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Differential alternative splicing of PACAP receptor in pituitary cell subpopulations
ELSEVIER
Molecularand CellularEndocrinology113(1995)131-135
Differential alternative splicing of PACAP receptor in pituitary cell subpopulations Pascale Vertongena, Brigitte Velkeniersh, Elisabeth Hooghe-Petersb, Patrick Robberecht” a ‘Laboratory of Biochemistry and Nutrition, Unioersit~Libre de Brurelles, Medical School, Building G/E, CP 611, 808 Route de Len&, B-1070 Brussels, Belgium bLaboratory of Pharmacology, Medical School, Vrije Universiteit Brussel, Brussels, Belgium
Received8 June
1995; accepted
12 June 1995
Abstract
The capability of rat pituitary cells to express receptors for pituitary adenylate cyclase activating polypeptide (PACAP) and was evaluated by binding studies and measurement of adenylate cyclase activity on whole gland preparations and by reverse transcriptase-polyrnerase chain reaction (RT-PCR) using specific primers on preparations from isolated cell populations enriched in PRL- and GH-producing cells. Data obtained on whole gland preparations indicated that selective PACAP receptors (PACAP Type I) predominated. The mRNA coding for PACAP Type I and for the non-selective PACAP receptor Type II VIP, (but not VIP,) were identified. The mRNA coding for four different spliced variants of the PACAP Type I receptor were detected. In PRL producing cells, three variants and the VIP, mRNA were detected, whereas in GH-producing cells the mRNA coding for the variant having a 28-amino acid insert (termed HOP) in the third intracellular loop was the only present. VIP
Keywords: Pituitary adenylate cyclase polypeptide (PACAP); Vasoactive intestinal polypeptide (VIP); Rat pituitary cell; PACAP receptor; Splice variant
1. Introduction Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the secretin/ glucagon/vasoactive intestinal peptide (VIP) family of peptides. It was originally isolated from ovine hypothalamus for its ability to stimulate adenylate cyclase activity of cultured rat pituitary cells (Arimura, 1992). Although its physiological role has not yet been determined, PACAP has been clearly shown to be a modulator of pituitary functions: (a) PACAP immunoreaction fiber’s were demonstrated in the median eminence in close contact with the hypophyseal portal capillaries (Arimura, 1992); (b) PACAP receptors are present on anterior pituitary cell membranes
*Corresponding
author,
0303-7207/95/$09.50 SSDZ 0303-7207(95)
Tel.: 32 2 555 6215; Fax: 32 2 555 6230.
0 1995 Elsevier 036264
Science
Ireland
(Gottschall et al., 1990); (c) PACAP increased cyclic AMP and intracellular calcium in several rat pituitary models (Canny, 1992; Koch, 1992; Rawlings, 1993; Schomerus, 1994). Data on the effects of PACAP on pituitary hormone release are conflicting. (a) In superfused rat pituitary cell culture, PACAP stimulated in a dose-dependent manner the release of GH, PRL and ACTH but had no effect on the secretion of FSH or TSH (Miyata, 1990). (b) In static rat primary cell cultures, PACAP increased, within 30 min, the number of cells secreting GH and the amount of hormone secreted (Goth et al., 19921, but Wei et al. (1993) observed only a transient increase of GH release. Long-term exposure to PACAP (3, 4 or 5 h) was ineffective on GH release (Miyata, 1990; Jarry et al., 1992, Wei et al., 1993). (c) In dispersed cells, PACAP alone had no effect on gonadotrophin secretion but potentiated LHRH effect (Culler and Paschall, 1991).
Ltd. All rights reserved.
132
P. Vertongen et al. /Molecular and Cellular Endocrinology 113 (1995) 131-135
(d) GH-, ACTH-, LH- and (Y-su b unit secretion was increased after 24 h of exposure (Hart et al., 1992). (e) Expression of GH mRNA was stimulated by PACAPafter 2, 24 and 48 h of incubation of cultured dispersed purified GH cells (Velkeniers, 1994). (f) In vivo, in rat, PACAP infusion increased the release of GH and PRL (Jarry et al., 1992); the latter effect could be at least partly due to a central effect mediated by VIP (Yamauchi et al., 1995). (g) PACAP was also able to stimulate IL-6 secretion from rat pituitary cell cultures probably through interaction with specific PACAP receptors on folliculostellate cells (Gottschall et al., 1994). PACAP effects may be mediated through interaction with at least three classes of receptors that also recognise VIP (PACAP I, PACAP II, VIP, and PACAP II, VIP, receptors - see Discussion). In the case of PACAP I receptors that acted through stimulation of G protein-dependent phospholipase C and adenylate cyclase activation, five variant forms have been identified (Spengler et al., 1993): there appear to be three distinct 28-amino acid inserts (termed HIP, HOP, and HOP,) and combination insert (termed HIP-HOP) in addition to the short form (termed normal). The aim of the present study was to identify by the RT-PCR technique, on rat purified pituitary cell populations, the capability of the cells to express a given receptor subtype. 2. Materials and methods 2.1. Cell preparation A single step procedure was performed to enrich PRL and GH cells from the rat anterior pituitary gland. After dissociation, cells were centrifuged on a Percoll gradient as previously described (Velkeniers et al., 1988). Three layers were obtained: the first lower density layer (5% GH cells, 5% GN cells, 5% ACTH cells) contained 85% PRL cells; .the second intermediate density layer contained also a majority of PRL cells (55%) which were contaminated by 25% GH cells; the third high density layer was enriched in GH cells to 93% and PRL cells represented only 3% of the total cell content. 2.2. Receptor mRNA identification Total RNA was extracted by the guanidium isothiocyanate and caesium chloride method. The total RNA was treated with DNAse and then reverse transcribed as previously described (Vertongen et al., 1994). The following primers were used: (1) (a)
For PACAP receptor cDNA: for normal and HOP or HIP receptor sense primer:5’-TTAACTTTGTCTTTT-
cDNA,
(b)
antisense primer:5’-TCCCCATCGGC-3’; CTC’ITGCTGACGTTCTC-3’; corresponding, respectively, to the 952-976 and 1118-1333 sequences of the PACAP Type I receptor cDNA (Spengler et al., 1993). for HIP and HIP-HOP receptor cDNA, sense primer:5’-CCCTCAGACCAGCATTCACC-3’; antisense primer:5’-TCCCTCTGCTGACGTTCTC-3’; corresponding, respectively, to the 45-65 sequence of the HIP cassette and 1118-133 of the PACAP Type I receptor cDNA.
(2) For VIP, receptor cDNA: sense primer:5’-GC-
(3)
CTG’ITCAGGAAGCTGCACTG-3’; antisense primer:5’-AGGTAGAGGCCCTCTACCAG-3’; corresponding, respectively, to the 556-577 and 762-781 sequences of the rat VIP, receptor cDNA (Ishihara et al., 1992). For VIP, receptor cDNA:sense primer:5’GTCACAGTACAAGAGGCTCGC-3’; antisense primer:5’-CCCTCATACAGAGCTGACAGTG3’; corresponding, respectively, to the 1025-1045 and 1389-1410 sequences of the VIP, receptor cDNA (Lutz et al., 1993).
Thirty cycles (94°C for 1 min, 60°C for 1 min, 72°C for 1 min and a final cycle of 10 min at 72°C) were used in PCR performed with the Goldstar DNA polymerase (Eurogentec, Seraing, Belgium) according to the manufacturer’s instructions. Five microliters of the PCR mixture were submitted to electrophoresis on an agarose gel (1,2%) and stained with ethidium bromide. 2,3. Receptor identification Adenylate cyclase was assayed according to the procedure of Salomon et al. (1974) as detailed previously. 1251-Ac-His’-PACAIQ-27) was prepared by the iodogen method and binding assays were performed as previously described (Robberecht et al., 1993). 3. Results and discussion 3.1. Identification of the receptorprotein for PACAP and VlP
Dose-effect curves of both molecular forms of PACAP (PACAPand PACP”-38) on adenylate cyclase activation were performed on anterior pituitary membrane preparations. Both PACAP forms were equipotent with an EC,, of 6 nM while VIP was lOO-fold less potent (Fig. 1A). PACAP curves developed on a wide range of concentrations (more than 2 log) and a small but signiticant increase of cyclic AMP in presence of VIP was
P. Vetiongen et al. /Molecular
and Cellzdar Endocrinology
presence of increasing concentrations of PACAP-27, PACAPand VIP (Fig. 1B). PACAPand PACAPwere equipotent (IC,, of 2 nM) while 1 PM VIP was necessary to inhibit 50% of the binding. As the dose-effect curves observed in total pituitary membranes could result from the interaction of the peptides with different receptor classes, the aim of the present study was to identify the types of receptors expressed in the pituitary gland and to test the hypothesis that receptor heterogeneity was due to the expression of different types of receptors on different cell types. PACAP effects are mediated through interaction with at least three classes of receptors. The PACAP Type I receptor recognizes both PACAP forms with the same affmity but has a 300- to lOOO-fold lower affinity for VIP; this receptor is found in anterior pituitary, brain, astrocytes, neuroblastoma cells, adrenal cells and in rat pancreatic cancerous cell line AR 4-2J (see review, Christophe, 1994). The PACAP Type II receptor corresponds to a mixture of two recently cloned receptors, VIP, and VIP, (Ishihara et al., 1992; Lutz et al., 1993). They recognized VIP and PACAP with a comparable affinity. The VIP, receptor differs from the VIP, by a 3-fold lower affinity for the peptides and by its capability to discriminate PACAPand PACAP(Svoboda et al., 1994). Both types of PACAP receptors are coupled to adenylate cyclase but only the selective PACAP I receptor may also increase inositol triphosphate synthesis and intracellular calcium concentrations.
INORMAL PITUITARY GLAND1 @
PACAP-
-10
-9
-8
-6
-7
[PEPTIDE] (log
133
113 (1995) 131-135
M)
3.2. Identification of VIPl, VIP2 and PACAP I receptors
Fig. 1. Upper panel: dose-effect curves of adenylate cyclase activation in membrane preparations from normal pituitary gland. The results were the means of three determinations. Lower panel: effect of increasing concentrations of unlabelled PACAP(01, PACAP(0) and VIP (A ) on ‘ZSI-[Acetyl-His’]PACAP-27 binding to membrane preparations of normal pituitary gland. The results were the mean of three determinations.
mRNA
VIP, receptor mRNA was never detected in pituitary cell preparations (total pituitary gland, whole cell culture, purified subpopulations) (Table 1). The VIP, receptor mRNA was weakly detected in the anterior pituitary and the corresponding total culture. In the purified subpopulations it was only detected in the layer 1. Five different isoforms of the PACAP Type I re-
noticed at low concentrations. The inhibition of tracer binding (competition curves) was measured in the Table I Sample
Total anterior pituitary Total culture Layer 1 Layer 2 Layer 3
PACAP receptor mRNA
VIP receptor mRNA
Normal
HOP
HIP
HIP-HOP
VIP,
VIP,
*i** i+* ff ff -
ff ff ++ ff +++
* +f _ _ -
+ _
_ _ _
(*> (*) + _
+f -
P. Vertongenet al. /Molecular and Celtuiar Endocrinology 113 (199s) 131-135
134
ceptor can be generated by an alternative splicing mechanism (Spengler et al., 1993; Svoboda et al., 1993). The different variants were arbitrarily named normal for the shortest forms; HOP, (presence of an additional 28 amino acid residues in the third intracytoplasmic loop), HOP, (equivalent to HOP, but with a deletion of the first HOP amino acid residue); HIP (addition of 28 amino acid residues in the same position than HOP but with a different sequence), and HIP-HOP (addition of 56 amino acid residues: 28 amino acids from HIP and 28 amino acids from HOP,, successively). In the anterior pituitary, and in the total culture, all the PACAP receptor mRNA isoforms were detected. In layer 1, which contains 85% of prolactin cells, the shortest form and the HOP form of the receptor were equally represented (Table 1, Fig. 2). In layer 2, which contains 55% of PRL cells and 25% of GH cells, the normal and the HOP isoform were equally represented, whereas the HIP and the HIP-HOP isoforms were not detected. In layer 3, which contains 93% GH cells, the HOP isoform of PACAP Type I receptor was the only form detected. Thus, in rat anterior pituitary, there is a cell type-specific alternative splicing of the mRNA coding for the PACAP Type I
receptors. We were unable to demonstrate that the corresponding receptor proteins were indeed expressed in the isolated cells: these cultured cells respond poorly to the peptides and dose-effect curves could not be validly interpreted. This could be due to receptor damage resulting from trypsinisation of the cells during the isolation procedure. The expression of different receptor subtypes in different cell populations might be physiologically relevant: (a) the HOP variant receptor had a 2-fold higher affinity for PACAP than the ‘normal receptor’; (b) after a prolonged exposure of CHO cells expressing the recombinant normal PACAP and PACAPHOP receptors, the number of receptors decreased and the response to PACAP was blunted. The ‘basal’ adenylate cyclase activity (in the absence of peptide) was, however, markedly increased after pretreatment of cells expressing the normal PACAP receptors, but not after pretreatment of cells expressing the PACAP-HOP receptors (Ciccarelli et al., 1994). Acknowledgements
Supported by Grant no 3.4502.95 from the Fonds de la Recherche Scientifique Medicale and Fonds A.
SAMPLE
PACAP
I
RECEPTOR
516 bp
mRNA 220 bp
Fig. 2. Visualization with ethidium bromide of the electrophoretic pattern in 1.2% agarose of molecular weight standards and RT-PCR products obtained with the specific pruners for normal and HOP or HIP PACAP receptor cDNA. The lower and the higher size bands represented the normal and HOP variant receptor cDNA respectively. The same results were obtained with three different RNA preparations.
P. Vertongen et al. /Molecular and Cellular Endocrinology 113 (1995) 131-135
Solvay et des Usines Solvay (Belgium) and by grant nr. 3.0023.94 from the Nationaal Fonds voor Wetenschappelijk Geneeskundig Onderzoek (F.G.W.O.). References Arimura, A. (1992) Regul. Pept. 37,287-303. Canny, B.J., Rawlings, S.R. and Leong, D.A. (1992) Endocrinology 130,211-215. Christophe, J. (1994) Am. J. Physiol. 266, G963-G971. Ciccarelli, E., Svoboda, M., De Neef, P., Di Paolo, E., Bollen, A., Dubeaux, C., Vilardaga, J.-P., Waelbroeck, M. and Robberecht, P. (1995) Eur. J. Pharmacol. 288, 259-267. Culler, M.D. and Paschall, C.S. (1991) Endocrinology 129, 2260-2262. Goth, M.I., Lyons, C.E., Cany, B.J. and Thomer, M.O. (1992) Endocrinology 130,939-944. Gottschall, P.E., Tatsuno, I., Miyata, A. and Arimura, A. (1990) Endocrinology 127,272-277. Gottschal!, P.E., Tatsuno, I. and Arimura, A. (1994) Brain Res. 637, 197-203. Hart, G.R., Gowing, H. and Burrin, J.M. (1992) J. Endocrinol. 134, 33-41. Ishihara, T., Shigemoto, R., Mori, K., Takahashi, K. and Nagata, S. (1992) Neuron 8, 811-819. Jarry, H., Leonhardt, S., Schmidt, W.E., Creutzfeldt, W. and Wuttke, W. (1992) Life Sci. 51, 823-830. Koch, B. and Lutz-Bucher, B. (1992) Regul. Pept. 38,45-53. Lutz, E.M., Sheward, W.J., West, K.M., Morrow, J.A., Fink, G. and Harmar, A.J. (1993) FEBS Lett. 334, 3-8. Miyata, A., Jiang, L., Dahl, R.D., Kitada, C., Kubo, K., Fujino, M.,
135
Minamino, N. and Arixnura, A. (1990) Biochem. Biophys. Res. Commun. 170,643~648. Rawlings, S.R., Canny, B.J. and Leong, D.A. (1993) Endocrinology 132, 1447-1452. Robberecht, P., Vertongen, P., Velkeniers, B., De Neef, P., Vergani, P., Raftopoulos, C., Brotchi, J., Hooghe-Peters, E. and Christophe, J. (1993) J. Clin. Endocrinol. Metab. 77: 1235-1239. Salomon, Y., Londos, C. and Rodbell, M. (1974) Anal. Biochem. 58, 541-548. Schomerus, E., Poch, A., Bunting, R., Mason, W.T. and McArdle, C.A. (1994) Endocrinology 134, 315-323. Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeburg, P.H. and Joumot, L. (1993) Nature 365, 170-175. Svoboda, M., Tastenoy, M., Ciccarelli, E., Stihenart, M. and Christophe, J. (1993) Biochem. Biophys. Res. Commun. 195, 881-888. Svoboda, M., Tastenoy, M., Van Rampelbergh, J., Goossens, J.-F., De Neef, P., Waelbroeck, M. and Robberecht, P. (1994) B&hem. Biophys. Res. Commun. 205, 1617-1624. Velkeniers, B., Hooghe-Peters, E.L., Hooghe, R., Belayew, A., Smets, G., Claeys, A., Robberecht, P. and Vanhaelst, L. (1988) Endocrinology 123, 1619-1630. Velkeniers, B., Zheng, L., Kazemzadeh, M., Robberecht, P., Vanhaelst, L. and Hooghe-Peters, E.L. (1994) J. Endocrinol. 143, l-11. Vertongen, P., Ciccarelli, E., Woussen-Colle, M.-C., De Neef, P., Robberecht, P. and Cauvin, A. (1994) Endocrinology 135, 1537-1542. Wei, L., Chan, W.W.-S., Butler, B. and Cheng, K. (1993) Biochem. Biophys. Res. Commun. 197, 1396-1401. Yamauchi, K., Murakami, Y., Nishiki, M., Tanaka, J., Koshimura, K. and Kato, Y. (1995) Neurosci. Lett. 189, 131-134.
Molecularand CellularEndocrinology113(1995)131-135
Differential alternative splicing of PACAP receptor in pituitary cell subpopulations Pascale Vertongena, Brigitte Velkeniersh, Elisabeth Hooghe-Petersb, Patrick Robberecht” a ‘Laboratory of Biochemistry and Nutrition, Unioersit~Libre de Brurelles, Medical School, Building G/E, CP 611, 808 Route de Len&, B-1070 Brussels, Belgium bLaboratory of Pharmacology, Medical School, Vrije Universiteit Brussel, Brussels, Belgium
Received8 June
1995; accepted
12 June 1995
Abstract
The capability of rat pituitary cells to express receptors for pituitary adenylate cyclase activating polypeptide (PACAP) and was evaluated by binding studies and measurement of adenylate cyclase activity on whole gland preparations and by reverse transcriptase-polyrnerase chain reaction (RT-PCR) using specific primers on preparations from isolated cell populations enriched in PRL- and GH-producing cells. Data obtained on whole gland preparations indicated that selective PACAP receptors (PACAP Type I) predominated. The mRNA coding for PACAP Type I and for the non-selective PACAP receptor Type II VIP, (but not VIP,) were identified. The mRNA coding for four different spliced variants of the PACAP Type I receptor were detected. In PRL producing cells, three variants and the VIP, mRNA were detected, whereas in GH-producing cells the mRNA coding for the variant having a 28-amino acid insert (termed HOP) in the third intracellular loop was the only present. VIP
Keywords: Pituitary adenylate cyclase polypeptide (PACAP); Vasoactive intestinal polypeptide (VIP); Rat pituitary cell; PACAP receptor; Splice variant
1. Introduction Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the secretin/ glucagon/vasoactive intestinal peptide (VIP) family of peptides. It was originally isolated from ovine hypothalamus for its ability to stimulate adenylate cyclase activity of cultured rat pituitary cells (Arimura, 1992). Although its physiological role has not yet been determined, PACAP has been clearly shown to be a modulator of pituitary functions: (a) PACAP immunoreaction fiber’s were demonstrated in the median eminence in close contact with the hypophyseal portal capillaries (Arimura, 1992); (b) PACAP receptors are present on anterior pituitary cell membranes
*Corresponding
author,
0303-7207/95/$09.50 SSDZ 0303-7207(95)
Tel.: 32 2 555 6215; Fax: 32 2 555 6230.
0 1995 Elsevier 036264
Science
Ireland
(Gottschall et al., 1990); (c) PACAP increased cyclic AMP and intracellular calcium in several rat pituitary models (Canny, 1992; Koch, 1992; Rawlings, 1993; Schomerus, 1994). Data on the effects of PACAP on pituitary hormone release are conflicting. (a) In superfused rat pituitary cell culture, PACAP stimulated in a dose-dependent manner the release of GH, PRL and ACTH but had no effect on the secretion of FSH or TSH (Miyata, 1990). (b) In static rat primary cell cultures, PACAP increased, within 30 min, the number of cells secreting GH and the amount of hormone secreted (Goth et al., 19921, but Wei et al. (1993) observed only a transient increase of GH release. Long-term exposure to PACAP (3, 4 or 5 h) was ineffective on GH release (Miyata, 1990; Jarry et al., 1992, Wei et al., 1993). (c) In dispersed cells, PACAP alone had no effect on gonadotrophin secretion but potentiated LHRH effect (Culler and Paschall, 1991).
Ltd. All rights reserved.
132
P. Vertongen et al. /Molecular and Cellular Endocrinology 113 (1995) 131-135
(d) GH-, ACTH-, LH- and (Y-su b unit secretion was increased after 24 h of exposure (Hart et al., 1992). (e) Expression of GH mRNA was stimulated by PACAPafter 2, 24 and 48 h of incubation of cultured dispersed purified GH cells (Velkeniers, 1994). (f) In vivo, in rat, PACAP infusion increased the release of GH and PRL (Jarry et al., 1992); the latter effect could be at least partly due to a central effect mediated by VIP (Yamauchi et al., 1995). (g) PACAP was also able to stimulate IL-6 secretion from rat pituitary cell cultures probably through interaction with specific PACAP receptors on folliculostellate cells (Gottschall et al., 1994). PACAP effects may be mediated through interaction with at least three classes of receptors that also recognise VIP (PACAP I, PACAP II, VIP, and PACAP II, VIP, receptors - see Discussion). In the case of PACAP I receptors that acted through stimulation of G protein-dependent phospholipase C and adenylate cyclase activation, five variant forms have been identified (Spengler et al., 1993): there appear to be three distinct 28-amino acid inserts (termed HIP, HOP, and HOP,) and combination insert (termed HIP-HOP) in addition to the short form (termed normal). The aim of the present study was to identify by the RT-PCR technique, on rat purified pituitary cell populations, the capability of the cells to express a given receptor subtype. 2. Materials and methods 2.1. Cell preparation A single step procedure was performed to enrich PRL and GH cells from the rat anterior pituitary gland. After dissociation, cells were centrifuged on a Percoll gradient as previously described (Velkeniers et al., 1988). Three layers were obtained: the first lower density layer (5% GH cells, 5% GN cells, 5% ACTH cells) contained 85% PRL cells; .the second intermediate density layer contained also a majority of PRL cells (55%) which were contaminated by 25% GH cells; the third high density layer was enriched in GH cells to 93% and PRL cells represented only 3% of the total cell content. 2.2. Receptor mRNA identification Total RNA was extracted by the guanidium isothiocyanate and caesium chloride method. The total RNA was treated with DNAse and then reverse transcribed as previously described (Vertongen et al., 1994). The following primers were used: (1) (a)
For PACAP receptor cDNA: for normal and HOP or HIP receptor sense primer:5’-TTAACTTTGTCTTTT-
cDNA,
(b)
antisense primer:5’-TCCCCATCGGC-3’; CTC’ITGCTGACGTTCTC-3’; corresponding, respectively, to the 952-976 and 1118-1333 sequences of the PACAP Type I receptor cDNA (Spengler et al., 1993). for HIP and HIP-HOP receptor cDNA, sense primer:5’-CCCTCAGACCAGCATTCACC-3’; antisense primer:5’-TCCCTCTGCTGACGTTCTC-3’; corresponding, respectively, to the 45-65 sequence of the HIP cassette and 1118-133 of the PACAP Type I receptor cDNA.
(2) For VIP, receptor cDNA: sense primer:5’-GC-
(3)
CTG’ITCAGGAAGCTGCACTG-3’; antisense primer:5’-AGGTAGAGGCCCTCTACCAG-3’; corresponding, respectively, to the 556-577 and 762-781 sequences of the rat VIP, receptor cDNA (Ishihara et al., 1992). For VIP, receptor cDNA:sense primer:5’GTCACAGTACAAGAGGCTCGC-3’; antisense primer:5’-CCCTCATACAGAGCTGACAGTG3’; corresponding, respectively, to the 1025-1045 and 1389-1410 sequences of the VIP, receptor cDNA (Lutz et al., 1993).
Thirty cycles (94°C for 1 min, 60°C for 1 min, 72°C for 1 min and a final cycle of 10 min at 72°C) were used in PCR performed with the Goldstar DNA polymerase (Eurogentec, Seraing, Belgium) according to the manufacturer’s instructions. Five microliters of the PCR mixture were submitted to electrophoresis on an agarose gel (1,2%) and stained with ethidium bromide. 2,3. Receptor identification Adenylate cyclase was assayed according to the procedure of Salomon et al. (1974) as detailed previously. 1251-Ac-His’-PACAIQ-27) was prepared by the iodogen method and binding assays were performed as previously described (Robberecht et al., 1993). 3. Results and discussion 3.1. Identification of the receptorprotein for PACAP and VlP
Dose-effect curves of both molecular forms of PACAP (PACAPand PACP”-38) on adenylate cyclase activation were performed on anterior pituitary membrane preparations. Both PACAP forms were equipotent with an EC,, of 6 nM while VIP was lOO-fold less potent (Fig. 1A). PACAP curves developed on a wide range of concentrations (more than 2 log) and a small but signiticant increase of cyclic AMP in presence of VIP was
P. Vetiongen et al. /Molecular
and Cellzdar Endocrinology
presence of increasing concentrations of PACAP-27, PACAPand VIP (Fig. 1B). PACAPand PACAPwere equipotent (IC,, of 2 nM) while 1 PM VIP was necessary to inhibit 50% of the binding. As the dose-effect curves observed in total pituitary membranes could result from the interaction of the peptides with different receptor classes, the aim of the present study was to identify the types of receptors expressed in the pituitary gland and to test the hypothesis that receptor heterogeneity was due to the expression of different types of receptors on different cell types. PACAP effects are mediated through interaction with at least three classes of receptors. The PACAP Type I receptor recognizes both PACAP forms with the same affmity but has a 300- to lOOO-fold lower affinity for VIP; this receptor is found in anterior pituitary, brain, astrocytes, neuroblastoma cells, adrenal cells and in rat pancreatic cancerous cell line AR 4-2J (see review, Christophe, 1994). The PACAP Type II receptor corresponds to a mixture of two recently cloned receptors, VIP, and VIP, (Ishihara et al., 1992; Lutz et al., 1993). They recognized VIP and PACAP with a comparable affinity. The VIP, receptor differs from the VIP, by a 3-fold lower affinity for the peptides and by its capability to discriminate PACAPand PACAP(Svoboda et al., 1994). Both types of PACAP receptors are coupled to adenylate cyclase but only the selective PACAP I receptor may also increase inositol triphosphate synthesis and intracellular calcium concentrations.
INORMAL PITUITARY GLAND1 @
PACAP-
-10
-9
-8
-6
-7
[PEPTIDE] (log
133
113 (1995) 131-135
M)
3.2. Identification of VIPl, VIP2 and PACAP I receptors
Fig. 1. Upper panel: dose-effect curves of adenylate cyclase activation in membrane preparations from normal pituitary gland. The results were the means of three determinations. Lower panel: effect of increasing concentrations of unlabelled PACAP(01, PACAP(0) and VIP (A ) on ‘ZSI-[Acetyl-His’]PACAP-27 binding to membrane preparations of normal pituitary gland. The results were the mean of three determinations.
mRNA
VIP, receptor mRNA was never detected in pituitary cell preparations (total pituitary gland, whole cell culture, purified subpopulations) (Table 1). The VIP, receptor mRNA was weakly detected in the anterior pituitary and the corresponding total culture. In the purified subpopulations it was only detected in the layer 1. Five different isoforms of the PACAP Type I re-
noticed at low concentrations. The inhibition of tracer binding (competition curves) was measured in the Table I Sample
Total anterior pituitary Total culture Layer 1 Layer 2 Layer 3
PACAP receptor mRNA
VIP receptor mRNA
Normal
HOP
HIP
HIP-HOP
VIP,
VIP,
*i** i+* ff ff -
ff ff ++ ff +++
* +f _ _ -
+ _
_ _ _
(*> (*) + _
+f -
P. Vertongenet al. /Molecular and Celtuiar Endocrinology 113 (199s) 131-135
134
ceptor can be generated by an alternative splicing mechanism (Spengler et al., 1993; Svoboda et al., 1993). The different variants were arbitrarily named normal for the shortest forms; HOP, (presence of an additional 28 amino acid residues in the third intracytoplasmic loop), HOP, (equivalent to HOP, but with a deletion of the first HOP amino acid residue); HIP (addition of 28 amino acid residues in the same position than HOP but with a different sequence), and HIP-HOP (addition of 56 amino acid residues: 28 amino acids from HIP and 28 amino acids from HOP,, successively). In the anterior pituitary, and in the total culture, all the PACAP receptor mRNA isoforms were detected. In layer 1, which contains 85% of prolactin cells, the shortest form and the HOP form of the receptor were equally represented (Table 1, Fig. 2). In layer 2, which contains 55% of PRL cells and 25% of GH cells, the normal and the HOP isoform were equally represented, whereas the HIP and the HIP-HOP isoforms were not detected. In layer 3, which contains 93% GH cells, the HOP isoform of PACAP Type I receptor was the only form detected. Thus, in rat anterior pituitary, there is a cell type-specific alternative splicing of the mRNA coding for the PACAP Type I
receptors. We were unable to demonstrate that the corresponding receptor proteins were indeed expressed in the isolated cells: these cultured cells respond poorly to the peptides and dose-effect curves could not be validly interpreted. This could be due to receptor damage resulting from trypsinisation of the cells during the isolation procedure. The expression of different receptor subtypes in different cell populations might be physiologically relevant: (a) the HOP variant receptor had a 2-fold higher affinity for PACAP than the ‘normal receptor’; (b) after a prolonged exposure of CHO cells expressing the recombinant normal PACAP and PACAPHOP receptors, the number of receptors decreased and the response to PACAP was blunted. The ‘basal’ adenylate cyclase activity (in the absence of peptide) was, however, markedly increased after pretreatment of cells expressing the normal PACAP receptors, but not after pretreatment of cells expressing the PACAP-HOP receptors (Ciccarelli et al., 1994). Acknowledgements
Supported by Grant no 3.4502.95 from the Fonds de la Recherche Scientifique Medicale and Fonds A.
SAMPLE
PACAP
I
RECEPTOR
516 bp
mRNA 220 bp
Fig. 2. Visualization with ethidium bromide of the electrophoretic pattern in 1.2% agarose of molecular weight standards and RT-PCR products obtained with the specific pruners for normal and HOP or HIP PACAP receptor cDNA. The lower and the higher size bands represented the normal and HOP variant receptor cDNA respectively. The same results were obtained with three different RNA preparations.
P. Vertongen et al. /Molecular and Cellular Endocrinology 113 (1995) 131-135
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