added ligand, but cyclic AMP accumulation was stimulated 2- to 5-fold by 100 nM chPTHrP(1-36) (ECs0 = 0.95 _+ 0.35 nM; n = 4). hPTH(3-34) did not affect cyclic AMP accu-

Fig. 3. In situ hybridization of PTtI/PTHrP receptor mRNA in cryostat sections of adult rat brain. A-F: Frontal sections at different anterior to posterior levels. G, H: Higher magnification of the cerebeUar region. B, H: Toluidine blue stained sections of (A) and (G), respectively. D, F: Autoradiography with 100-fold excess of unlabeled oligonucleotides to indicate nonspecific hybridization (for details see Material and methods) on ,sections adjacent to section (C) and (El, respectively. Black bars represent 1 mm. Hippocampal area (HI), dentate gyrus (DG), cortex (Cx), mesencephalic nucleus of the trigeminal nerve (me5l, molecular cell layer (moll, granular cell layer (gr), lateral reticular nucleus (LRt) and Purkinje cell layer of the cerebellum (PC).

M. E ggenberger et al. / Molecular Brain Research 36 (1996) 127-136

134

mulation at concentrations of up to 10 /.tM. These findings provide evidence for low level expression of PTH/PTHrP receptors in non-transfected SK-N-MC cells. In stably transfected SK-N-MC cells specific binding of 125 pM [1251]chPTHrP(1-36) reached equilibrium after 3 h at 15°C and amounted to 9.2 _+ 0.6 fmol [t25I]chPTHrP per well. Binding in the presence of 1 /zM chPTHrP(1-36) was less than 5% of total binding and considered to be non-specific. Binding of [ 125I]chPTHrP was displaced with similar IC50 by hPTH(1-84), hPTHrP(I-34) and chPTHrP(1-36). Binding inhibitory potencies of hPTH(1-34) and hPTH(3-34) were 2-fold higher (IC5o = 2.0 _+ 0.1 nM; n = 3; P < 0.01) and 10-fold lower (IC5o = 38.3_+ 9.6 nM; n - 3 ; P < 0 . 0 1 ) , respectively, as compared to hPTH(1-84) (IC5o = 4.0 _+ 0.6 nM; n -- 3) (Fig. 5). Basal cyclic AMP levels in stably transfected SK-N-MC cells amounted to 1.8 + 0 . 6 pmol/well. With 100 nM chPTHrP(I-36) cyclic AMP accumulation was maximally stimulated 40-60-fold. hPTH(1-34), chPTHrP(1-36), hPTHrP(I-34) and hPTH(i-84) stimulated cyclic AMP accumulation with similar potency. The ECs, values of hPTH(1-84) (0.19 + 0.06 nM; n = 3), hPTH(I-34) (0.09 + 0.01 nM; n = 3), and of chPTHrP(I-36) (0.09 + 0.01 nM; n = 4) were 12-fold lower ( P < 0.05) and that of hPTHrP(I-34) 4-fold lower ( P > 0.1) in transfected than in non-transfected cells. 1 /xM hPTH(3-34), a partial agonist of PTH action, marginally raised cyclic AMP accumulation. In 12 single, non-transfected SK-N-MC cells 100 nM hPTH(I-34) did not affect [Ca2+]i. Out of 48 stably transfected SK-N-MC cells, 16 (33%) had raised [Ca2' ]i in response to 100 nM hPTH(l-34) (Fig. 6). The percentage of responding cells was in the range of PTH/PTHrP receptor positive cells as estimated in parallel microscopically by single cell a u t o r a d i o g r a p h y using 750

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[1251]chPTHrP(I-36). With 100 nM hPTH(3-34) [Ca 2÷ ]i remained unchanged in 12 single, stably transfected SK-NMC cells.

4. Discussion

The structure of a PTH/PTHrP receptor of the human cerebellum is identical to that previously reported in traditional target organs of PTH, the kidney and bone [23,24]. The uniform size of 2.3 kb mRNA identified in kidney, liver and several human brain regions provides additional evidence for the expression of a single type PTH/PTHrP receptor in human tissues so far. This contrasts with the structural heterogeneity of splice variants of other members of the same receptor subfamily, such as of human, porcine and rat calcitonin [12,19,25,31], human corticotropin-releasing hormone [2] and rat pituitary adenylyl cyclase-activating peptide [27]. Among those, two rat calcitonin receptor subtypes differ in a 37 amino acid insert in the putative second extracellular domain. This affects ligand specificity and binding kinetics, and the longer form is predominantly expressed in the brain [12,25]. Northern blot analysis of mRNA from different human brain regions indicates widespread, low level expression of the PTH/PTHrP receptor. We have been unable to detect PTH/PTHrP receptor encoding mRNA in different preparations of the human cerebellum. Low receptor density sufficient for recognition by PCR may not have allowed us to identify the receptor on Northern blot analysis. But using in situ hybridization in the rat brain, the PTH/PTHrP receptor mRNA was predominantly present in the Purkinje cell layer of the cerebellum. Other regions included the mesencephalic nucleus of the trigeminal nerve, the reticulotegmental nucleus, the lateral reticular nucleus and the granular cell layer of the cerebellum. The results correspond to those of Weaver et al. [29] using in situ hybridization of rat brain tissue sections. Moreover, Northern blot analysis has revealed PTH/PTHrP receptor encoding mRNA in the rat cerebellum and in astrocytes enriched from the cerebral cortex [11,28]. Biologically active N-terminal PTH and PTHrP fragments bind to so far identified PTH/PTHrP receptors of different species with similar affinity and stimulate both G-protein linked adenylyl cyclase and phospholipase C. PTH and PTHrP are, furthermore, expressed in the rat brain, but, as revealed by in situ hybridization histochemistry, PTH is mainly localized in the hypothalamus, whereas PTHrP is much more widely distributed [5,20,29]. PTHrP may have some neuronal function [30]. To investigate for the first time PTH/PTHrP receptor ligand interactions in a cell line of neuronal origin, we have studied binding and second messenger activation of intact human PTH and of N-terminal PTH and PTHrP fragments in human neuroblastoma SK-N-MC cells. A 2- to 4-fold PTH and PTHrP dependent stimulation of cyclic AMP

M. Eggenberger et al. / Molecular Brain Research 36 (19961 127-136

accumulation in the absence of detectable specific [t2-~l]chPTHrP(1-361 binding and of [Ca2+]i responses provided evidence for low level endogenous PTH/PTHrP receptor expression in these cells. Stable transfection of SK-N-MC cells with the cloned cDNA was required to reveal PTH/PTHrP receptor binding inhibition and raised [Ca~" ]~, properties similar to those previously reported in COS-1 cells transiently transfected with human PTH/PTHrP receptor encoding cDNA [24]. Intact human PTH(1-84) and N-terminal fragments of human PTH and PTHrP inhibit [l_~.Si]chPTHrP(l_36) binding and stimulate cyclic AMP accumulation similarly. The observed 12-fold lower ECs0 values and the increased maximal stimulation of cyclic AMP accumulation in stably transfected SK-NMC cells as compared to the wild type are attributed to overexpression of PTH/PTHrP receptors. Much like [Nle 8'tS,Tyr34 ]bPTH(3-34)NH ~ [22], hPTH(3-34) inhibits [1_,5I]chPTHrP( 1-361 receptor binding and is a partial agonist in the SK-N-MC neuroblastoma cells as shown here. In conclusion, a PTH/PTHrP receptor has been identified in the human cerebellum. The receptor has the structure of the PTH/PTHrP receptors in human kidney and bone. The functional properties investigated in SK-N-MC cells of neuronal origin are indistinguishable from those in the monkey kidney cell lines COS-1 and COS-7. Both, PTH and PTHrP, are potential ligands in the brain but with a distinct regional distribution. Brain specific overexpression of cloned cDNAs encoding PTH/PTHrP receptors and of corresponding ligands may help to elucidate their physiological role in the central nervous system.

Acknowledgements This study was supported by the Swiss National Science Foundation Grant 32-28279.90, the Kanton of Zurich, the Schweizerischer Verein Balgrist, and by Ciba, Basel, Switzerland.

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