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Properties of the VIP-PACAP type II receptor stably expressed in CHO cells
ELSEVIER
Regulatory Peptides 54 (1994) 397-407
Properties of the VIP-PACAP type II receptor stably expressed in CHO cells Ernesto Ciccarelli a, Jean-Pierre Vilardaga b, Philippe De Neef a, Emmanuel Di Paolo Magali Waelbroeck a, Alex Bollen b, Patrick Robberecht a,,
a,b,
aDepartment of Biochemistry and Nutrition, Faculty of Medicine, Universit~ Libre de Bruxelles, 808 Route de Lennik, B-1070 Brussels, Belgium bDepartment of Applied Genetics, Faculty of Sciences, Universitd Libre de Bruxelles, Brussels, Belgium
Received 25 May 1994; revised version received and accepted 8 August 1994
Abstract
The VIP receptor cloned from rat lung (VIP 1 receptor from the group of the PACAP-VIP type II receptors) was inserted into a mammalian expression vector and stably transfected into Chinese hamster ovary cells (CHO). Two clones were selected, expressing respectively a high (850 + 50 fmol/mg protein, for clone 3) and a low (100 + 30 fmol/mg protein for clone 16) number of receptors. Both clones had the same apparent K d value of binding for VIP and related peptides. The receptor expressed had the same binding properties as the natural VIP receptor, judged from the relative potency of VIP and PACAP analogues and fragments. The ECso value of adenylate cyclase activation were 3 to 10 fold lower in clone 3 than in 16. The values observed in clone 16 were closer to the binding K d values. The differences between the two clones were explained by the existence of spare receptors in clone 3, since: (a) the relative efficacy of some fragments were lower in clone 16 than in clone 3; (b) pretreatment of the cells with VIP reduced the number of receptors in both clones and increased the ECso value for VIP in clone 3 but decreased peptide efficacy in clone 16 without significant change of the ECso value. Keywords." VIP; PACAP; PACAP type II receptor; Chinese hamster ovary cell; Transfection
* Corresponding author. Fax: + 32 2 5556230. Abbreviations: PACAP, pituitary adenylate cyclase activating peptide; VIP, vasoactive intestinal peptide; CHO, Chinese hamster ovary cells; PHI, peptide histidine isoleucinamide-(1-27), PHI-GIy, peptide histidine isoleucinamide-(1-27)-glycine;PHV, peptide histidine valine-(1-42); Kd, equilibrium dissociation constant of the radioligand; ECso, concentration producing half-maximal stimulation of ~t . adenylate cyclase; Ki, concentration of antagonist that requires a 2-fold increase of agonist concentration for observing the same biological effect. 0167-0115/94/$7.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 0 1 1 5 ( 9 4 ) 0 0 0 7 6 - X
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E. Ciccarelli et al. / Regulatory Peptides 54 (1994) 397-407
1. Introduction
2. Material and methods
VIP/PHI/PACAP receptors have been subdivided into two groups: group I receptors are highly selective for PACAP and group II receptors do not discriminate PACAP from VIP. Three receptors were cloned recently and belong to the seven helices receptor family: (1) two VIP receptors (VIP 1 receptors [1] and VIP 2 receptors [2]), belonging to the PACAP type II receptors group [ 3 ] do not discriminate between VIP and PACAP. The VIP 1 receptor was cloned in rat [1] and human [4], the VIP2 receptor in rat tissue only. (2) A PACAP receptor [5-7] corresponding to the PACAP type I receptor [3] highly selective for PACAP was cloned in rat tissues. This receptor, however, may exist in several forms differing in the sequence of the third intracellular loop [ 5 ]. Despite the rapid progress in this field, several questions remain unsolved. For instance: is the socalled 'helodermin-preferring receptor' described on human cell lines and related to the PACAP type II receptor [8-9] a third VIP receptor? Are the low affinity binding sites described in most tissues original molecular entities or low affinity states of the already cloned receptors? Is the molecular heterogeneity due to a variable degree of receptor glycosylation [10] responsible for a functional heterogeneity? Are the discrepancies which are often observed between binding data and functional studies due to receptor heterogeneity or to an amplification process [11]? Since receptors of different subtypes may coexist in tissues and cell lines, most of these questions were previously difficult to solve. The Chinese hamster ovary cells (CHO cells) do not express constitutively any receptor for the VIP/PHI/PACAP peptides family. CHO clones transfected with a defined receptor type and stably expressing different receptor densities are useful tools to study these questions. We used this experimental model to study the type II receptor (VIP~) from rat.
2.1. Construction of the expression plasmid, transfection, selection and expression in CHO cells The DNA coding for the rat VIP/PACAP receptor was excised from the plasmid pCDM8 kindly provided by Prof. Nagata, Osaka, (Japan) by digestion with Hind III and Xho I, and cloned into the Hind III and Xho I sites of the mammalian expression Vector pNIV187, a derivative of pRc/RSV (Invitrogen) that contains the selectable neomycin phospho-transferase gene, and an expression cassette for dihydrofolate reductase (dhfr), as described in Connors et al. [12]. The resulting recombinant plasmid was transfected into the CHO cell line DG44 [13] by electroporation using a gene pulser. Approximately 107 cells were preincubated on ice for 30 min with 25/~g DNA in 0.8 ml 7 mM sodium phosphate buffer (pH 7.4) containing 272 mM sucrose and 1 mM MgC12. Electroporation was performed at 600 V and 3 /~F. After electroporation, cells were kept on ice for 10 min, added to 10 ml of culture medium and harvested in 96 well plates. Cells were maintained in or-minimal essential medium (~MEM T), supplemented with 10~o FCS, 2 mM L-gtutamine, 100 ~tg/ml penicillin and 100 /~g/ml streptomycin with an atmosphere of 95 ~o air, 5 j°o CO2 at 37°C. 48 h after transfection the G148 resistant cells were selected by the addition of geneticin to a final concentration of 450/~g/ml. Selection was done in 96 well plates with 2000-5000 ceils per well. To produce sufficient amounts of cells for receptor characterization, the clones were then grown to confluency in six-well plates of 35 mm diameter. The clones used in the present study thereafter referred to as CHO-VIPr-c3 (clone 3) and CHOVIPr-cl6 (clone 16) were obtained without amplification and were selected on the basis of a high and low binding capacity of 125I-VIP, respectively (see Results). Geneticin at 0.5 mg/ml was maintained in the culture medium of the stock culture. Subcultures
E. Ciccarelli et al. / Regulatory Peptides 54 (1994) 397-407
prepared for membrane purification were done in a medium without geneticin.
2.2. Membrane preparation, receptor identification and adenylate cyclase activity Cells were detached with a rubber policeman and pelleted by low speed centrifugation, the supernatant was discarded and the cells lysed in 1 mM NaHCO 3 solution and immediate freezing in liquid nitrogen. After thawing, the lysate was first centrifuged at 800 x g for 10 min and the supernatant further centrifuged at 20,000 x g for 10 min. The pellet, resuspended in 1 mM NaHCO3 was used immediately as a crude membrane preparation. [ 125I]VIP was labelled by the chloramin T method as described [14] and purified by adsorption on a cellulose column eluted with discontinuous gradient of BSA. Tracer specific binding radioactivity was 1.0 mCi/mmol. Binding of the tracer to the membranes was performed as described [15]. Saturation curves were done with increasing concentrations of tracer and competition curves with a fixed tracer concentration (equal to one fourth of the value of tracer Ka) and increasing concentrations of unlabelled peptides. In all cases, the non specific binding was defined as the residual binding in the presence of 1/~M VIP. Binding was performed at 37°C in a 20 mM Tris-maleate, 2 mM MgC12, 0.1 mg/ml bacitracin, 1~o BSA (pH 7.4) buffer, 3 to 30 /zg protein were used per assay. The bound was separated from the free radioactivity by filtration through glass fiber filters G F / C presoaked for 24 h in 0.1 ~o polyethyleneimine and rinsed three times with a 20 mM (pH 7.4) phosphate buffer containing 1 ~o BSA. Adenylate cyclase activity was determined by the Salomon et al. procedure [ 16]. Membrane protein (3 to 15 #g) was incubated in a total volume of 60 #1 containing 0.5 mM [~-32p]ATP, 10 ktM GTP, 5 mM MgC12, 0.5 mM EGTA, 1 mM cAMP, 1 mM theophylline, 10 mM phosphoenolpyruvate, 30 /zg/ml pyruvate kinase and 30 mM Tris-HC1 at a final pH
399
of 7.5. The reaction was initiated by adding membranes and was terminated after a 12 min incubation at 37°C by adding 0.5 ml cAMP and 20,000 cpm [8-3H]cAMP. cAMP was separated from ATP by two successive chromatographies on Dowex 50-W X8 and neutral alumina.
2.3. Pretreatment of the cells Pretreatment of the cells with VIP was done on confluent cells. Attached cells were then washed twice with the culture medium without foetal calf serum, then harvested with a rubber policeman and centrifuged at a low speed. The pellet was washed once and finally the cells were lysed with 1 mM NaHCO 3 and immediate freezing in liquid nitrogen. The lysate was stored at -80°C until membrane preparation.
2.4. Materials Restriction endonuclease, T 4 DNA ligase, Escherichia coli DNA polymerase I (Klenow) were purchased from Amersham, Boehringer-Mannheim, New England Biolab and Gibco-BRL, respectively. Neomycin (Geneticin G416), cell culture medium ( ~ M E M + ) , L-glutamine, penicillin, streptomycin and foetal calf serum (FCS) were from GIBCOBRL. All the peptides were synthesized in the Laboratory by A. Vandermeers, M.-C. Vandermeers-Piret and P. Gourlet by the solid phase methodology.
3. Results
3.1. Binding kinetics 125I-VIP bound rapidly and reversibly to the membranes from both clones: steady state was achieved in both cases after 10 min incubation at 37°C and maintained for at least 30 min (data not shown). Dissociation was studied in clone 3 only and was induced by addition of 10 -6 M VIP 15 min after
400
E. Ciccarelli et al. / Regulator), Peptides 54 (1994) 397-407
exposure of the membranes to the tracer. A rapid dissociation of 70~o of the bound tracer occurred with a t~ of 5 + 1 min (mean + S.E.M. of 3 determinations) and was reduced to 2 + 1 min in the presence of 10-5 M GTP. The second, slowly dissociating component represented 30~o of the labelled sites. It was not further investigated (data not shown).
3.2. Scatchard analysis of the binding to clones 3 and 16 Saturation curves performed with increasing concentrations of 125I-VIP were transformed as Scatchard plots and were compatible with the presence of a single class of binding sites (Fig. 1). The density of binding sites (Bmax) in clones 3 and 6 were
o
0.4
0.3
o
3
tic kt_
i
0.2-
O. I-
CHO-VIPR-c16
BOUND
850 + 50 and 100 _+30 fmol/mg protein, respectively ( m e a n + S . E . M . of 4 determinations). The K d were not different in both clones: 0.5 + 0.1 and 0.3 + 0.1 nM (mean + S.E.M. of 4 determinations).
3.3. Selectivity of the binding sites labelled with l:51VIP The selectivity of the binding sites was tested by inhibition of tracer binding in the presence of increasing concentrations of unlabelled peptides. Inhibition curves were identical in both clones. All the curves were compatible with the presence of a single class of binding sites. Tracer binding was inhibited with the same potency (K d 2 nM) by VIP, PACAP-27 and PACAP-38; PHI, but also PHI-Gly and PHV (data not shown), were 5-fold less potent; helodermin and secretin were 15- and 125-fold less potent than VIP; glucagon was inactive (Table 1 and Fig. 2). We documented the importance of His i and the N-terminal portion of the peptides for receptor recognition by studying the capacity of acetyl-His lVIP and acetyl-HisLPACAP-27, PheLVIP and Phe1-PACAP-27, D-Hisa-VIP as well as VIP and PACAP fragments to inhibit tracer binding. Peptide potency was reduced in all cases (Table 1, Fig. 2). The same modification, substitution, or deletion made respectively on VIP or PACAP affected differently the peptide potency: [Phe ~]VIP was 10-fold more potent than [Phet]PACAP-27, PACAP(2-27) was 4-fold more potent than VIP(2-27) but 2-fold less potent than PACAP(2-38).
3.4. Coupling of the receptors to adenylate cyclase
(fmoles)
Fig. 1. Scatchard analysis of saturation curves obtained by incubating 25 #g of membrane protein from clone 3 and clone 16 in the presence of increasing concentrations of [ ~25I]VIP in a total volume of 120 #1. Non-specific binding was obtained in the presence of 1 MM VIP. One representative of 4.
Basal as well as the G p p [ N H ] p , NaF, forskolin, GTP, and VIP stimulated adenylate cyclase activities were significantly higher in clone 3 than in clone 16 (Table 2). In both clones, the dose-response curves of enzyme stimulation by the peptides developed on two logarithms. The ECso values were 2.5- to 10-fold lower in clone 3 than in clone 16. The ECso values
401
E. Ciccarelli et aL / Regulatory Peptides 54 (1994) 397-407 Table 1 Characteristics of the receptor expressed in CHO-VIP R-c3 and VIP R-cl6 (clones 3 and 16) Peptides binding
Kd
Adenylate cyclase activation Clone 3
VIP PACAP-27 PACAP-38 PHI Helodermin Secretin Acetyl-His
2+0.3 2_+0.3 2_+0.2 10 + 1.0 30 -+ 5 250 + 15 15 _+3 8 _+3 30+5 300 _+20 200 _+30 200 + 50 3000 -+ 500 1500 + 500 30 + 10 2000 + 500 800 + 300 80 _+20 800 _+100 800 + 200
Clone 16
ECs0 value
Eft
ECs0 value
Eff
0.4+0.1 0.4_+0.1 0.4_+0.1 0.8 + 0.5 1 -+0.3 40 + 10 0.8 _+0.5 0.6 + 0.3 6+2 20 + 4 20 + 6 15 + 3 200 + 60 400 + 50 5+3 200 -+ 100 200 _+50 50 _+10 200 _+50 300 + 50
1.0 1,1 + 0.1 0,9+0.1 1,2 + 0.2 0,8 -+0.2 0,9 + 0.3 0.9 + 0.2 1.1 + 0.2 0.8-+0.3 0.8 + 0.3 0.7 + 0.4 0.9 + 0.2 0.2* + 0.1 0.1" _+0.1 0.9 + 0.3 0.5* + 0;2 0.2* _+0.1 0.8 + 0.3 0.6* + 0.2 0.2* -+ 0.1
2+0.2 1 +0.3 1 +0.3 6 + 1.0 10 + 2 150 + 20 3+ 1 2+ 1 30+8 150 + 50 100 + 30 100 + 30 20 + 5 1000 _+200 200 _+80 200 _+100 -
1.0 0.9+0.1 1.0+ 0.1 1.1 + 0.2 0.9 + 0.3 0.8 + 0.3 1.0 + 0.2 1.1 + 0.2 0.9+0.2 0.7 + 0.3 0.7 + 0.3 0.6* + 0.2 0* 0* 0.9 + 0.2 0.1" _+0.2 1.0 + 0.2 0.1" + 0.2 -
The K d of binding and the ECho value of adenylate cyclase activation (both in nM) were derived from competition and dose-effect curves. The competition curves for the two cell lines were superimposable and the results were pooled and are expressed as the mean of at least four determinations. ECso value were the mean of at least three determinations, efficacy (Eft) refers to the ratio between the maximum effect of a peptide and the maximum effect of 1/~M VIP. * Indicated the values of efficacy significantly different from 1.0 (P< 0.5).
o f c l o n e 16 w e r e c l o s e t o t h e K d v a l u e s o f b i n d i n g ( T a b l e 1). I n b o t h c l o n e s , t h e p e p t i d e s t e s t e d ( e x c e p t V I P a n d P A C A P f r a g m e n t s ) w e r e as efficient as V I P ( s a m e m a x i m a l a c t i v i t y ) ( T a b l e 1). V I P ( 2 - 2 8 ) w a s as efficient as V I P in c l o n e 3 b u t w a s a p a r t i a l a g o n i s t in c l o n e 16 ( m a x i m u m a c t i v i t y w a s 60~o o f t h a t o f V I P ) . V I P ( 3 - 2 8 ) a n d ( 4 - 2 8 ) w e r e p a r t i a l a g o n i s t s in c l o n e 3 b u t h a d n o d e t e c t a b l e s t i m u l a t o r y effect in c l o n e 16 (Fig. 3). I n b o t h c l o n e s , t h e V I P d o s e r e s p o n s e c u r v e w a s s h i f t e d t o t h e r i g h t in t h e p r e s ence of VIP(4-28) but reached the same maximum
v a l u e (Fig. 4). T h e c a l c u l a t e d K i (Fig. 5), a s s u m i n g c o m p e t i t i v e i n h i b i t i o n , w a s 800 100 n M ( m e a n S.E.M. of three determinations). PACAP(2-27) and P A C A P ( 2 - 3 8 ) w e r e as efficient as V I P a n d P A C A P in c l o n e s 3 a n d 16. P A C A P ( 3 - 2 7 ) a n d ( 3 - 3 8 ) w e r e p a r t i a l a g o n i s t s o n b o t h c l o n e s ( r e a c h i n g 50 a n d 60~o o f m a x i m u m a c t i v i t y in c l o n e 3 a n d 1 0 ~ in c l o n e 16). F r a g m e n t s ( 4 - 2 7 ) a n d ( 4 - 3 8 ) w e r e p a r tial a g o n i s t s in c l o n e 3 (25 a n d 15~o o f m a x i m u m a c t i v i t y ) b u t d i d n o t a c t i v a t e t h e a d e n y l a t e c y c l a s e in c l o n e 16.
E. Ciccarelli et al. I Regulator), Peptides 54 (1994) 397-407
402
Table 2
CHO-VIPR-c3ANDc16 ®
I00- ~
~
P
H
General characteristics of adenylate cyclase activity in clone 3 and clone 16 membranes
I
\\ \\
'-x
Stimulus
Adenylate cyclase activity
//'a,\ \ ~i "~ Secretin
,>
None Gpp[NH]p (10-4 M) Naf (10-2 M) Forskolin GTP (10 '5 ~a) GTP (10-5 M)+VIP (10 5M)
® 1oo-~1~,~3
~ tAc-Hish rIP
3 50
VIP
~.?~k
~ ~
(Ac-HisI) PACAP-"~ , o ~ .
-I0
.
-9
.
-fl (PEPTIOE]
[D-Hiisl) s I)VIP
Clone 3
Clone 16
8 -+2 310_+12 234_+ 15 60 _+6 20+2 250_+8
6 +_1 196_+8 174_+ 8 47 + 5 13 _+2 176_+9
The values were given in pmol of cyclic AMP formed per min and per mg membranes protein and were the mean of _+8 determinations.
"N..~ . , . . . ' N ~
.
-7
-6
-5
(log M)
Fig. 2. (Upper panel) Dose dependent inhibition of [125I]VIP binding by unlabelled VIP (O), PACAP-27 (,x), PACAP-38 (F-l), PHI (A), helodermin ( 0 ) and secretin ( x ). (Lower panel) Dose dependent inhibition of [12sI]VIP binding by unlabelled VIP (C)), [Ac-Hisa]PACAP (,), [Ac-Hisl]VIP (@), [Phel]VIP (v) and [D-Hisl]VIP (m). In both panels the results were the mean of at least 4 experiments obtained on clone 3 or 16 and the results were expressed in yo of tracer specifically bound.
3.5. Effect of VIP pretreatment on VIP receptors and adenylate cyclase activation P r e t r e a t m e n t o f the cells for 24 h with V I P red u c e d the binding c a p a c i t y o f the m e m b r a n e s . This was quantified in clone 3 only where S c a t c h a r d analysis a n d c o m p l e t e c o m p e t i t i o n curves could be validly analysed. The tracer affinity as well as the K d o f unlabelled V I P were u n c h a n g e d after V I P pretreatment ( d a t a not shown). T h e m a x i m u m binding was 106~'o + 1 0 , 8 1 ~ o + 5 , 37~/o+3 and 25~o+__5 (mean + S . E . M . o f 5 determinations) o f control (untreated cells) after p r e t r e a t m e n t with 0.1, 1.0, 10 and 100 n M V I P for 24 h, respectively.
The c o n s e q u e n c e s o f V I P pretreatment on adenylate cyclase activity were evaluated in both clones: Table 3 s u m m a r i z e s the adenylate cyclase activity o f clones 3 a n d 6 after pretreatment with VIP. In clone 3, there was a progressive decrease in V I P potency (shift to the right o f the dose-effect curve) a n d an
®
CHO-VIPR-c3
®
CHO-VIPR-c~5
'c 300 o L cl ~ 200 i c E c:z
I00-I !
u
-iO "g
50
28 27 26 25
~ g )
f
-10 -9
[PEPTIDE}(log M)
-8
-7
-6
-5
Fig. 3. Doseeffect curves of VIP (@), VIP(2-28) (0), VIP(3-
28) (~), and VIP(4-28) ([~) stimulated adenylate cyclase activity in membranes from clone 3 (panel A) and clone 16 (panel B). The results expressed in pmol cyclic AMP produced/min/mg protein were the mean of 3 experiments.
E. Occarelli et al. I Regulatory Peptides 54 (1994) 397-407 300.
IC
CHO-VIPR-cI5
CHO-VIPR-c3 Control
.~~
o
~ 200
#k
~"iO0I
oE
S ~ loo
/
j
+3.M.
~'
50-
o
y
Ic
I
rI_lio -'g
-'a
-'7
[VIP} (log M)
-'B
-
Sv;pW;s, HI,
-lO
-'9 [VIP]
-'B
-'7
-'6
(log M)
Fig. 4. Dose effect curves of VIP stimulated adenylate cyclase activity in clone 3 (panel A) and clone 16 (panel B) membranes in absence (open symbols) or presence (black symbols) of 3/xM VIP(4-28). The results expressed in pmol cyclic AMP produced min/mg protein were the mean of 3 experiments.
increase in the basal and Gpp[NH]p, NaF, forskolin, and GTP-stimulated activity after preincubation with 10 and 100 nM VIP. In clone 16, there was a progressive decrease in the maximum effect of VIP but no significant change in peptide potency. There was no significant change in the basal and Gpp[NH]p, NaF, forskolin and GTP-stimulated activity.
4. Discussion
The seven helices receptor cloned by Ishihara et al. [1] had the characteristics of a high affinity VIP receptor on both its binding selectivity, its ability to stimulate adenylate cyclase activity and its tissue distribution. It did not discriminate between VIP and PACAP and had the characteristics of a PACAP type II receptor according to the proposed classification of Shivers et al. [3]. PACAP type I receptors were recently cloned and corresponded to high affinity PACAP receptors, discriminating between PACAP and VIP. In the present work we transfected the DNA sequence of Ishihara's receptor in CHO cells that did not express constitutively the
403
receptor. We selected and studied two clones that expressed different receptor densities. The clone expressing the highest receptor density also had a higher adenylate cyclase activity as judged by the basal and Gpp[NH]p, NaF, GTP, and forskolin stimulation. The reason for that difference (number of G sites, number of catalycal units, intrinsic activity of the receptor?) was not further investigated. There was no difference in the binding properties of the receptor expressed in the two clones. We confirmed that the construction was indeed a PACAP type II receptor considering the order of potency of the peptides (VIP = PACAP-27 and 3 8 > P H I > helodermin). As observed on membrane preparations from cells or tissues with VIP receptors, secretin cross reacted with the transfected receptor but was 100-fold less potent than VIP [17]. This contrasts with the higher selectivity of the secretin receptor that recognizes VIP with a 1000- to 5000-fold lower potency than secretin [18,19]. As also observed with the naturally occurring receptors, the N-terminus of VIP was essential for a high affinity recognition of the cloned receptor subtype [20,21]. N-Acetylation of His l, substitution 1-His by the D enantiomer or by phenylalanine, deletion of His 1 reduced VIP potency, 7-, 100-, 15- and 100-fold respectively. This allowed us to discriminate between this receptor subtype and the 'helodermin-preferring receptor' which is more tolerant to histidine modifications [8,9]. Surprisingly, although VIP, PACAP-27 and PACAP-38 had the same ability to recognize the receptor, N-acetyl-HisLpACAP-27, PheI-PACAP-27 and PACAP[2-27] had, respectively, a 2-fold higher, a 10-fold lower and a 7-fold higher potency than the corresponding VIP analogues. PACAP(2-38) was 3-fold less potent than PACAP(2-27) but 2-fold more potent than VIP. Further N-terminal deletions of VIP and PACAP's reduced dramatically peptide potency: VIP(3-28) and (4-28), PACAP(3-27), (4-27) or (4-38) had a low affinity in the micromolar range. The different affinities of similarly modified VIP and PACAP derivatives were intriguing but could be
404
E. Ciccarelli et al. ,; Regulatory Peptides 54 (1994) 397-407
(~
CHO-VIPR-c3
(~)
CHO-VIPR-cI6
400"
24 Hours pretreetment IC
~ aooo.
150-
T .5 E
• 200-
o
None
o • •
10-I0 M VIP I0 -g M VlP 10-8 M VIP iO-7 M VIP
100-
o LU
N 100.
50
r,r Q.
(d
~Li0
dg
:e
~7
2B
o
-10
$9
S8
27
76
[VIP] (log M)
Fig. 5. Dose effect curves of VIP stimulated adenylate cyclase activity in membranes from clone 3 (left panel) and clone 16 (right panel) treated without (O) or with 10- lo (~), 10- 9 (F-l), 10- s (O), 10- 7 ( A ) M VIP for 24 h. The results were the mean of 3 experiments and were expressed in pmol cyclic AMP produced min/mg protein.
due to a different conformation of the N-terminal part of the ligands: it has been suggested that the VIP histidine 1 interacted with phenylalanine 6 creating a small N-terminal loop [22,23]. The PACAP N-terminal structure is apparently less ordered [2425]. The transfected receptor was coupled to adenylate cyclase. Several arguments suggested that in CHO cells, the occupancy of a limited number of receptors was sufficient to activate maximally the adenylate cyclase. (1) In clone 3, the ECs0 value of adenylate cyclase activation was, for all the ligands tested, lower than the corresponding ECso value observed in clone 16, despite a similar Kd of binding. (2) VIP(2-28) was a full agonist in clone 3, but a partial agonist in clone 16; PACAP(3-27) and (338) were partial agonists in clone 3, but were inactive in clone 16. (3) Pretreatment of both clones with increasing
concentrations of VIP that reduced - probably by internalization - the receptor number, decreased peptide potency in clone 3 without noticeable change in peptide efficacy, but reduced only peptide efficacy in clone 16. These observations were typical of the results expected if spare receptors exist in clone 3 but not in clone 16 [26-28]. We recently observed similar results in CHO cells stably transfected with the recombinant secretin receptor [29]. The efficient coupling of the VIP receptor to adenylate cyclase and the high number of receptors in clone 3 probably explained the fact that VIP pretreatment with high doses of VIP (10 and 100 nM) induced a persistent activation of the enzyme, even though the washing procedure was fully sufficient to clear the receptors in clone 16. The existence of spareness is of interest to clarify the definition of partial agonists and antagonists: for instance, VIP(2-28) was previously reported to be
405
E. Ciccarelli et al. / Regulatory Peptides 54 (1994) 397-407
Table 3 Adenylate cyclase activity (expressed in pmol cAMP formed/min/mg membrane protein) of membranes from clones 3 and 16 after pretreatment with increasing concentrations of VIP Stimulus
None Gpp[NH]p 10 - 4 Naf 10 - 2 M Forskolin 10 -4 M GTP 10 -3 M GTP 10 4 M +VIP 10 - 6 M ECso
Clone 3 Pretreatment with VIP 0
0.1 nM
1 nM
10 nM
100 nM
10_+3 366 _+20 264 -+20 74 _+8 26_+8 292_+ 15 0.2_+0.1
11_+3 381 _+25 282 -+ 15 79 + 5 25_+4 316_+ 18 0.2_+0.1
11_+3 396 + 20 294 -+ 18 72 -+8 25+4 320_+20 0.3_+0.1
39*_+5 666* + 35 383* -+20 159" -+ 12 63*+6 346*_+20 2.0_+0.3
43*_+5 747* + 40 416" _+30 188" _+20 77*+ l0 344*_+30 3.0_+0.3
6+2 163_+ 12 164 _+12 37 _+5 11_+2 119_+8 2.0 _+0.4
4+ 1 154_+8 157 _+9 35 _+6 10+3 70*_+4 3.0 _+0.3
5+2 155_+8 157 _+8 35 _+4 10_+2 51"_+3 3.0 _+0.4
7+2 174_+8 190 _+7 43 _+9 10_+3 40*_+3 3.0 _+0.4
Clone 16 None Gpp[NH]p 10 4 Naf 10 2 M Forskolin 10 -4 M GTP 10 -5 M G T P 10 - 4 M + V I P 10 6 M ECs0 VIP
6+3 166_+20 159 _+18 59 _+7 12_+3 135_+8 2.0 _+0.4
The results were the mean + S.E.M. of three experiments. * Indicate the values significantly different (P< 0.05) from the control, untreated clones. The ECs0 value (in nM) for VIP activation was also given.
o n rat tissues a partial a g o n i s t [21]; this p r o p e r t y a t t r i b u t e d to an intrinsic p r o p e r t y o f the m o l e c u l e w a s p r o b a b l y a c o n s e q u e n c e o f the b a l a n c e b e t w e e n the intrinsic activity o f t h e m o l e c u l e , the n u m b e r o f r e c e p t o r s , a n d their c o u p l i n g efficacy in t h e tissue studied. T h e p r e s e n t results d e m o n s t r a t e d t h a t the N-terminally deleted fragments of VIP and PACAP m u s t b e c o n s i d e r e d as partial a g o n i s t s w i t h an effic a c y d e p e n d i n g , inter alia o n t h e n u m b e r o f r e c e p t o r s e x p r e s s e d at the cell surface.
Acknowledgements T h i s w o r k w a s s u p p o r t e d by a S c i - C T 9 1 - 0 6 3 2 S c i e n c e P l a n f r o m the C o m m i s s i o n o f t h e E u r o p e a n C o m m u n i t i e s , by G r a n t s 3.4525.91 a n d 3.4514.94
f r o m the F o n d s de la R e c h e r c h e Scientifique M 6 d i cale (Belgium). W e t h a n k Dr. S. N a g a t a ( O s a k a B i o s c i e n c e Institute, O s a k a , J a p a n ) for their k i n d gift o f t h e V I P r e c e p t o r in t h e p C D M 8 p l a s m i d .
References [ 1] Ishihara, T., Shigemoto, R., Mori, K., Takahashi, K. and Nagata, S., Functional expression and tissue distribution of a novel receptor for vasoactive intestinal polypeptide, Neuron, 8 (1992) 811-819. [ 2] Lutz, E.M., Sheward, W.J., West, K.M., Morrow, J.A., Fink, G. and Harmar, A.J., The VIP 2 receptor: molecular characterisation of a cDNA encoding a novel receptor for vasoactive intestinal peptide, FEBS Lett., 334 (1993) 3-8. [ 3] Shivers, B.D., GOrcs, T.J., Gottschall, P.E. and Arimura, A., Two high affinity binding sites for pituitary adenylate
406
[ 4]
[ 5]
[ 6]
[ 7]
[ 8]
[ 9]
[10]
[ 11 ]
[12]
[13]
[ 14]
E. Ciccarelli et al. / Regulatory Peptides 54 (1994) 397-407
cyclase-activating polypeptide have different tissue distributions, Endocrinology, 128 (1991) 3055-3065. Sreedharan, S.P., Patel, D.R., Huang, J.-X. and Goetzl, E.J., Cloning and functional expression of a human neuroendocrine vasoactive intestinal peptide receptor, Biochem. Biophys. Res. Commun., 193 (1993) 546-553. Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeburg, P.H. and Journot, L., Differential signal transduetion by five splice variants of the PACAP receptor, Nature, 365 (1993) 170-175. Svoboda, M., Tastenoy, M., Ciccarelli, E., Sti~venart, M. and Christophe, J., Cloning of a splice variant of the pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptor, Biochem. Biophys. Res. Commun., 195 (1993) 881-888. Pisegna, J.R. and Wank, S.A., Molecular cloning and functional expression of the pituitary adenylate cyclase-activating polypeptide type I receptor, Proc. Natl. Acad. Sci. USA, 90 (1993) 6345-6349. Robberecht, P., De Neef, P., Gourlet, P., Cauvin, A., Coy, D.H. and Christophe, J., Pharmacological characterization of the novel helodermin/VIP receptor present in human SUP-TI lymphoma cell membranes, Regul. Pept., 26 (1989) 117-126. Gourlet, P., De Neef, P., Woussen-Colle, M-C., Vandermeers, A., Vandermeers-Piret, M-C., Robberecht, P. and Christophe J., The activation of adenylate cyclase by pituitary adenylate cyclase activating polypeptide (PACAP) via helodermin-preferring VIP receptors in human SUP -T1 lymphoblastic membranes, Biochim. Biophys. Acta, 1066 (1991) 245-251. Fabre, C., El Battari, A,, Karamanos, Y., Couvineau, A., Salomon, R., Laburthe, M., Marvaldi, J., Pichon, J. and Luis, J., Glycosylation of VIP receptors: A molecular basis for receptor heterogeneity, Peptides, 14 (1993) 483-489. Robberecht, P., Cauvin, A., Gourlet, P. and Christophe, J., Heterogeneity of VIP receptors. In Pauwels, R., Lefebvre, R.A., and Herman, A.G. (Eds.), Second Workshop on Nonadrenergic Noncholinergic Mechanisms, Arch. Int. Pharmacodyn. Ther. Vol. 303, 1990, pp. 51-66. Connors, R.W., Sweet, R.W., Noveral, J.P., Pfarr, D.S., Trill, J.J., Shebuski, R.J., Berkowitz, B.A., Williams, D., Franklin, S. and Reff, M.E., DHFR coamplifieation oft-Pa in DHFR + bovine endothelial cells: in vitro characterization of the purified serine protease, DNA, 7 (1988) 651-661. Urlaub, G. and Chasin, L.A., Isolation of chinese hamster cell mutants deficient in dihydrofolate reductase activity, Proc. Natl. Acad. Sci. USA, 77 (1980) 4216-4220. Robberecht, P., Waelbroeck, M., Camus, J.-C., De Neef, P. and Christophe, J., Importance of disulfide bonds in receptors for vasoactive intestinal peptide and seeretin in rat
[15]
[16]
[17]
[ 18]
[19]
[20]
[21]
[22]
[23]
[24]
pancreatic plasma membranes, Biochim. Biophys. Acta, 773 (1984) 271-278. Robberecht, P., Coy, D.H., De Neef, P., Camus, J.-C., Cauvin, A., Waelbroeck, M. and Christophe, J., [D-Phe4]Peptide histidine-isoleucinamide ([D-Phe4]PHI), a highly selective vasoactive-intestinal-peptide (VIP) agonist, discriminates VIP-preferring from secretin-preferring receptors in rat pancreatic membranes, Eur. J. Biochem., 165 (1987) 243-249. Salomon, Y., Londos, C. and Rodbell, M., A highly sensitive adenylate cyclase assay, Anal. Biochem.. 58 (1974) 541-548. Waelbroeck, M., Robberecht, P., De Neef, P., Chatelain, P. and Christophe, J., Binding of vasoactive intestinal peptide and its stimulation of adenylate cyclase through two classes of receptors in rat liver membranes. Effects of 12 secretin analogues and 2 secretin fragments, Biochim. Biophys. Acta. 678 (1981) 83-90. Gossen, D., Tastenoy, M., Robberecht, P., and Christophe, J., Secretin receptors in the neuroglioma hybrid cell line NG 108-15. Characterization and regulation of their expression, Eur. J. Biochem., 193 (1990) 149-154. Jensen, R.T., Charlton, C.G., Adachi, H., Jones, S.W., O'Donohue, T.L. and Gardner, J.D., Use of ~251-secretin to identify and characterize high-affinity secretin receptors on pancreatic acini, Am. J. Physiol., 245 (1983) G186G195. Robberecht, P., Coy, D.H., De Neef, P., Camus, J.-C., Cauvin, A., Waelbroeck, M. and Christophe, J., Interaction of vasoactive intestinal peptide (VIP) and N-terminally modified VIP analogs with rat pancreatic, hepatic and pituitary membranes, Eur. J. Biochem., 159 (1986) 45-49. Couvineau, A., Rouyer-Fessard, C., Fournier, A., St-Pierre, S., Pipkorn, R. and Laburthe, M., Structural requirements for VIP interaction with specific receptors in human and rat intestinal membranes: effect of nine partial sequences, Biochem. Biophys. Res. Commun., 121 (1984) 493-498. Fournier, A. Saunders, J.K., Boulanger, Y. and St. Pierre, S.A., Conformation analysis of vasoactive intestinal peptide and related fragments, Ann. N.Y. Acad. Sci., 527 (1988) 51-67. Goossens, J.F., Pommery, N., Lohez, M., Pommery, J., Helbecque, N., Cotelle, P., Lhermitte, M. and Henichart, J.P., Antagonistic effect of vasoactive intestinal peptide fragment, vasoaetive intestinal peptide(1-11), on guinea pig trachea smooth muscle relaxation, Mol. Pharmaeol., 41 (1991) 104-109. Inooka, H., Endo, S., Kitada, C., Mizuta, E. and Fujino, M., Pituitary adenylate cyclase activating polypeptide (PACAP) with 27 residues, Int. J. Pept. Prot. Res., 40 (1992) 456-464.
E. Ciccarelli et al. / Regulatory Peptides 54 (1994) 397-407
[25] Wray, V., Kakoschke, C., Nokihara, K. and Naruse, S., Solution structure of pituitary adenylate cyclase activating polypeptide by nuclear magnetic resonance spectroscopy, Biochemistry, 32 (1993) 5832-5841. [26] Arii~ns, E.J., Van Rossum, J.M. and Koopman, P.C., Receptor reserve and threshold phenomena. I. Theory and experiments with autonomic drugs tested in isoled organs, Arch. Int. Pharmacodyn., 127 (1960) 459-478. [27] Van Rossum, J.M. and Afians, E.J., Receptor-reserve and threshold-phenomena. II. Theories on drug-action and a
407
quantitative approach to spare receptors and threshold values, Arch. Int. Pharmacodyn., 136 (1962) 385-413. [28] Kenakin, T.P., The classification of drugs and drug receptors in isolated tissues, Pharmacol. Rev., 36 (1984) 165222. [29] Vilardaga, J.P., Ciccarelli, E., Dubeaux, C., De Neef, P., Bollen, A. and Robberecht, P., Properties and regulation of the coupling to adenylate cyclase of the secretin receptor stably transfected in CHO cells, Mol. Pharmacol., 45 (1994) 1022-1028.
Regulatory Peptides 54 (1994) 397-407
Properties of the VIP-PACAP type II receptor stably expressed in CHO cells Ernesto Ciccarelli a, Jean-Pierre Vilardaga b, Philippe De Neef a, Emmanuel Di Paolo Magali Waelbroeck a, Alex Bollen b, Patrick Robberecht a,,
a,b,
aDepartment of Biochemistry and Nutrition, Faculty of Medicine, Universit~ Libre de Bruxelles, 808 Route de Lennik, B-1070 Brussels, Belgium bDepartment of Applied Genetics, Faculty of Sciences, Universitd Libre de Bruxelles, Brussels, Belgium
Received 25 May 1994; revised version received and accepted 8 August 1994
Abstract
The VIP receptor cloned from rat lung (VIP 1 receptor from the group of the PACAP-VIP type II receptors) was inserted into a mammalian expression vector and stably transfected into Chinese hamster ovary cells (CHO). Two clones were selected, expressing respectively a high (850 + 50 fmol/mg protein, for clone 3) and a low (100 + 30 fmol/mg protein for clone 16) number of receptors. Both clones had the same apparent K d value of binding for VIP and related peptides. The receptor expressed had the same binding properties as the natural VIP receptor, judged from the relative potency of VIP and PACAP analogues and fragments. The ECso value of adenylate cyclase activation were 3 to 10 fold lower in clone 3 than in 16. The values observed in clone 16 were closer to the binding K d values. The differences between the two clones were explained by the existence of spare receptors in clone 3, since: (a) the relative efficacy of some fragments were lower in clone 16 than in clone 3; (b) pretreatment of the cells with VIP reduced the number of receptors in both clones and increased the ECso value for VIP in clone 3 but decreased peptide efficacy in clone 16 without significant change of the ECso value. Keywords." VIP; PACAP; PACAP type II receptor; Chinese hamster ovary cell; Transfection
* Corresponding author. Fax: + 32 2 5556230. Abbreviations: PACAP, pituitary adenylate cyclase activating peptide; VIP, vasoactive intestinal peptide; CHO, Chinese hamster ovary cells; PHI, peptide histidine isoleucinamide-(1-27), PHI-GIy, peptide histidine isoleucinamide-(1-27)-glycine;PHV, peptide histidine valine-(1-42); Kd, equilibrium dissociation constant of the radioligand; ECso, concentration producing half-maximal stimulation of ~t . adenylate cyclase; Ki, concentration of antagonist that requires a 2-fold increase of agonist concentration for observing the same biological effect. 0167-0115/94/$7.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0 1 6 7 - 0 1 1 5 ( 9 4 ) 0 0 0 7 6 - X
398
E. Ciccarelli et al. / Regulatory Peptides 54 (1994) 397-407
1. Introduction
2. Material and methods
VIP/PHI/PACAP receptors have been subdivided into two groups: group I receptors are highly selective for PACAP and group II receptors do not discriminate PACAP from VIP. Three receptors were cloned recently and belong to the seven helices receptor family: (1) two VIP receptors (VIP 1 receptors [1] and VIP 2 receptors [2]), belonging to the PACAP type II receptors group [ 3 ] do not discriminate between VIP and PACAP. The VIP 1 receptor was cloned in rat [1] and human [4], the VIP2 receptor in rat tissue only. (2) A PACAP receptor [5-7] corresponding to the PACAP type I receptor [3] highly selective for PACAP was cloned in rat tissues. This receptor, however, may exist in several forms differing in the sequence of the third intracellular loop [ 5 ]. Despite the rapid progress in this field, several questions remain unsolved. For instance: is the socalled 'helodermin-preferring receptor' described on human cell lines and related to the PACAP type II receptor [8-9] a third VIP receptor? Are the low affinity binding sites described in most tissues original molecular entities or low affinity states of the already cloned receptors? Is the molecular heterogeneity due to a variable degree of receptor glycosylation [10] responsible for a functional heterogeneity? Are the discrepancies which are often observed between binding data and functional studies due to receptor heterogeneity or to an amplification process [11]? Since receptors of different subtypes may coexist in tissues and cell lines, most of these questions were previously difficult to solve. The Chinese hamster ovary cells (CHO cells) do not express constitutively any receptor for the VIP/PHI/PACAP peptides family. CHO clones transfected with a defined receptor type and stably expressing different receptor densities are useful tools to study these questions. We used this experimental model to study the type II receptor (VIP~) from rat.
2.1. Construction of the expression plasmid, transfection, selection and expression in CHO cells The DNA coding for the rat VIP/PACAP receptor was excised from the plasmid pCDM8 kindly provided by Prof. Nagata, Osaka, (Japan) by digestion with Hind III and Xho I, and cloned into the Hind III and Xho I sites of the mammalian expression Vector pNIV187, a derivative of pRc/RSV (Invitrogen) that contains the selectable neomycin phospho-transferase gene, and an expression cassette for dihydrofolate reductase (dhfr), as described in Connors et al. [12]. The resulting recombinant plasmid was transfected into the CHO cell line DG44 [13] by electroporation using a gene pulser. Approximately 107 cells were preincubated on ice for 30 min with 25/~g DNA in 0.8 ml 7 mM sodium phosphate buffer (pH 7.4) containing 272 mM sucrose and 1 mM MgC12. Electroporation was performed at 600 V and 3 /~F. After electroporation, cells were kept on ice for 10 min, added to 10 ml of culture medium and harvested in 96 well plates. Cells were maintained in or-minimal essential medium (~MEM T), supplemented with 10~o FCS, 2 mM L-gtutamine, 100 ~tg/ml penicillin and 100 /~g/ml streptomycin with an atmosphere of 95 ~o air, 5 j°o CO2 at 37°C. 48 h after transfection the G148 resistant cells were selected by the addition of geneticin to a final concentration of 450/~g/ml. Selection was done in 96 well plates with 2000-5000 ceils per well. To produce sufficient amounts of cells for receptor characterization, the clones were then grown to confluency in six-well plates of 35 mm diameter. The clones used in the present study thereafter referred to as CHO-VIPr-c3 (clone 3) and CHOVIPr-cl6 (clone 16) were obtained without amplification and were selected on the basis of a high and low binding capacity of 125I-VIP, respectively (see Results). Geneticin at 0.5 mg/ml was maintained in the culture medium of the stock culture. Subcultures
E. Ciccarelli et al. / Regulatory Peptides 54 (1994) 397-407
prepared for membrane purification were done in a medium without geneticin.
2.2. Membrane preparation, receptor identification and adenylate cyclase activity Cells were detached with a rubber policeman and pelleted by low speed centrifugation, the supernatant was discarded and the cells lysed in 1 mM NaHCO 3 solution and immediate freezing in liquid nitrogen. After thawing, the lysate was first centrifuged at 800 x g for 10 min and the supernatant further centrifuged at 20,000 x g for 10 min. The pellet, resuspended in 1 mM NaHCO3 was used immediately as a crude membrane preparation. [ 125I]VIP was labelled by the chloramin T method as described [14] and purified by adsorption on a cellulose column eluted with discontinuous gradient of BSA. Tracer specific binding radioactivity was 1.0 mCi/mmol. Binding of the tracer to the membranes was performed as described [15]. Saturation curves were done with increasing concentrations of tracer and competition curves with a fixed tracer concentration (equal to one fourth of the value of tracer Ka) and increasing concentrations of unlabelled peptides. In all cases, the non specific binding was defined as the residual binding in the presence of 1/~M VIP. Binding was performed at 37°C in a 20 mM Tris-maleate, 2 mM MgC12, 0.1 mg/ml bacitracin, 1~o BSA (pH 7.4) buffer, 3 to 30 /zg protein were used per assay. The bound was separated from the free radioactivity by filtration through glass fiber filters G F / C presoaked for 24 h in 0.1 ~o polyethyleneimine and rinsed three times with a 20 mM (pH 7.4) phosphate buffer containing 1 ~o BSA. Adenylate cyclase activity was determined by the Salomon et al. procedure [ 16]. Membrane protein (3 to 15 #g) was incubated in a total volume of 60 #1 containing 0.5 mM [~-32p]ATP, 10 ktM GTP, 5 mM MgC12, 0.5 mM EGTA, 1 mM cAMP, 1 mM theophylline, 10 mM phosphoenolpyruvate, 30 /zg/ml pyruvate kinase and 30 mM Tris-HC1 at a final pH
399
of 7.5. The reaction was initiated by adding membranes and was terminated after a 12 min incubation at 37°C by adding 0.5 ml cAMP and 20,000 cpm [8-3H]cAMP. cAMP was separated from ATP by two successive chromatographies on Dowex 50-W X8 and neutral alumina.
2.3. Pretreatment of the cells Pretreatment of the cells with VIP was done on confluent cells. Attached cells were then washed twice with the culture medium without foetal calf serum, then harvested with a rubber policeman and centrifuged at a low speed. The pellet was washed once and finally the cells were lysed with 1 mM NaHCO 3 and immediate freezing in liquid nitrogen. The lysate was stored at -80°C until membrane preparation.
2.4. Materials Restriction endonuclease, T 4 DNA ligase, Escherichia coli DNA polymerase I (Klenow) were purchased from Amersham, Boehringer-Mannheim, New England Biolab and Gibco-BRL, respectively. Neomycin (Geneticin G416), cell culture medium ( ~ M E M + ) , L-glutamine, penicillin, streptomycin and foetal calf serum (FCS) were from GIBCOBRL. All the peptides were synthesized in the Laboratory by A. Vandermeers, M.-C. Vandermeers-Piret and P. Gourlet by the solid phase methodology.
3. Results
3.1. Binding kinetics 125I-VIP bound rapidly and reversibly to the membranes from both clones: steady state was achieved in both cases after 10 min incubation at 37°C and maintained for at least 30 min (data not shown). Dissociation was studied in clone 3 only and was induced by addition of 10 -6 M VIP 15 min after
400
E. Ciccarelli et al. / Regulator), Peptides 54 (1994) 397-407
exposure of the membranes to the tracer. A rapid dissociation of 70~o of the bound tracer occurred with a t~ of 5 + 1 min (mean + S.E.M. of 3 determinations) and was reduced to 2 + 1 min in the presence of 10-5 M GTP. The second, slowly dissociating component represented 30~o of the labelled sites. It was not further investigated (data not shown).
3.2. Scatchard analysis of the binding to clones 3 and 16 Saturation curves performed with increasing concentrations of 125I-VIP were transformed as Scatchard plots and were compatible with the presence of a single class of binding sites (Fig. 1). The density of binding sites (Bmax) in clones 3 and 6 were
o
0.4
0.3
o
3
tic kt_
i
0.2-
O. I-
CHO-VIPR-c16
BOUND
850 + 50 and 100 _+30 fmol/mg protein, respectively ( m e a n + S . E . M . of 4 determinations). The K d were not different in both clones: 0.5 + 0.1 and 0.3 + 0.1 nM (mean + S.E.M. of 4 determinations).
3.3. Selectivity of the binding sites labelled with l:51VIP The selectivity of the binding sites was tested by inhibition of tracer binding in the presence of increasing concentrations of unlabelled peptides. Inhibition curves were identical in both clones. All the curves were compatible with the presence of a single class of binding sites. Tracer binding was inhibited with the same potency (K d 2 nM) by VIP, PACAP-27 and PACAP-38; PHI, but also PHI-Gly and PHV (data not shown), were 5-fold less potent; helodermin and secretin were 15- and 125-fold less potent than VIP; glucagon was inactive (Table 1 and Fig. 2). We documented the importance of His i and the N-terminal portion of the peptides for receptor recognition by studying the capacity of acetyl-His lVIP and acetyl-HisLPACAP-27, PheLVIP and Phe1-PACAP-27, D-Hisa-VIP as well as VIP and PACAP fragments to inhibit tracer binding. Peptide potency was reduced in all cases (Table 1, Fig. 2). The same modification, substitution, or deletion made respectively on VIP or PACAP affected differently the peptide potency: [Phe ~]VIP was 10-fold more potent than [Phet]PACAP-27, PACAP(2-27) was 4-fold more potent than VIP(2-27) but 2-fold less potent than PACAP(2-38).
3.4. Coupling of the receptors to adenylate cyclase
(fmoles)
Fig. 1. Scatchard analysis of saturation curves obtained by incubating 25 #g of membrane protein from clone 3 and clone 16 in the presence of increasing concentrations of [ ~25I]VIP in a total volume of 120 #1. Non-specific binding was obtained in the presence of 1 MM VIP. One representative of 4.
Basal as well as the G p p [ N H ] p , NaF, forskolin, GTP, and VIP stimulated adenylate cyclase activities were significantly higher in clone 3 than in clone 16 (Table 2). In both clones, the dose-response curves of enzyme stimulation by the peptides developed on two logarithms. The ECso values were 2.5- to 10-fold lower in clone 3 than in clone 16. The ECso values
401
E. Ciccarelli et aL / Regulatory Peptides 54 (1994) 397-407 Table 1 Characteristics of the receptor expressed in CHO-VIP R-c3 and VIP R-cl6 (clones 3 and 16) Peptides binding
Kd
Adenylate cyclase activation Clone 3
VIP PACAP-27 PACAP-38 PHI Helodermin Secretin Acetyl-His
2+0.3 2_+0.3 2_+0.2 10 + 1.0 30 -+ 5 250 + 15 15 _+3 8 _+3 30+5 300 _+20 200 _+30 200 + 50 3000 -+ 500 1500 + 500 30 + 10 2000 + 500 800 + 300 80 _+20 800 _+100 800 + 200
Clone 16
ECs0 value
Eft
ECs0 value
Eff
0.4+0.1 0.4_+0.1 0.4_+0.1 0.8 + 0.5 1 -+0.3 40 + 10 0.8 _+0.5 0.6 + 0.3 6+2 20 + 4 20 + 6 15 + 3 200 + 60 400 + 50 5+3 200 -+ 100 200 _+50 50 _+10 200 _+50 300 + 50
1.0 1,1 + 0.1 0,9+0.1 1,2 + 0.2 0,8 -+0.2 0,9 + 0.3 0.9 + 0.2 1.1 + 0.2 0.8-+0.3 0.8 + 0.3 0.7 + 0.4 0.9 + 0.2 0.2* + 0.1 0.1" _+0.1 0.9 + 0.3 0.5* + 0;2 0.2* _+0.1 0.8 + 0.3 0.6* + 0.2 0.2* -+ 0.1
2+0.2 1 +0.3 1 +0.3 6 + 1.0 10 + 2 150 + 20 3+ 1 2+ 1 30+8 150 + 50 100 + 30 100 + 30 20 + 5 1000 _+200 200 _+80 200 _+100 -
1.0 0.9+0.1 1.0+ 0.1 1.1 + 0.2 0.9 + 0.3 0.8 + 0.3 1.0 + 0.2 1.1 + 0.2 0.9+0.2 0.7 + 0.3 0.7 + 0.3 0.6* + 0.2 0* 0* 0.9 + 0.2 0.1" _+0.2 1.0 + 0.2 0.1" + 0.2 -
The K d of binding and the ECho value of adenylate cyclase activation (both in nM) were derived from competition and dose-effect curves. The competition curves for the two cell lines were superimposable and the results were pooled and are expressed as the mean of at least four determinations. ECso value were the mean of at least three determinations, efficacy (Eft) refers to the ratio between the maximum effect of a peptide and the maximum effect of 1/~M VIP. * Indicated the values of efficacy significantly different from 1.0 (P< 0.5).
o f c l o n e 16 w e r e c l o s e t o t h e K d v a l u e s o f b i n d i n g ( T a b l e 1). I n b o t h c l o n e s , t h e p e p t i d e s t e s t e d ( e x c e p t V I P a n d P A C A P f r a g m e n t s ) w e r e as efficient as V I P ( s a m e m a x i m a l a c t i v i t y ) ( T a b l e 1). V I P ( 2 - 2 8 ) w a s as efficient as V I P in c l o n e 3 b u t w a s a p a r t i a l a g o n i s t in c l o n e 16 ( m a x i m u m a c t i v i t y w a s 60~o o f t h a t o f V I P ) . V I P ( 3 - 2 8 ) a n d ( 4 - 2 8 ) w e r e p a r t i a l a g o n i s t s in c l o n e 3 b u t h a d n o d e t e c t a b l e s t i m u l a t o r y effect in c l o n e 16 (Fig. 3). I n b o t h c l o n e s , t h e V I P d o s e r e s p o n s e c u r v e w a s s h i f t e d t o t h e r i g h t in t h e p r e s ence of VIP(4-28) but reached the same maximum
v a l u e (Fig. 4). T h e c a l c u l a t e d K i (Fig. 5), a s s u m i n g c o m p e t i t i v e i n h i b i t i o n , w a s 800 100 n M ( m e a n S.E.M. of three determinations). PACAP(2-27) and P A C A P ( 2 - 3 8 ) w e r e as efficient as V I P a n d P A C A P in c l o n e s 3 a n d 16. P A C A P ( 3 - 2 7 ) a n d ( 3 - 3 8 ) w e r e p a r t i a l a g o n i s t s o n b o t h c l o n e s ( r e a c h i n g 50 a n d 60~o o f m a x i m u m a c t i v i t y in c l o n e 3 a n d 1 0 ~ in c l o n e 16). F r a g m e n t s ( 4 - 2 7 ) a n d ( 4 - 3 8 ) w e r e p a r tial a g o n i s t s in c l o n e 3 (25 a n d 15~o o f m a x i m u m a c t i v i t y ) b u t d i d n o t a c t i v a t e t h e a d e n y l a t e c y c l a s e in c l o n e 16.
E. Ciccarelli et al. I Regulator), Peptides 54 (1994) 397-407
402
Table 2
CHO-VIPR-c3ANDc16 ®
I00- ~
~
P
H
General characteristics of adenylate cyclase activity in clone 3 and clone 16 membranes
I
\\ \\
'-x
Stimulus
Adenylate cyclase activity
//'a,\ \ ~i "~ Secretin
,>
None Gpp[NH]p (10-4 M) Naf (10-2 M) Forskolin GTP (10 '5 ~a) GTP (10-5 M)+VIP (10 5M)
® 1oo-~1~,~3
~ tAc-Hish rIP
3 50
VIP
~.?~k
~ ~
(Ac-HisI) PACAP-"~ , o ~ .
-I0
.
-9
.
-fl (PEPTIOE]
[D-Hiisl) s I)VIP
Clone 3
Clone 16
8 -+2 310_+12 234_+ 15 60 _+6 20+2 250_+8
6 +_1 196_+8 174_+ 8 47 + 5 13 _+2 176_+9
The values were given in pmol of cyclic AMP formed per min and per mg membranes protein and were the mean of _+8 determinations.
"N..~ . , . . . ' N ~
.
-7
-6
-5
(log M)
Fig. 2. (Upper panel) Dose dependent inhibition of [125I]VIP binding by unlabelled VIP (O), PACAP-27 (,x), PACAP-38 (F-l), PHI (A), helodermin ( 0 ) and secretin ( x ). (Lower panel) Dose dependent inhibition of [12sI]VIP binding by unlabelled VIP (C)), [Ac-Hisa]PACAP (,), [Ac-Hisl]VIP (@), [Phel]VIP (v) and [D-Hisl]VIP (m). In both panels the results were the mean of at least 4 experiments obtained on clone 3 or 16 and the results were expressed in yo of tracer specifically bound.
3.5. Effect of VIP pretreatment on VIP receptors and adenylate cyclase activation P r e t r e a t m e n t o f the cells for 24 h with V I P red u c e d the binding c a p a c i t y o f the m e m b r a n e s . This was quantified in clone 3 only where S c a t c h a r d analysis a n d c o m p l e t e c o m p e t i t i o n curves could be validly analysed. The tracer affinity as well as the K d o f unlabelled V I P were u n c h a n g e d after V I P pretreatment ( d a t a not shown). T h e m a x i m u m binding was 106~'o + 1 0 , 8 1 ~ o + 5 , 37~/o+3 and 25~o+__5 (mean + S . E . M . o f 5 determinations) o f control (untreated cells) after p r e t r e a t m e n t with 0.1, 1.0, 10 and 100 n M V I P for 24 h, respectively.
The c o n s e q u e n c e s o f V I P pretreatment on adenylate cyclase activity were evaluated in both clones: Table 3 s u m m a r i z e s the adenylate cyclase activity o f clones 3 a n d 6 after pretreatment with VIP. In clone 3, there was a progressive decrease in V I P potency (shift to the right o f the dose-effect curve) a n d an
®
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~ g )
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-8
-7
-6
-5
Fig. 3. Doseeffect curves of VIP (@), VIP(2-28) (0), VIP(3-
28) (~), and VIP(4-28) ([~) stimulated adenylate cyclase activity in membranes from clone 3 (panel A) and clone 16 (panel B). The results expressed in pmol cyclic AMP produced/min/mg protein were the mean of 3 experiments.
E. Occarelli et al. I Regulatory Peptides 54 (1994) 397-407 300.
IC
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-'B
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Fig. 4. Dose effect curves of VIP stimulated adenylate cyclase activity in clone 3 (panel A) and clone 16 (panel B) membranes in absence (open symbols) or presence (black symbols) of 3/xM VIP(4-28). The results expressed in pmol cyclic AMP produced min/mg protein were the mean of 3 experiments.
increase in the basal and Gpp[NH]p, NaF, forskolin, and GTP-stimulated activity after preincubation with 10 and 100 nM VIP. In clone 16, there was a progressive decrease in the maximum effect of VIP but no significant change in peptide potency. There was no significant change in the basal and Gpp[NH]p, NaF, forskolin and GTP-stimulated activity.
4. Discussion
The seven helices receptor cloned by Ishihara et al. [1] had the characteristics of a high affinity VIP receptor on both its binding selectivity, its ability to stimulate adenylate cyclase activity and its tissue distribution. It did not discriminate between VIP and PACAP and had the characteristics of a PACAP type II receptor according to the proposed classification of Shivers et al. [3]. PACAP type I receptors were recently cloned and corresponded to high affinity PACAP receptors, discriminating between PACAP and VIP. In the present work we transfected the DNA sequence of Ishihara's receptor in CHO cells that did not express constitutively the
403
receptor. We selected and studied two clones that expressed different receptor densities. The clone expressing the highest receptor density also had a higher adenylate cyclase activity as judged by the basal and Gpp[NH]p, NaF, GTP, and forskolin stimulation. The reason for that difference (number of G sites, number of catalycal units, intrinsic activity of the receptor?) was not further investigated. There was no difference in the binding properties of the receptor expressed in the two clones. We confirmed that the construction was indeed a PACAP type II receptor considering the order of potency of the peptides (VIP = PACAP-27 and 3 8 > P H I > helodermin). As observed on membrane preparations from cells or tissues with VIP receptors, secretin cross reacted with the transfected receptor but was 100-fold less potent than VIP [17]. This contrasts with the higher selectivity of the secretin receptor that recognizes VIP with a 1000- to 5000-fold lower potency than secretin [18,19]. As also observed with the naturally occurring receptors, the N-terminus of VIP was essential for a high affinity recognition of the cloned receptor subtype [20,21]. N-Acetylation of His l, substitution 1-His by the D enantiomer or by phenylalanine, deletion of His 1 reduced VIP potency, 7-, 100-, 15- and 100-fold respectively. This allowed us to discriminate between this receptor subtype and the 'helodermin-preferring receptor' which is more tolerant to histidine modifications [8,9]. Surprisingly, although VIP, PACAP-27 and PACAP-38 had the same ability to recognize the receptor, N-acetyl-HisLpACAP-27, PheI-PACAP-27 and PACAP[2-27] had, respectively, a 2-fold higher, a 10-fold lower and a 7-fold higher potency than the corresponding VIP analogues. PACAP(2-38) was 3-fold less potent than PACAP(2-27) but 2-fold more potent than VIP. Further N-terminal deletions of VIP and PACAP's reduced dramatically peptide potency: VIP(3-28) and (4-28), PACAP(3-27), (4-27) or (4-38) had a low affinity in the micromolar range. The different affinities of similarly modified VIP and PACAP derivatives were intriguing but could be
404
E. Ciccarelli et al. ,; Regulatory Peptides 54 (1994) 397-407
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Fig. 5. Dose effect curves of VIP stimulated adenylate cyclase activity in membranes from clone 3 (left panel) and clone 16 (right panel) treated without (O) or with 10- lo (~), 10- 9 (F-l), 10- s (O), 10- 7 ( A ) M VIP for 24 h. The results were the mean of 3 experiments and were expressed in pmol cyclic AMP produced min/mg protein.
due to a different conformation of the N-terminal part of the ligands: it has been suggested that the VIP histidine 1 interacted with phenylalanine 6 creating a small N-terminal loop [22,23]. The PACAP N-terminal structure is apparently less ordered [2425]. The transfected receptor was coupled to adenylate cyclase. Several arguments suggested that in CHO cells, the occupancy of a limited number of receptors was sufficient to activate maximally the adenylate cyclase. (1) In clone 3, the ECs0 value of adenylate cyclase activation was, for all the ligands tested, lower than the corresponding ECso value observed in clone 16, despite a similar Kd of binding. (2) VIP(2-28) was a full agonist in clone 3, but a partial agonist in clone 16; PACAP(3-27) and (338) were partial agonists in clone 3, but were inactive in clone 16. (3) Pretreatment of both clones with increasing
concentrations of VIP that reduced - probably by internalization - the receptor number, decreased peptide potency in clone 3 without noticeable change in peptide efficacy, but reduced only peptide efficacy in clone 16. These observations were typical of the results expected if spare receptors exist in clone 3 but not in clone 16 [26-28]. We recently observed similar results in CHO cells stably transfected with the recombinant secretin receptor [29]. The efficient coupling of the VIP receptor to adenylate cyclase and the high number of receptors in clone 3 probably explained the fact that VIP pretreatment with high doses of VIP (10 and 100 nM) induced a persistent activation of the enzyme, even though the washing procedure was fully sufficient to clear the receptors in clone 16. The existence of spareness is of interest to clarify the definition of partial agonists and antagonists: for instance, VIP(2-28) was previously reported to be
405
E. Ciccarelli et al. / Regulatory Peptides 54 (1994) 397-407
Table 3 Adenylate cyclase activity (expressed in pmol cAMP formed/min/mg membrane protein) of membranes from clones 3 and 16 after pretreatment with increasing concentrations of VIP Stimulus
None Gpp[NH]p 10 - 4 Naf 10 - 2 M Forskolin 10 -4 M GTP 10 -3 M GTP 10 4 M +VIP 10 - 6 M ECso
Clone 3 Pretreatment with VIP 0
0.1 nM
1 nM
10 nM
100 nM
10_+3 366 _+20 264 -+20 74 _+8 26_+8 292_+ 15 0.2_+0.1
11_+3 381 _+25 282 -+ 15 79 + 5 25_+4 316_+ 18 0.2_+0.1
11_+3 396 + 20 294 -+ 18 72 -+8 25+4 320_+20 0.3_+0.1
39*_+5 666* + 35 383* -+20 159" -+ 12 63*+6 346*_+20 2.0_+0.3
43*_+5 747* + 40 416" _+30 188" _+20 77*+ l0 344*_+30 3.0_+0.3
6+2 163_+ 12 164 _+12 37 _+5 11_+2 119_+8 2.0 _+0.4
4+ 1 154_+8 157 _+9 35 _+6 10+3 70*_+4 3.0 _+0.3
5+2 155_+8 157 _+8 35 _+4 10_+2 51"_+3 3.0 _+0.4
7+2 174_+8 190 _+7 43 _+9 10_+3 40*_+3 3.0 _+0.4
Clone 16 None Gpp[NH]p 10 4 Naf 10 2 M Forskolin 10 -4 M GTP 10 -5 M G T P 10 - 4 M + V I P 10 6 M ECs0 VIP
6+3 166_+20 159 _+18 59 _+7 12_+3 135_+8 2.0 _+0.4
The results were the mean + S.E.M. of three experiments. * Indicate the values significantly different (P< 0.05) from the control, untreated clones. The ECs0 value (in nM) for VIP activation was also given.
o n rat tissues a partial a g o n i s t [21]; this p r o p e r t y a t t r i b u t e d to an intrinsic p r o p e r t y o f the m o l e c u l e w a s p r o b a b l y a c o n s e q u e n c e o f the b a l a n c e b e t w e e n the intrinsic activity o f t h e m o l e c u l e , the n u m b e r o f r e c e p t o r s , a n d their c o u p l i n g efficacy in t h e tissue studied. T h e p r e s e n t results d e m o n s t r a t e d t h a t the N-terminally deleted fragments of VIP and PACAP m u s t b e c o n s i d e r e d as partial a g o n i s t s w i t h an effic a c y d e p e n d i n g , inter alia o n t h e n u m b e r o f r e c e p t o r s e x p r e s s e d at the cell surface.
Acknowledgements T h i s w o r k w a s s u p p o r t e d by a S c i - C T 9 1 - 0 6 3 2 S c i e n c e P l a n f r o m the C o m m i s s i o n o f t h e E u r o p e a n C o m m u n i t i e s , by G r a n t s 3.4525.91 a n d 3.4514.94
f r o m the F o n d s de la R e c h e r c h e Scientifique M 6 d i cale (Belgium). W e t h a n k Dr. S. N a g a t a ( O s a k a B i o s c i e n c e Institute, O s a k a , J a p a n ) for their k i n d gift o f t h e V I P r e c e p t o r in t h e p C D M 8 p l a s m i d .
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cyclase-activating polypeptide have different tissue distributions, Endocrinology, 128 (1991) 3055-3065. Sreedharan, S.P., Patel, D.R., Huang, J.-X. and Goetzl, E.J., Cloning and functional expression of a human neuroendocrine vasoactive intestinal peptide receptor, Biochem. Biophys. Res. Commun., 193 (1993) 546-553. Spengler, D., Waeber, C., Pantaloni, C., Holsboer, F., Bockaert, J., Seeburg, P.H. and Journot, L., Differential signal transduetion by five splice variants of the PACAP receptor, Nature, 365 (1993) 170-175. Svoboda, M., Tastenoy, M., Ciccarelli, E., Sti~venart, M. and Christophe, J., Cloning of a splice variant of the pituitary adenylate cyclase-activating polypeptide (PACAP) type I receptor, Biochem. Biophys. Res. Commun., 195 (1993) 881-888. Pisegna, J.R. and Wank, S.A., Molecular cloning and functional expression of the pituitary adenylate cyclase-activating polypeptide type I receptor, Proc. Natl. Acad. Sci. USA, 90 (1993) 6345-6349. Robberecht, P., De Neef, P., Gourlet, P., Cauvin, A., Coy, D.H. and Christophe, J., Pharmacological characterization of the novel helodermin/VIP receptor present in human SUP-TI lymphoma cell membranes, Regul. Pept., 26 (1989) 117-126. Gourlet, P., De Neef, P., Woussen-Colle, M-C., Vandermeers, A., Vandermeers-Piret, M-C., Robberecht, P. and Christophe J., The activation of adenylate cyclase by pituitary adenylate cyclase activating polypeptide (PACAP) via helodermin-preferring VIP receptors in human SUP -T1 lymphoblastic membranes, Biochim. Biophys. Acta, 1066 (1991) 245-251. Fabre, C., El Battari, A,, Karamanos, Y., Couvineau, A., Salomon, R., Laburthe, M., Marvaldi, J., Pichon, J. and Luis, J., Glycosylation of VIP receptors: A molecular basis for receptor heterogeneity, Peptides, 14 (1993) 483-489. Robberecht, P., Cauvin, A., Gourlet, P. and Christophe, J., Heterogeneity of VIP receptors. In Pauwels, R., Lefebvre, R.A., and Herman, A.G. (Eds.), Second Workshop on Nonadrenergic Noncholinergic Mechanisms, Arch. Int. Pharmacodyn. Ther. Vol. 303, 1990, pp. 51-66. Connors, R.W., Sweet, R.W., Noveral, J.P., Pfarr, D.S., Trill, J.J., Shebuski, R.J., Berkowitz, B.A., Williams, D., Franklin, S. and Reff, M.E., DHFR coamplifieation oft-Pa in DHFR + bovine endothelial cells: in vitro characterization of the purified serine protease, DNA, 7 (1988) 651-661. Urlaub, G. and Chasin, L.A., Isolation of chinese hamster cell mutants deficient in dihydrofolate reductase activity, Proc. Natl. Acad. Sci. USA, 77 (1980) 4216-4220. Robberecht, P., Waelbroeck, M., Camus, J.-C., De Neef, P. and Christophe, J., Importance of disulfide bonds in receptors for vasoactive intestinal peptide and seeretin in rat
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pancreatic plasma membranes, Biochim. Biophys. Acta, 773 (1984) 271-278. Robberecht, P., Coy, D.H., De Neef, P., Camus, J.-C., Cauvin, A., Waelbroeck, M. and Christophe, J., [D-Phe4]Peptide histidine-isoleucinamide ([D-Phe4]PHI), a highly selective vasoactive-intestinal-peptide (VIP) agonist, discriminates VIP-preferring from secretin-preferring receptors in rat pancreatic membranes, Eur. J. Biochem., 165 (1987) 243-249. Salomon, Y., Londos, C. and Rodbell, M., A highly sensitive adenylate cyclase assay, Anal. Biochem.. 58 (1974) 541-548. Waelbroeck, M., Robberecht, P., De Neef, P., Chatelain, P. and Christophe, J., Binding of vasoactive intestinal peptide and its stimulation of adenylate cyclase through two classes of receptors in rat liver membranes. Effects of 12 secretin analogues and 2 secretin fragments, Biochim. Biophys. Acta. 678 (1981) 83-90. Gossen, D., Tastenoy, M., Robberecht, P., and Christophe, J., Secretin receptors in the neuroglioma hybrid cell line NG 108-15. Characterization and regulation of their expression, Eur. J. Biochem., 193 (1990) 149-154. Jensen, R.T., Charlton, C.G., Adachi, H., Jones, S.W., O'Donohue, T.L. and Gardner, J.D., Use of ~251-secretin to identify and characterize high-affinity secretin receptors on pancreatic acini, Am. J. Physiol., 245 (1983) G186G195. Robberecht, P., Coy, D.H., De Neef, P., Camus, J.-C., Cauvin, A., Waelbroeck, M. and Christophe, J., Interaction of vasoactive intestinal peptide (VIP) and N-terminally modified VIP analogs with rat pancreatic, hepatic and pituitary membranes, Eur. J. Biochem., 159 (1986) 45-49. Couvineau, A., Rouyer-Fessard, C., Fournier, A., St-Pierre, S., Pipkorn, R. and Laburthe, M., Structural requirements for VIP interaction with specific receptors in human and rat intestinal membranes: effect of nine partial sequences, Biochem. Biophys. Res. Commun., 121 (1984) 493-498. Fournier, A. Saunders, J.K., Boulanger, Y. and St. Pierre, S.A., Conformation analysis of vasoactive intestinal peptide and related fragments, Ann. N.Y. Acad. Sci., 527 (1988) 51-67. Goossens, J.F., Pommery, N., Lohez, M., Pommery, J., Helbecque, N., Cotelle, P., Lhermitte, M. and Henichart, J.P., Antagonistic effect of vasoactive intestinal peptide fragment, vasoaetive intestinal peptide(1-11), on guinea pig trachea smooth muscle relaxation, Mol. Pharmaeol., 41 (1991) 104-109. Inooka, H., Endo, S., Kitada, C., Mizuta, E. and Fujino, M., Pituitary adenylate cyclase activating polypeptide (PACAP) with 27 residues, Int. J. Pept. Prot. Res., 40 (1992) 456-464.
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[25] Wray, V., Kakoschke, C., Nokihara, K. and Naruse, S., Solution structure of pituitary adenylate cyclase activating polypeptide by nuclear magnetic resonance spectroscopy, Biochemistry, 32 (1993) 5832-5841. [26] Arii~ns, E.J., Van Rossum, J.M. and Koopman, P.C., Receptor reserve and threshold phenomena. I. Theory and experiments with autonomic drugs tested in isoled organs, Arch. Int. Pharmacodyn., 127 (1960) 459-478. [27] Van Rossum, J.M. and Afians, E.J., Receptor-reserve and threshold-phenomena. II. Theories on drug-action and a
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