Neuroscience Letter~'. 137 (1992) 19 23 c" 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00
19
NSL 08448
Investigation of the interaction of VIP binding sites with VIP and PACAP in human brain Katsuyuki Suda, David M. Smith, M o h a m m a d A. Ghatei and Stephen R. Bloom Department o/Medicine, Royal Postgraduate Medical School. Hammcrsmith Hospital. Lomhm ( UK; (Received 8 October 1991: Revised version received 29 November 1991: Accepted 3 December 1991)
Key words." PACAP receptor: Vasoactive intestinal polypeptide receptor: Human brain: Chemical cross-linking We have compared the binding of ['>l]vasoactive intestinal polypcptide (VlP) to human brain membranes with that of [t>I]PACAP27. [*-'q]VlP was displaced by PACAP27, VIP and two synthetic peptides, peptide-1 (N-terminal PACAP27/C-terminal VIP) and peptide-2 (N-terminal VIP/Cterminal PACAP27), but the IC~0 of PACAP27 and peptide-I were 10 20 times lower than those of VIP and peptide-2. [':51]PACAP27 was readily displaced by PACAP27 and peptide-l, with an ICs, of less than 1 nM, but poorly by VIP and peptide-2. Chemical cross-linking revealed that both labels were bound to polypeptides of M~ 66,000 and M~ 50,000. The resnlts indicate that in human brain membranes both binding sites have a higher affinity to the N-terminal sequence of PACAP27, and VIP binding sites prefer PACAP27 to VIP itselF.
Pituitary adenylate cyclase activating polypeptide (PACAP) is a 38 amino acid peptide (PACAP38) of ovine hypothalamic origin. It was originally isolated on the basis of its stimulation of adenylate cyclase in cultured rat anterior pituitary cells [15]. Recently, PACAP receptors have been identified in some cultured cell lines [3, 4], rat astrocytes [20], rat tissues [10, 12], bovine brain [17] and human brain [19]. All PACAP receptors except those in rat lung were highly specific for PACAP and of very low affinity for vasoactive intestinal polypeptide (VIP) despite the structural similarity with VIE On the other hand, in a recent report, bovine brain VIP receptors have been shown to crossreact with PACAP [17]. Both PACAP and VIP receptors are thought to be linked via G, to stimulate adenylate cyclase [3], and PACAP and VIP have several pharmacological effects in common, e.g. vasodilation [1]. It is therefore possible that PACAP can act both through PACAP receptors and VIP receptors. Therefore, in human brain membranes, we examined the interaction with VIP and PACAP binding sites of VIP, PACAP and two synthetic peptides which were N-terminal VIP/C-terminal PACAP27 and N-terminal PACAP27/C-terminal VIE PACAP1-27NH2 (PACAP27), the N-terminal amidated 27 residue derivative of PACAP1-38NH~ (PACAP38), was synthesized by Peptide Products (UK). Correspondence: S.R. Bloom, Francis Fraser 2 Lab, Dept. of Medicine, R.RM.S., Hammersmith Hospital, Du Cane Road, London, W12, ONN, UK.
VIP was synthesized by IAF Biochem (UK). Peptide-I consisted of PACAP(I-14) in the N-terminus and VIP(15-28) in the C-terminus. Peptide-2 consisted of VIP(I-14) in the N- terminus and PACAP(15-27) in the C-terminus. Both peptide-I and peptide-2 were synthesized by the Palliard Chemical Co Ltd (UK) (see Table I). VIP and PACAP27 were iodinated by the iodogen (1,3,4,6-tetrachloro-3c~-6c~-diphenylglycoluril, Pierce, Rockford. Illinois, USA) method [7], as previously described [19]. Brain membranes were prepared from five regions of human brain (cortex, cerebellum, basal ganglia, hypothalamus and brainstem), as previously described [19]. The postmortem samples were obtained from subjects who were without history of neurological or psychiatric disorder by Dr. J.K. Murphy (York District Hospital, York. UK) and were stored at -70°C. Membrane protein concentration [3 6 mg/ml] was measured by the Biuret method [9]. For the VIP binding assay, the membranes (100 jag) were incubated at 34°C for 60 min with 0.2 nM [1251]VIP (500 cps) in 25 mM Tris-HC1 buffer pH 7.4 containing 1% BSA. 4 mM MgCI2, 2 mM EDTA and 0.25 mg/ml bacitracin. In the PACAP binding assay, the membranes (60 jag) were incubated at 37°C for 90 min with 0.25 nM [1251]PACAP27 (500 cps) in 50 mM Tris-HCl buffer pH 7.6 containing 3% BSA. The final assay volume was 0.5 ml. Non-specific binding was determined in the presence of 0.2 jaM PACAP27 or 0.2 jaM VIR The receptorlabelled peptide complex was separated from free la-
20
TAB[.I 1 C O M P O S I T I O N O t : T H E A M I N O A C I D S E Q U E N C E O | : PACAP27. VIP. P E P T 1 D E - I A N D P E P T I D E - 2
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Peptide-I Peptide-2
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Sequence of P A C A P 2 7 is underlined: o. ovine: h. h u m a n ; p. porcine.
belled peptide by centrifugation at 15,000 × g for 2 min. The pellets were washed at 4°C with 0.5 ml of the binding assay buffer and counted in a 7" counter. FPLC analysis of PACAP and VIP label in the supernatants using a Pep RPC C~s column (Pharmacia, Milton Keynes, Bucks UK) eluted with a water/acetonitrile gradient in 0.1% TFA of 20-45% acetonitrile in 30 min showed that less than 10% of the label was degraded during the assay (results not shown). Specific binding was calculated as total binding minus non-specific binding. Chemical cross-linking of [~25I]VIP and [~2SI]PACAP27 to human brain membranes was performed by incubating brain membranes (100 200 ~g) with 1 nM [~25I]PACAP27 or 2 nM [~25I]VIP in the presence and the absence of 0.2 ktM PACAP and 0.2 ~M VIE Membranes were centrifuged at 15,000 × g for 2 rain and washed twice at 4°C in 0.5 ml of binding assay buffer. After resuspension of pellets in 50 mM Tris-HC1 pH 7.4 (500 pl), disuccinimidyl suberate (Pierce) dissolved in dimethyl sulfoxide was added at a final concentration of I mM. After 30-min incubation at room temperature, samples were centrifuged at 15,000 x g for 2 min and washed twice with 0.5 ml of Tris-HC1 buffer. Pellets were resuspended in 40 ~1 of Laemmli sample buffer, boiled for 2 min, and then subjected to SDS polyacrylamide (7,5%) gel electrophoresis [19]. Autoradiographs were obtained at -70°C for 1 3 weeks. The association of [~2Sl]VIP and [~25I]PACAP27 to human brain membranes were time and temperature sensitive. The specific binding of [t25I]PACAP27 increased more rapidly at 37°C than at 4°C and reached a steady state after 80 min [19]. The time course of [~25I]VIP showed a similar pattern reaching equilibrium at 34°C after 60 min (see Fig. 1.). The maximal specific binding of [12sI]VIP and [~2Sl]PACAP27 was 7% and 19% of total counts added and the non-specific binding of [~2q]V1P and [J25I]PACAP was approximately 30% and 15% of this maximal total binding. The binding characteristics of receptors from the five regions of the brain were very similar and interchangeable in all these experiments [19]. The displacement curves of [12q]VIP and [~251]PACAP27 in the presence of increasing concentrations of PACAP27, VIR peptide-I (PACAP27/V1P) and
peptide-2 (VIP/PACAP27) are shown in Fig. 2 and Fig. 3. [125I]VIP binding was inhibited by all four peptides. The ICs0 of PACAP27 (0.08 + 0.02 nM, mean + S.E.M., n=4) was similar to that of peptide 1 (0,07 _+ 0.01 nM). The ICs0 of VIP (1.50 + 0.49 riM) and peptide-2 (0.70 _+ 0,09 nM) were 10-20 times higher than that of PACAP27 and peptide-1. On the other hand, both PACAP27 and peptide-1 were able to displace [~25I]PACAP27 with high affinity. The ICs0 of PACAP27 and peptide-1 were 0.79 _+ 0.34 nM and 0.61 _+ 0.14 nM (mean _+ S.E.M., n=3), respectively, but VIP and peptide-2 displaced very little label with a 1,000-fold higher ICs0 than PACAP27 and peptide- 1. The distribution of [~25I]VIP binding sites and [~25I]PACAP binding sites are shown in Fig. 4. [~25I]VIP binding was highest in cortex, whilst [J25I]PACAP binding was highest in hypothalamus and brainstem. The density of [~25I]VIP binding was 8-17 times less than that of [~2sI]PACAP binding in all five regions. Chemical cross-linking studies revealed that both PACAP27 and VIP labels were bound to polypeptide chains of M,. 66,000 (69,000-3,000 mot. wt. for both
~5 ¢,j
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120
time (min) Fig. I. Effect of i n c u b a t i o n time and t e m p e r a t u r e on association of [~:~I]VIP to h u m a n brain m e m b r a n e s . B i n d i n g assays were p e r f o r m e d as described in the text with 0.2 n M [~251]VIP at 34°C (O) a n d 4°C ( ) . Values are p e r c e n t a g e o f m a x i m a l specific b i n d i n g a n d m e a n + S.E.M. o f three experiments.
21
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~I]PACAP were not observed in the presence of 0.2/aM PACAP27, but they were unaffected in the presence of 0.2/aM VIP (Fig. 5). We have previously demonstrated the existence of PACAP binding sites in human brain [19]. The present study was performed to establish the binding portion of the PACAP27 and VIP molecules to their receptors and the interaction of both peptides with their receptors. Displacement of [~25I]VIP by peptide-I (PACAP27/ VIP), which has the N-terminal sequence of PACAP27,
c"
-8
10
-6
Fig. 2. Inhibition of [t>l]PACAP27 (0.25 nM) binding in human brain membranes by increasing concentrations of PACAP27 ( ) , V1P (A), peptide-1 ( t ) and peptide-2 (,). Values are expressed as percentage of specific binding and mean +_ S.E.M. of three experiments.
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Fig. 3. Inhibition of [~:5l]VlP (0.2 nM) binding in human brain membranes by PACAP27 (V), VIP (A), peptide-1 (I) and peptide-2 ( ) . Values are expressed as percentage of specific binding and mean _+ S.E.M. of four experiments.
o
0 CO
BG
HY
BS
CE
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Fig. 4. Distribution of[P>I]PACAP27 (0.25 nMI and [t:'l [VI P (I).2 nM) binding sites to human brain membranes. Binding as~,ay,, x~erc pcrformed in triplicate with membranes IYom each brahl tissue. ('(), cortex; BG. Basal ganglia: HY, hypolhalamus: BS. bl'mnstenl: and CE. cerebellum Values are expressed as mean * S.tLM. of four cxpcriII/ellts.
and PACAP27 were similar. Peptide-2 (VlP/PACAP27), with the C-terminal sequence of PACAP27, and VIP were both of lower affinity, suggesting these peplides depend for binding on the N-terminal sequences. Surprisingly, the N-terminal sequence of PACAP27 had a 10-20 times higher affinity to the binding sites of VIP than that of VIP itself. Peptide-I (PACAP27/VlP) displaced [~:~I]PACAP27 with ahnost the same affinity as PACAP27, but peptide-2 lVIPiPACAP27) displaced [L'5I]PACAP2? poorly, as did VIP. This indicates that PACAP binding sites are very specilic for the N-terminal sequence of PACAP. Taken together these results may indicate the presence of two types of PACAP receptors in human brain membranes, one receptor type being specific for PACAP and another able to bind both PACAP and VIP but, showing a greater altinit? l\)r PACAP. These two binding sites may correspond to the type I (specific PACAP) and type 11 (binding both PACAP and VIP) described by Shivers et al. [1~1 ira rat tissues, except that since they used o n b [~"I]PACAP they were only able to describe type I receptors m the brain. In this study using [L"I]VIP we have demonstrated the presence of type 11 receptors in the brain. We were unable to demonstrate by these methods receptors in human brain membranes which were specitic t\)r VI P and did not bind PACAR Chemical cross-linking studies showed that VIP binding sites were of the same reel. wt. as PACAP binding sites. Therefore it is possible that VIP binding sites might be a subset of PA('AP binding sitcs in human brain. The distribution of VIP binding sites, with highest
1
VIP
2
3
4
5
6
*
Fig. 5. Autoradiograph illustrating cross-linking of [~>I]PACAP27 (lane 1 3) and [~'sI]VIP(lane 4-6) to human brain cortex membranes. Cross-linking was performed as described in the text using human cortex membranes in the presence and absence of 0.2 ruM PACAP27 or 0.2gM VIP. The positions of molecular weight standards are shown on the left side of the figure. The autoradiograph is represenlive of two experiments. density in cortex and lower density in hypothalamus and cerebellum, was similar to that previously described in the rat brain [16, 22]. The distribution and density of PACAP binding sites were different from those of VIP binding sites. High densities of PACAP binding sites were present in all the central nervous system regions examined, particularly hypothalamus and brainstem and they were 8 17 times more dense than those of VIR This finding is in accordance with that of Tatsuno et al. [21] which demonstrated five times greater binding of [125I]PACAP than [12sI]VIP in rat astrocytes. The difference in distribution and densities of PACAP and VIP receptors suggests that the two peptides may have different biological significance. We observed that the concentration of immunoreactive PACAP38 in the human brain was less than 7 pmol/g wet weight (unpublished data), much less (approximately one tenth) than previously reported for VIP immunoreactivity [6, 8, 1 1]. The high affinity of PACAP and VIP receptors for PACAP means that even if PACAP were present together with 10-fold excess VIR PACAP would still compete successfully for binding sites. However, it is possible that VIP and PACAP are anatomically separated, i.e. confined to separate local areas, and thus do not interact. VIP has
been reported to regulate cortical blood h o w [5] and cortical energy metabolism [13], relate to the sleep mechanism [14] and protect neurons l¥om the envelope protein of HIV [2]. The biological significance of PACAP in the human central nervous system remains to be clarified, although there is a report that PACAP also prevented murine neuronal death induced by the gp 120 protein of HIV virus [20]. These two peptides probably act not only on postsynaptic binding sites but also, as local hormones, on binding sites on neighbouring neurons since it is thought that neuropeptides diffuse widely in comparison with the non-peptide neurotransmitters (e.g. acetylcholine), the latter being confined closely to their release sites. Thus PACAP may be able to modulate neurons with VIP receptors in addition to the action through its own receptor. The present study shows that the densities of [125I]PACAP binding sites are much higher than those of [L~q]VIE Both binding sites have a higher affinity for the N-terminal sequence of PACAR with [L~sI]PACAP binding sites being totally specific for the N-terminal sequence of PACAP and not significantly influenced by VIR PACAP crossreacts with VIP binding sites with a higher affinity than VIE suggesting that in the human central nervous system, PACAP may act not only through its receptor but also through VIP receptors. wishes to thank the Nuffield Foundation and M R C for their support. D.M.S.
Abbreviations
VIE vasoactive intestinal polypeptide; PACAP38, full 38 residue peptide of pituitary adenylate cyclase activating polypeptide; PACAP27, amidated first 27 residue derivative of PACAP38; ICso, the concentration of competing ligand that inhibits binding by 50%: G~, the guanine nucleotide regulatory protein which mediates the stimulation of adenylate cyclase. 1 Arimura, A., Katsuura, G., Gottschall, RE. and Dahl, RR:, Biological actions of a novel hypothalamic polypeptide with 38 residues, 71st Annual Meeting of the Endocrine Society Seattle WA, 1986, 263 pp. (abstract). 2 Brenneman, D.E., Westbrook, G.L., Fitzgerald, S.P., Ennist, D.L., Elkins, K.L, Ruff, M.R. and Pert, C.B., Neuronal cell killing by the envelope protein of HIV and its prevention by vasoactive intestinal peptide, Nature, 335 (1988) 639 642. 3 Buscail, L., Goulet, R, Cauvin, A., DeNeet\ P., Gossen, D., Arimura, A., Miyata, A., Coy, D.H., Robberecht, R and Christophe, J., Presence of highly selective receptors for PACAP (pituitary adenylate cyclase activating polypeptide) in membranes from the rat pancreatic acinar cell line AR4-2J, FEBS Lett., 262 (1990) 77-8 l. 4 Cauvin, A., Buscail, L., Goulet, E, DeNeef, R, Gossen, D., Arimura, A., Miyata, A., Coy, D.H., Robberecht, P. and
23
5
6
7
8
9
10
II
12
13
Christophe, J., The novel VIP-like hypothalamic polypeptide PACAP interacts with high affinity receptors in the human neuroblastoma cell line NB-OK, Peptides, 1 I (19901 773 777. Eckenstein, F. and Baughman, R,W., Two types of cholinergic innervation in cortex, one co-localised with vasoactive intestinal polypeptide, Nature, 309 (19841153 155. Fahrenkrug, J. and Emson, P.C., Characterization and regional distribution of peptides derived from the vasoactive intestinal peptide precursor in the normal human brain, J. Neurochem., 53 (19891 1142 1148. Frackcr. P.J. and Speck, J.C., Protein and cell membrane iodinations with a sparingly soluble chloroamide. 1,3.4,6,-tetrachloro3~, 6~, diphenyl-glycoluril, Biochem. Biophys, Res. Commun., 80 (1978)~49 S57. Ghatei, M.A., Bloom, S.R., t, angevin, H., McGregor, G.P., Lee, Y.C., Adrian. T.E., O'Shaughnessy, D.J., Blank, M.A. and Uttenthai, L.O., Regional distribution of bombesin and seven other regulatory peptides m the human brain, Brain Res., 293 (1984) 101 109. Gornall, A.G., Bardawill, C.,I. and David, M.M., Determination of serum proteins by means of the biuret reaction, J. Biol. Chem., 177 (1949) 751 766. Goitschall, P.E., Tatsuno, I., Miyata, A. and Arimura, A., Characterisation and distribution of binding sites for the fiypothalamic peptide, pituitary adenylate cyclase activating polypeptide, Endocrinology, 127 11990) 272 277. Jegou. S., .lavoy-Agid, F,, Delbende, C., Tranchand-Bunel, D., Coy, D.H., Agid, Y. and Vaudry, H., Regional distribution of vasoactive intestinal peptide in brains from normal and Parkinsonian subjects, Peptides, 9 (1988) 787 793. Lam, H-C., Takahashi, K., Ghatei, M.A., Kanse, S.M., Polak, J.M. and Bloom, S.R., Binding sites of a novel neuropeptide pituitary adenylate cyclase activating polypeptide in the rat brain, Eur. J. Biochem., 193(1990) 725 729. Magistretti. P.J., Morrison, J.H., Shoemaker, W.J., Sapin, V. and Bloom, F.E., Vasoactive intestinal polypepfide induces glycogenolysis in mouse cortical slices, a possible regulatory mechanism for the local control of energy metabolism. Proc. Natl. Acad. Sci. USA, 78 (19811 6535 6539.
14 MagistrettL RR., VIP neurons in the cerebral cortex, Trends Pfiarmacol. Sci., 11 (1990)250 254. 15 Miyata, A., Arimura, A., Dahl, R.R., Minamino. N.. Uehara, A., Jiang, L., Culler, M.D. and Coy, D.H., Isolation of a novel 38 residue hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells, Biochem. Biophys. Res. Commun., 164 (1989) 567 574. 16 Ogawa, N., Mizuno. S.. Mort, A., Nukina, I. and Yanaihara, N,, Properties and distribution of vasoactive intestinal polypeptide receptors in the rat brain, Peptides, 6 (19841 103 109. 17 Ohtaki, T.. Walanabe, T.. lshibashi. Y., Kitada, C., Tsuda, M., Gottschall, P.E., Arimura. A. and Eujino, M.. Molecular identification of receptor l\~r pituitary adenylate cyclase activating polypeptide. Biochem. Biophys. Res. Commun., 171 (1990) g38 844. 18 Shivers, B.D., Gores. T.J., Gottschall, P.E. and Arimura, A., Two high affinity binding sites for pituitary adenylate cyclase-actiwifing polypeptide have different tissue distributions. Endocrinology, 128 (1991) 3055 3065. 19 Suda, K., Smith, D.M., Ghaiei. M.A., Murphy. J K . and Bloom, S.R., Investigation and characterisation of receptors t\~r PACAP (pituitary adenylate cyclase activating polypepiidel in human brain by radioligand binding and chemical cross-linking, J. ('lin. Endocr. Metab., 72 ( 1991 ) 958-964. 20 Tatsuno. I., Gottschall, RE.. Koves. K. and Arimura. A.. Demonstration of specific binding for pituitary adenylate cyclase activating polypeptide (PACAP) in rat astrocytcs, Biochem. Biophys. Res. Commun., 168 (19901 1027-1033. 21 Tatsuno, I., Goltschall. P.E. and Arimura. A., Specific binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) in rat cultured astrocytes: molecular identification and interaction with vasoactive intestinal peptide (V1PI, Peptides, 12 (19911 617 621. 22 Taylor, D.P. and Pert, C.B., Vasoactive intestinal polypeptide: specific binding to rat brain membranes, Proc, Natl. Acad. Sci. USA, 76 (1979) 660 664.
19
NSL 08448
Investigation of the interaction of VIP binding sites with VIP and PACAP in human brain Katsuyuki Suda, David M. Smith, M o h a m m a d A. Ghatei and Stephen R. Bloom Department o/Medicine, Royal Postgraduate Medical School. Hammcrsmith Hospital. Lomhm ( UK; (Received 8 October 1991: Revised version received 29 November 1991: Accepted 3 December 1991)
Key words." PACAP receptor: Vasoactive intestinal polypeptide receptor: Human brain: Chemical cross-linking We have compared the binding of ['>l]vasoactive intestinal polypcptide (VlP) to human brain membranes with that of [t>I]PACAP27. [*-'q]VlP was displaced by PACAP27, VIP and two synthetic peptides, peptide-1 (N-terminal PACAP27/C-terminal VIP) and peptide-2 (N-terminal VIP/Cterminal PACAP27), but the IC~0 of PACAP27 and peptide-I were 10 20 times lower than those of VIP and peptide-2. [':51]PACAP27 was readily displaced by PACAP27 and peptide-l, with an ICs, of less than 1 nM, but poorly by VIP and peptide-2. Chemical cross-linking revealed that both labels were bound to polypeptides of M~ 66,000 and M~ 50,000. The resnlts indicate that in human brain membranes both binding sites have a higher affinity to the N-terminal sequence of PACAP27, and VIP binding sites prefer PACAP27 to VIP itselF.
Pituitary adenylate cyclase activating polypeptide (PACAP) is a 38 amino acid peptide (PACAP38) of ovine hypothalamic origin. It was originally isolated on the basis of its stimulation of adenylate cyclase in cultured rat anterior pituitary cells [15]. Recently, PACAP receptors have been identified in some cultured cell lines [3, 4], rat astrocytes [20], rat tissues [10, 12], bovine brain [17] and human brain [19]. All PACAP receptors except those in rat lung were highly specific for PACAP and of very low affinity for vasoactive intestinal polypeptide (VIP) despite the structural similarity with VIE On the other hand, in a recent report, bovine brain VIP receptors have been shown to crossreact with PACAP [17]. Both PACAP and VIP receptors are thought to be linked via G, to stimulate adenylate cyclase [3], and PACAP and VIP have several pharmacological effects in common, e.g. vasodilation [1]. It is therefore possible that PACAP can act both through PACAP receptors and VIP receptors. Therefore, in human brain membranes, we examined the interaction with VIP and PACAP binding sites of VIP, PACAP and two synthetic peptides which were N-terminal VIP/C-terminal PACAP27 and N-terminal PACAP27/C-terminal VIE PACAP1-27NH2 (PACAP27), the N-terminal amidated 27 residue derivative of PACAP1-38NH~ (PACAP38), was synthesized by Peptide Products (UK). Correspondence: S.R. Bloom, Francis Fraser 2 Lab, Dept. of Medicine, R.RM.S., Hammersmith Hospital, Du Cane Road, London, W12, ONN, UK.
VIP was synthesized by IAF Biochem (UK). Peptide-I consisted of PACAP(I-14) in the N-terminus and VIP(15-28) in the C-terminus. Peptide-2 consisted of VIP(I-14) in the N- terminus and PACAP(15-27) in the C-terminus. Both peptide-I and peptide-2 were synthesized by the Palliard Chemical Co Ltd (UK) (see Table I). VIP and PACAP27 were iodinated by the iodogen (1,3,4,6-tetrachloro-3c~-6c~-diphenylglycoluril, Pierce, Rockford. Illinois, USA) method [7], as previously described [19]. Brain membranes were prepared from five regions of human brain (cortex, cerebellum, basal ganglia, hypothalamus and brainstem), as previously described [19]. The postmortem samples were obtained from subjects who were without history of neurological or psychiatric disorder by Dr. J.K. Murphy (York District Hospital, York. UK) and were stored at -70°C. Membrane protein concentration [3 6 mg/ml] was measured by the Biuret method [9]. For the VIP binding assay, the membranes (100 jag) were incubated at 34°C for 60 min with 0.2 nM [1251]VIP (500 cps) in 25 mM Tris-HC1 buffer pH 7.4 containing 1% BSA. 4 mM MgCI2, 2 mM EDTA and 0.25 mg/ml bacitracin. In the PACAP binding assay, the membranes (60 jag) were incubated at 37°C for 90 min with 0.25 nM [1251]PACAP27 (500 cps) in 50 mM Tris-HCl buffer pH 7.6 containing 3% BSA. The final assay volume was 0.5 ml. Non-specific binding was determined in the presence of 0.2 jaM PACAP27 or 0.2 jaM VIR The receptorlabelled peptide complex was separated from free la-
20
TAB[.I 1 C O M P O S I T I O N O t : T H E A M I N O A C I D S E Q U E N C E O | : PACAP27. VIP. P E P T 1 D E - I A N D P E P T I D E - 2
h/pVlP
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Peptide-I Peptide-2
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D
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Sequence of P A C A P 2 7 is underlined: o. ovine: h. h u m a n ; p. porcine.
belled peptide by centrifugation at 15,000 × g for 2 min. The pellets were washed at 4°C with 0.5 ml of the binding assay buffer and counted in a 7" counter. FPLC analysis of PACAP and VIP label in the supernatants using a Pep RPC C~s column (Pharmacia, Milton Keynes, Bucks UK) eluted with a water/acetonitrile gradient in 0.1% TFA of 20-45% acetonitrile in 30 min showed that less than 10% of the label was degraded during the assay (results not shown). Specific binding was calculated as total binding minus non-specific binding. Chemical cross-linking of [~25I]VIP and [~2SI]PACAP27 to human brain membranes was performed by incubating brain membranes (100 200 ~g) with 1 nM [~25I]PACAP27 or 2 nM [~25I]VIP in the presence and the absence of 0.2 ktM PACAP and 0.2 ~M VIE Membranes were centrifuged at 15,000 × g for 2 rain and washed twice at 4°C in 0.5 ml of binding assay buffer. After resuspension of pellets in 50 mM Tris-HC1 pH 7.4 (500 pl), disuccinimidyl suberate (Pierce) dissolved in dimethyl sulfoxide was added at a final concentration of I mM. After 30-min incubation at room temperature, samples were centrifuged at 15,000 x g for 2 min and washed twice with 0.5 ml of Tris-HC1 buffer. Pellets were resuspended in 40 ~1 of Laemmli sample buffer, boiled for 2 min, and then subjected to SDS polyacrylamide (7,5%) gel electrophoresis [19]. Autoradiographs were obtained at -70°C for 1 3 weeks. The association of [~2Sl]VIP and [~25I]PACAP27 to human brain membranes were time and temperature sensitive. The specific binding of [t25I]PACAP27 increased more rapidly at 37°C than at 4°C and reached a steady state after 80 min [19]. The time course of [~25I]VIP showed a similar pattern reaching equilibrium at 34°C after 60 min (see Fig. 1.). The maximal specific binding of [12sI]VIP and [~2Sl]PACAP27 was 7% and 19% of total counts added and the non-specific binding of [~2q]V1P and [J25I]PACAP was approximately 30% and 15% of this maximal total binding. The binding characteristics of receptors from the five regions of the brain were very similar and interchangeable in all these experiments [19]. The displacement curves of [12q]VIP and [~251]PACAP27 in the presence of increasing concentrations of PACAP27, VIR peptide-I (PACAP27/V1P) and
peptide-2 (VIP/PACAP27) are shown in Fig. 2 and Fig. 3. [125I]VIP binding was inhibited by all four peptides. The ICs0 of PACAP27 (0.08 + 0.02 nM, mean + S.E.M., n=4) was similar to that of peptide 1 (0,07 _+ 0.01 nM). The ICs0 of VIP (1.50 + 0.49 riM) and peptide-2 (0.70 _+ 0,09 nM) were 10-20 times higher than that of PACAP27 and peptide-1. On the other hand, both PACAP27 and peptide-1 were able to displace [~25I]PACAP27 with high affinity. The ICs0 of PACAP27 and peptide-1 were 0.79 _+ 0.34 nM and 0.61 _+ 0.14 nM (mean _+ S.E.M., n=3), respectively, but VIP and peptide-2 displaced very little label with a 1,000-fold higher ICs0 than PACAP27 and peptide- 1. The distribution of [~25I]VIP binding sites and [~25I]PACAP binding sites are shown in Fig. 4. [~25I]VIP binding was highest in cortex, whilst [J25I]PACAP binding was highest in hypothalamus and brainstem. The density of [~25I]VIP binding was 8-17 times less than that of [~2sI]PACAP binding in all five regions. Chemical cross-linking studies revealed that both PACAP27 and VIP labels were bound to polypeptide chains of M,. 66,000 (69,000-3,000 mot. wt. for both
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~I]PACAP were not observed in the presence of 0.2/aM PACAP27, but they were unaffected in the presence of 0.2/aM VIP (Fig. 5). We have previously demonstrated the existence of PACAP binding sites in human brain [19]. The present study was performed to establish the binding portion of the PACAP27 and VIP molecules to their receptors and the interaction of both peptides with their receptors. Displacement of [~25I]VIP by peptide-I (PACAP27/ VIP), which has the N-terminal sequence of PACAP27,
c"
-8
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Fig. 2. Inhibition of [t>l]PACAP27 (0.25 nM) binding in human brain membranes by increasing concentrations of PACAP27 ( ) , V1P (A), peptide-1 ( t ) and peptide-2 (,). Values are expressed as percentage of specific binding and mean +_ S.E.M. of three experiments.
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Fig. 4. Distribution of[P>I]PACAP27 (0.25 nMI and [t:'l [VI P (I).2 nM) binding sites to human brain membranes. Binding as~,ay,, x~erc pcrformed in triplicate with membranes IYom each brahl tissue. ('(), cortex; BG. Basal ganglia: HY, hypolhalamus: BS. bl'mnstenl: and CE. cerebellum Values are expressed as mean * S.tLM. of four cxpcriII/ellts.
and PACAP27 were similar. Peptide-2 (VlP/PACAP27), with the C-terminal sequence of PACAP27, and VIP were both of lower affinity, suggesting these peplides depend for binding on the N-terminal sequences. Surprisingly, the N-terminal sequence of PACAP27 had a 10-20 times higher affinity to the binding sites of VIP than that of VIP itself. Peptide-I (PACAP27/VlP) displaced [~:~I]PACAP27 with ahnost the same affinity as PACAP27, but peptide-2 lVIPiPACAP27) displaced [L'5I]PACAP2? poorly, as did VIP. This indicates that PACAP binding sites are very specilic for the N-terminal sequence of PACAP. Taken together these results may indicate the presence of two types of PACAP receptors in human brain membranes, one receptor type being specific for PACAP and another able to bind both PACAP and VIP but, showing a greater altinit? l\)r PACAP. These two binding sites may correspond to the type I (specific PACAP) and type 11 (binding both PACAP and VIP) described by Shivers et al. [1~1 ira rat tissues, except that since they used o n b [~"I]PACAP they were only able to describe type I receptors m the brain. In this study using [L"I]VIP we have demonstrated the presence of type 11 receptors in the brain. We were unable to demonstrate by these methods receptors in human brain membranes which were specitic t\)r VI P and did not bind PACAR Chemical cross-linking studies showed that VIP binding sites were of the same reel. wt. as PACAP binding sites. Therefore it is possible that VIP binding sites might be a subset of PA('AP binding sitcs in human brain. The distribution of VIP binding sites, with highest
1
VIP
2
3
4
5
6
*
Fig. 5. Autoradiograph illustrating cross-linking of [~>I]PACAP27 (lane 1 3) and [~'sI]VIP(lane 4-6) to human brain cortex membranes. Cross-linking was performed as described in the text using human cortex membranes in the presence and absence of 0.2 ruM PACAP27 or 0.2gM VIP. The positions of molecular weight standards are shown on the left side of the figure. The autoradiograph is represenlive of two experiments. density in cortex and lower density in hypothalamus and cerebellum, was similar to that previously described in the rat brain [16, 22]. The distribution and density of PACAP binding sites were different from those of VIP binding sites. High densities of PACAP binding sites were present in all the central nervous system regions examined, particularly hypothalamus and brainstem and they were 8 17 times more dense than those of VIR This finding is in accordance with that of Tatsuno et al. [21] which demonstrated five times greater binding of [125I]PACAP than [12sI]VIP in rat astrocytes. The difference in distribution and densities of PACAP and VIP receptors suggests that the two peptides may have different biological significance. We observed that the concentration of immunoreactive PACAP38 in the human brain was less than 7 pmol/g wet weight (unpublished data), much less (approximately one tenth) than previously reported for VIP immunoreactivity [6, 8, 1 1]. The high affinity of PACAP and VIP receptors for PACAP means that even if PACAP were present together with 10-fold excess VIR PACAP would still compete successfully for binding sites. However, it is possible that VIP and PACAP are anatomically separated, i.e. confined to separate local areas, and thus do not interact. VIP has
been reported to regulate cortical blood h o w [5] and cortical energy metabolism [13], relate to the sleep mechanism [14] and protect neurons l¥om the envelope protein of HIV [2]. The biological significance of PACAP in the human central nervous system remains to be clarified, although there is a report that PACAP also prevented murine neuronal death induced by the gp 120 protein of HIV virus [20]. These two peptides probably act not only on postsynaptic binding sites but also, as local hormones, on binding sites on neighbouring neurons since it is thought that neuropeptides diffuse widely in comparison with the non-peptide neurotransmitters (e.g. acetylcholine), the latter being confined closely to their release sites. Thus PACAP may be able to modulate neurons with VIP receptors in addition to the action through its own receptor. The present study shows that the densities of [125I]PACAP binding sites are much higher than those of [L~q]VIE Both binding sites have a higher affinity for the N-terminal sequence of PACAR with [L~sI]PACAP binding sites being totally specific for the N-terminal sequence of PACAP and not significantly influenced by VIR PACAP crossreacts with VIP binding sites with a higher affinity than VIE suggesting that in the human central nervous system, PACAP may act not only through its receptor but also through VIP receptors. wishes to thank the Nuffield Foundation and M R C for their support. D.M.S.
Abbreviations
VIE vasoactive intestinal polypeptide; PACAP38, full 38 residue peptide of pituitary adenylate cyclase activating polypeptide; PACAP27, amidated first 27 residue derivative of PACAP38; ICso, the concentration of competing ligand that inhibits binding by 50%: G~, the guanine nucleotide regulatory protein which mediates the stimulation of adenylate cyclase. 1 Arimura, A., Katsuura, G., Gottschall, RE. and Dahl, RR:, Biological actions of a novel hypothalamic polypeptide with 38 residues, 71st Annual Meeting of the Endocrine Society Seattle WA, 1986, 263 pp. (abstract). 2 Brenneman, D.E., Westbrook, G.L., Fitzgerald, S.P., Ennist, D.L., Elkins, K.L, Ruff, M.R. and Pert, C.B., Neuronal cell killing by the envelope protein of HIV and its prevention by vasoactive intestinal peptide, Nature, 335 (1988) 639 642. 3 Buscail, L., Goulet, R, Cauvin, A., DeNeet\ P., Gossen, D., Arimura, A., Miyata, A., Coy, D.H., Robberecht, R and Christophe, J., Presence of highly selective receptors for PACAP (pituitary adenylate cyclase activating polypeptide) in membranes from the rat pancreatic acinar cell line AR4-2J, FEBS Lett., 262 (1990) 77-8 l. 4 Cauvin, A., Buscail, L., Goulet, E, DeNeef, R, Gossen, D., Arimura, A., Miyata, A., Coy, D.H., Robberecht, P. and
23
5
6
7
8
9
10
II
12
13
Christophe, J., The novel VIP-like hypothalamic polypeptide PACAP interacts with high affinity receptors in the human neuroblastoma cell line NB-OK, Peptides, 1 I (19901 773 777. Eckenstein, F. and Baughman, R,W., Two types of cholinergic innervation in cortex, one co-localised with vasoactive intestinal polypeptide, Nature, 309 (19841153 155. Fahrenkrug, J. and Emson, P.C., Characterization and regional distribution of peptides derived from the vasoactive intestinal peptide precursor in the normal human brain, J. Neurochem., 53 (19891 1142 1148. Frackcr. P.J. and Speck, J.C., Protein and cell membrane iodinations with a sparingly soluble chloroamide. 1,3.4,6,-tetrachloro3~, 6~, diphenyl-glycoluril, Biochem. Biophys, Res. Commun., 80 (1978)~49 S57. Ghatei, M.A., Bloom, S.R., t, angevin, H., McGregor, G.P., Lee, Y.C., Adrian. T.E., O'Shaughnessy, D.J., Blank, M.A. and Uttenthai, L.O., Regional distribution of bombesin and seven other regulatory peptides m the human brain, Brain Res., 293 (1984) 101 109. Gornall, A.G., Bardawill, C.,I. and David, M.M., Determination of serum proteins by means of the biuret reaction, J. Biol. Chem., 177 (1949) 751 766. Goitschall, P.E., Tatsuno, I., Miyata, A. and Arimura, A., Characterisation and distribution of binding sites for the fiypothalamic peptide, pituitary adenylate cyclase activating polypeptide, Endocrinology, 127 11990) 272 277. Jegou. S., .lavoy-Agid, F,, Delbende, C., Tranchand-Bunel, D., Coy, D.H., Agid, Y. and Vaudry, H., Regional distribution of vasoactive intestinal peptide in brains from normal and Parkinsonian subjects, Peptides, 9 (1988) 787 793. Lam, H-C., Takahashi, K., Ghatei, M.A., Kanse, S.M., Polak, J.M. and Bloom, S.R., Binding sites of a novel neuropeptide pituitary adenylate cyclase activating polypeptide in the rat brain, Eur. J. Biochem., 193(1990) 725 729. Magistretti. P.J., Morrison, J.H., Shoemaker, W.J., Sapin, V. and Bloom, F.E., Vasoactive intestinal polypepfide induces glycogenolysis in mouse cortical slices, a possible regulatory mechanism for the local control of energy metabolism. Proc. Natl. Acad. Sci. USA, 78 (19811 6535 6539.
14 MagistrettL RR., VIP neurons in the cerebral cortex, Trends Pfiarmacol. Sci., 11 (1990)250 254. 15 Miyata, A., Arimura, A., Dahl, R.R., Minamino. N.. Uehara, A., Jiang, L., Culler, M.D. and Coy, D.H., Isolation of a novel 38 residue hypothalamic polypeptide which stimulates adenylate cyclase in pituitary cells, Biochem. Biophys. Res. Commun., 164 (1989) 567 574. 16 Ogawa, N., Mizuno. S.. Mort, A., Nukina, I. and Yanaihara, N,, Properties and distribution of vasoactive intestinal polypeptide receptors in the rat brain, Peptides, 6 (19841 103 109. 17 Ohtaki, T.. Walanabe, T.. lshibashi. Y., Kitada, C., Tsuda, M., Gottschall, P.E., Arimura. A. and Eujino, M.. Molecular identification of receptor l\~r pituitary adenylate cyclase activating polypeptide. Biochem. Biophys. Res. Commun., 171 (1990) g38 844. 18 Shivers, B.D., Gores. T.J., Gottschall, P.E. and Arimura, A., Two high affinity binding sites for pituitary adenylate cyclase-actiwifing polypeptide have different tissue distributions. Endocrinology, 128 (1991) 3055 3065. 19 Suda, K., Smith, D.M., Ghaiei. M.A., Murphy. J K . and Bloom, S.R., Investigation and characterisation of receptors t\~r PACAP (pituitary adenylate cyclase activating polypepiidel in human brain by radioligand binding and chemical cross-linking, J. ('lin. Endocr. Metab., 72 ( 1991 ) 958-964. 20 Tatsuno. I., Gottschall, RE.. Koves. K. and Arimura. A.. Demonstration of specific binding for pituitary adenylate cyclase activating polypeptide (PACAP) in rat astrocytcs, Biochem. Biophys. Res. Commun., 168 (19901 1027-1033. 21 Tatsuno, I., Goltschall. P.E. and Arimura. A., Specific binding sites for pituitary adenylate cyclase activating polypeptide (PACAP) in rat cultured astrocytes: molecular identification and interaction with vasoactive intestinal peptide (V1PI, Peptides, 12 (19911 617 621. 22 Taylor, D.P. and Pert, C.B., Vasoactive intestinal polypeptide: specific binding to rat brain membranes, Proc, Natl. Acad. Sci. USA, 76 (1979) 660 664.
