Abstract
A vasoactive intestinal polypeptide (VIP) analog, acylated on the amino-terminal histidine by hexanoic acid (C6-VIP), behaved as a VPAC2 preferring agonist in binding and functional studies on human VIP receptors, and radioiodinated C6-VIP was a suitable ligand for binding studies on wild-type and chimeric receptors. We evaluated the properties of C6-VIP, its analog AcHis1-VIP, and the VPAC2-selective agonist Ro 25-1553 on the wild-type VPAC1 and VPAC2 receptors and on the chimeric receptors exchanging the different domains between both receptors. VIP had a normal affinity and efficacy on the chimeras starting with the amino-terminal VPAC2 receptor sequence. The binding and functional profile of these chimeric receptors suggested that the high affinity of Ro 25-1553 for VPAC2 receptors is supported by the amino-terminal extracellular domain, whereas the ability to prefer C6-VIP over VIP is supported by the VPAC2 fifth transmembrane (TM5)-EC3 receptor domain. These results further support the hypothesis that the central and carboxyl-terminal regions of the peptide (modified in RO 25-1553) recognize the extracellular amino-terminal region domain, whereas the amino-terminal VIP amino acids bind to the TM receptor core. VIP had a reduced affinity and efficacy on the N-VPAC1/VPAC2 and on the N→EC2-VPAC1/VPAC2 chimeric receptors. C6-VIP behaved as a high-affinity agonist on these constructions. The antagonists [AcHis1,d-Phe2,Lys15,Arg16,Leu27]VIP(3-7)/GRF(8-27) and VIP(5-27) had comparable affinities for the wild-type receptors and for the two latter chimeras, supporting the hypothesis that these chimeras were properly folded but unable to reach the high-agonist-affinity, active receptor conformation in response to VIP binding.
The 28-amino-acid neuropeptide vasoactive intestinal polypeptide (VIP) acts through two distinct receptors named VPAC1 and VPAC2 (Harmar et al., 1998). These two G protein-coupled, seven-TM-helix receptors have only 51% similarity (Couvineau et al., 1994; Svoboda et al., 1994) but recognize with a comparable, high-affinity VIP and the pituitary adenylate cyclase-activating polypeptide (PACAP). VPAC1 and VPAC2 receptors are differentially distributed in tissues (Ishihara et al., 1992; Usdin et al., 1994; Vertongen et al., 1997) and cell lines (Vertongen et al., 1996).
Two selective VPAC2 receptors agonists were recently discovered: Ro 25-1553 (Gourlet et al., 1997a) and Ro 25-1392 (Xia et al., 1997) are cyclic analogs that differ from VIP by acetylation of the amino terminus, some mutations in the core of the peptide, the presence of a lactam bridge, and two additional lysine residues in the carboxyl terminus. There are no known selective high-affinity antagonist for the VPAC2 receptors.
Two selective VPAC1 receptor agonists have also been recently developed: one of these agonists is a derivative of the chicken secretin molecule, and the second, a hybrid peptide with the VIP amino terminus and a growth hormone-releasing peptide (GRF)-like carboxyl-terminal sequence (Gourlet et al., 1997b). A selective VPAC1 receptor antagonist was also obtained through the single substitution of the VIP/GRF hybrid peptide (Gourlet et al., 1997c).
The group of Gozes recently described lipophilic VIP derivatives (Gozes and Fridkin, 1992; Gozes et al., 1994) and observed that the lipophilic VIP derivative obtained through the amidation of the amino terminus of VIP with an 18-carbon fatty acid (stearyl VIP) was 100-fold more potent than VIP in promoting neuronal cell survival in dissociated spinal cord cells (Gozes et al., 1995) but did not increase cAMP levels. We observed that this compound recognizes the recombinant rat and human VPAC1 and VPAC2 receptors with an affinity comparable to that of VIP and with a preference for the VPAC2 over the VPAC1receptor but behaves like a partial agonist on adenylate cyclase stimulation (Gourlet et al., 1998). Related compounds like myristoyl- and palmitoyl-VIP also had a low intrinsic activity and behaved as VPAC2-preferring partial agonists and/or competitive antagonists. These results suggested that acylation of the amino terminus might contribute to the VPAC2receptor selectivity of lipophilic VIP derivatives, Ro 25-1553 and Ro 25-1392, but that the length of the alkyl chain acylating the amino-terminal histidine residue may influence the intrinsic activity of the peptides.
These results prompted us to synthesize other derivatives, acylated at the amino terminus but with a shorter acyl chain. The hexanoyl-VIP (C6-VIP) described in the present study had a 4-fold lower and 3-fold higher affinity than VIP for the human VPAC1 and VPAC2 receptors, respectively, and stimulated maximally the adenylate cyclase activity. The binding and functional properties of this new VPAC2 preferring agonist were compared with the properties of Ro 25-1553 and acetyl-His1- (AcHis1)-VIP. By testing the ability of these agonists to recognize chimeric receptors, consisting of the interchange of the different domains of one receptor subtype grafted onto the core of the other receptor subtype, we were able to establish that the VPAC2 selectivities of Ro 25-1553 and C6-VIP were supported by different domains of the receptors.
Materials and Methods
All of the experiments were conducted on Chinese hamster ovary (CHO) cell membranes expressing the recombinant wild-type human VPAC1, VPAC2, or mutated receptors. The cell lines expressing the VPAC1and VPAC2 receptors have been detailed previously (Ciccarelli et al., 1994; Svoboda et al., 1994). All chimeric receptors were constructed by the PCR overlap extension strategy. Briefly, cDNA fragments overlapping at their 5′ or 3′ extremity were generated using human VPAC1 and VPAC2receptor cDNA as templates and appropriate chimeric primers. After purification of these fragments using the High Pure PCR Product Purification Kit (Boehringer-Mannheim Biochemica, Mannheim, Germany), they were used in a round of PCR overlap extension. The use of a phosphorylated forward primer surrounding the ATG initiation codon allowed us to obtain a 5′ hemiphosphorylated cDNA fragment. This particularity combined with the presence of a 3′-A overhang resulting from the terminal transferase activity of Taq polymerase, allowing the unidirectional cloning of the chimeric receptors in pCR 3.1-Uni (InVitrogen, San Diego, CA), suitable for both prokaryotic and eukaryotic expressions. The first series of chimeric receptor was generated by the substitution of different domains in the VPAC2 receptor by the counterpart of VPAC1 receptor. N-VPAC1/VPAC2 chimera was generated by combining codons 1 to 143 of the VPAC1 receptor with codons 128 to 438 of the VPAC2 receptor. The N→EC1-VPAC1/VPAC2combines codons 1 to 216 of the VPAC1 receptor with codons 204 to 438 of VPAC2 receptor. The receptor chimeric N→EC2-VPAC1/VPAC2combines the codons 1 to 293 of VPAC1 receptor with the codons 281 to 438 of VPAC2 receptor. The latest chimeric receptor of this series (N→EC3-VPAC1/VPAC2) was generated by combining codons 1 to 373 of the VPAC1 receptor with the codons 361 to 438 of the VPAC2 receptor. The other series of chimeric receptor was generated by the substitution of the different domain in the VPAC1 receptor by the counterpart of VPAC2 receptor. The N-VPAC2/VPAC1 construction combined the VPAC2 receptor codons 1 to 127 with the 144 to 457 VPAC1 receptor codons. The N→EC1-VPAC2/VPAC1construction was generated by combining the codons 1 to 203 of VPAC2 receptor and the codons 217 to 457 of VPAC1 receptor. The N→EC2-VPAC2/VPAC1chimera combined the codons 1 to 280 of the VPAC2receptor with the codons 297 to 457 of VPAC1receptor. And the last chimeric receptor (N→EC3-VPAC2/VPAC1) has the codons 1 to 360 of the VPAC2 receptor and the codons 374 to 457 of the VPAC1 receptor. The polymerase chain reactions were performed using the Expand Long Template system (Boehringher, Mannheim) in the Geneamp 2450 thermocycler (Perkin-Elmer Cetus, Norwalk, CT). All sequences were verified using ABI Prism By Dye Terminator Cycle Sequencing Ready Reaction Kit (PE Applied Biosystems, Norwalk, CT).
The methodology for cell transfection, as well as the cell culture medium, has been detailed previously (Van Rampelbergh et al., 1996). The cell clones expressing the different constructions were selected by testing the ability of the 10 μM VIP to stimulate the adenylate cyclase. One of the constructs (N→EC2-VPAC1/VPAC2) could not be identified by this method, so we also attempted to detect its expression in binding studies with 125I-VIP,125I-C6-VIP, and125I-VIP1 antagonist with negative results.
Membranes were prepared from scraped cells lysed in 1 mM NaHCO3 solution and immediate freezing in liquid nitrogen. After thawing, the lysate was first centrifuged at 4°C for 10 min at 400g; the supernatant was further centrifuged at 20,000g for 10 min. The pellet, resuspended in 1 mM NaHCO3, was used immediately as a crude membrane fraction.
Binding studies were performed as described previously (Gourlet et al., 1997a) using either 125I-VIP,125I-Ro 25-1553, or125I-C6-VIP. The three tracers were radiolabeled similarly and had comparable specific radioactivity (Gourlet et al., 1997b). In all cases, the nonspecific binding was defined as residual binding in the presence of 1 μM VIP. Binding was performed at 37°C in a total volume of 120 μl containing 20 mM Tris-maleate, 2 mM MgCl2, 0.1 mg/ml bacitracin, and 1% BSA (pH 7.4) buffer. From 3 to 30 μg of protein was used per assay. Bound and free radioactivity were separated by filtration through glass-fiber GF/C filters presoaked for 24 h in 0.01% polyethyleneimine and rinsed three times with a 20 mM (pH 7.4) sodium phosphate buffer containing 1% BSA.
Adenylate cyclase activity was determined according to the procedure ofSalomon et al. (1974). Membrane proteins (3–15 μg) were incubated in a total volume of 60 μl containing 0.5 mM [α-32P]ATP, 10 μM GTP, 5 mM MgCl2, 0.5 mM EGTA, 1 mM cAMP, 1 mM theophylline, 10 mM phospho(enol)pyruvate, 30 μg/ml pyruvate kinase, and 30 mM Tris · HCl at final pH 7.8.
The peptides used were synthesized in our laboratory as described previously (Gourlet et al., 1997b, 1998). The 1-hydroxybenzotriazole derivative of hexanoic acid was coupled to the amino terminus of VIP before cleavage and deprotection. The peptide purity was assessed by capillary electrophoresis, and the conformity was assessed by electrospray mass spectrometry.
All competition curves and dose-effect curves were analyzed by a nonlinear regression program (Prism; GraphPAD, San Diego, CA). The differences between the IC50, EC50, and maximal efficacy values were tested for statistical significance by Student's t test;P < .05 was accepted as being statistically significant.
Results
Properties of C6-VIP on Recombinant VPAC1 and VPAC2 Receptors.
On VPAC2 receptors, the inhibition curves were identical when 125I-VIP and125I-Ro 25-1553 were used as tracers (data not shown). AcHis1-VIP and C6-VIP were 6- to 4-fold less potent than VIP in binding studies on human recombinant VPAC1receptors, but 2- to 3-fold more potent than VIP in binding studies on recombinant VPAC2 receptors, respectively (Table1). Ro 25-1553 was 1000-fold less potent and 4-fold more potent than VIP on the VPAC1 and VPAC2 receptors, respectively.
C6-VIP increased adenylate cyclase activity on membranes expressing the VPAC1 and VPAC2 receptors. The maximal stimulation through both receptors was identical to that of VIP (Figs. 1 and2); C6-VIP was 6-fold less potent and 20-fold more potent than VIP on the VPAC1 and VPAC2 receptors, respectively. As previously reported (Gourlet et al., 1997a), Ro 25-1553 was a partial agonist on the VPAC1 receptors (Fig. 1). AcHis1-VIP was comparable to C6-VIP on the VPAC1receptors but had a lower potency than C6-VIP on the VPAC2 receptors (Table 1).
Thus, C6-VIP, like Ro 25-1553, was a superagonist compared with VIP on the VPAC2 receptors.
Contribution of Amino-Terminal Portion and TM5 to EC3Region of Receptor to VPAC2 Receptor Preference of Ro 25-1553 and C6-VIP, Respectively.
In a first set of experiments, we evaluated the properties of a chimeric receptor consisting in the amino-terminal (extracellular) VPAC2 receptor domain followed by the seven-TM-helix domain of the VPAC1 receptor (N-VPAC2/VPAC1). On that chimeric receptor, IC50 values of binding inhibition were 0.5, 3.0, 5.0, and 5.0 nM for VIP, AcHis1-VIP, C6-VIP, and Ro 25-1553, respectively (Fig. 1B and Table 1). Thus, the IC50 values of VIP, AcHis1-VIP, and C6-VIP were comparable to their VPAC1 receptor IC50 values, and that of Ro 25-1553 was comparable to its VPAC2 receptor IC50 value. In addition, the maximal stimulatory effect of Ro 25-1553 was higher than those of VIP, AcHis1-VIP, and C6-VIP. Thus, the amino-terminal domain of the VPAC2receptor was essential for high-affinity Ro 25-1553 recognition but not for that of C6-VIP. In an attempt to identify the receptor region that supported the preferential C6-VIP > VIP recognition by the VPAC2 receptor, we tested additional chimeric receptors increasing the contribution of the VPAC2 receptor sequence (Figs. 1C and 2, A and B). As shown in Figs. 1 and 2, chimeric receptors with the amino-terminal to EC2-VPAC2receptor sequence (Fig. 2A) had, like VPAC1receptor, a preference for VIP over C6-VIP. In contrast, the chimeric receptor with the amino-terminal to EC3-VPAC2 receptor sequence (Fig. 2B) preferred (like the VPAC2 receptor; Fig. 2C) C6-VIP over VIP. These results suggested that the hexanoyl anchoring point is somewhere between TM5 and EC3.
In a second set of experiments, we attempted to express the “mirror image” chimeric receptors, starting with the VPAC1 and followed by the complementary VPAC2 receptor sequence (Fig.3). The binding and functional properties of the last chimeric receptor (N→EC3-VPAC1/VPAC2) were identical with wild-type VPAC1 receptor (Fig. 3C). This result supported the hypothesis that the TM7 and carboxyl-terminal receptor region did not participate in the recognition of either Ro 25-1553 or C6-VIP. We were unable to identify any clone expressing the N→EC2-VPAC1/VPAC2chimeric receptor (not shown) or to perform125I-VIP binding studies on the amino-terminal VPAC1/VPAC2 and N→EC1-VPAC1/VPAC2chimeric receptors. The EC50 value of VIP on these two chimeras for adenylate cyclase stimulation was close to 50 nM, significantly higher than its EC50 value for both wild-type receptors (Fig. 3, D and E, and Table 1). Ro 25-1553 was a partial agonist on these chimeras with an EC50value of 1 μM. AcHis1-VIP behaved as VIP (Table1). C6-VIP, in contrast, had a higher affinity and efficacy than VIP on these two chimeric receptors (Fig. 3, D and E, and Table 1).
We anticipated that radioiodinated C6-VIP might be an appropriate ligand to study the binding properties of these two chimeric receptor. Specific125I-C6-VIP binding to the chimeric receptors was indeed measurable, and competition curves yielded IC50 values of 3, 100, 100, and 3000 nM for C6-VIP, VIP, AcHis1-VIP, and Ro 25-1553, respectively, in N-VPAC1/VPAC2 chimeric receptor (Fig. 3A and Table 1) and 5, 80, 80, and 200 mM for C6-VIP, VIP, AcHis1-VIP, and Ro 25-1553, respectively, in N→EC1-VPAC1/VPAC2chimeric receptors (Fig. 3B and Table 1).125I-C6-VIP was also used as a tracer to characterize the VPAC1, VPAC2, and N-VPAC2/VPAC1 receptors. The competition curves for the four unlabeled peptides were not different from those obtained with 125I-VIP or125I-Ro 25-1553 (data not shown).
The observations that VIP had a lower affinity for chimeras beginning with the VPAC1 receptor sequence and that it behaved as a partial agonist with respect to C6-VIP on these constructions suggested that the chimeric receptors were either misfolded or unable to reach the active receptor conformation in response to VIP. To discriminate between these two hypotheses, we measured the affinity of two VIP receptor antagonists, anticipating that a misfolded receptor would have a lower affinity for such ligand, whereas a nonactivatable receptor should have a lower affinity for agonist only (see Discussion).
[AcHis1,d-Phe2,Lys15,Arg16,Leu27]VIP(3-7)/GRF(8-27) and the VPAC2 preferring VIP fragment VIP(5-28) antagonized the effect of VIP on adenylate cyclase stimulation through the wild-type and chimeric receptors. These values were confirmed in functional assays: the VIP(5-28) and the VPAC1antagonist K i values are summarized in Table 1. At a concentration of 1 μM, the VPAC1antagonist did not significantly affect the VIP dose-effect curves on VPAC2 and on N-VPAC2/VPAC1 receptors.
Discussion
The natural ligands VIP and PACAP(1-27) have a clear preference for VPAC1 over VPAC2receptors. In contrast, Ro 25-1553 and Ro 25-1392 are highly selective for VPAC2 receptors. They differ from VIP by the acetylation of the amino-terminal histidine, several mutations in the central part of the molecule, a lactam ring between residues 21 and 25, and a basic carboxyl-terminal tail. Each modification could a priori contribute to the high affinity for the VPAC2receptor and the low affinity for the VPAC1receptor.
The following experimental data support the hypothesis that acylation of the amino terminus of these peptides probably does contribute to their VPAC2 selectivity. 1) AcHis1-VIP was reported to have a 2-fold higher affinity than VIP on the “helodermin preferring receptor” from the SUP-T1 lymphoblastic cell line (Svoboda et al., 1994), a receptor subsequently identified as the human VPAC2receptor (Robberecht et al., 1996), and a 5-to 10-fold lower affinity than VIP on the cloned rat VPAC1 receptor (Gourlet et al., 1996a) (these data were confirmed in the present report on recombinant human VPAC1 and VPAC2 receptors: Table 1). 2) Three fatty acyl derivatives of VIP (myristoyl-, palmitoyl-, and stearyl-[N,Leu17]VIP), obtained through the conjugation of a fatty acid to the amino terminus of the peptide chain by an amide bond, also had a lower and higher affinity than VIP for the human VPAC1 and VPAC2receptors, respectively. These three modified peptides were, however, partial agonists on the VPAC1 and VPAC2 receptors. Their intrinsic activity was in fact extremely low on the VPAC2 receptors, where they behaved like partial agonists (Gourlet et al., 1998). These results prompted us to test the properties of an amino-terminally acylated VIP derivative modified with hexanoic acid. The C6-VIP derivative had, compared with VIP, a 3-fold higher and a 4-fold lower IC50 value in binding studies on VPAC1 and VPAC2 receptors, respectively. It behaved as a full and selective VPAC2 agonist on adenylate cyclase stimulation.
We then attempted to identify the domains of the receptor implicated in the high- and low-affinity interaction of Ro 25-1553 and C6-VIP for the VPAC2 and VPAC1 receptors, respectively. The results obtained with the first N-VPAC2/VPAC1 chimera, in the amino-terminal part of the VPAC2 receptor grafted onto the VPAC1 receptor TM domain, indicated clearly that the VPAC receptor amino-terminal domain is responsible for selective Ro 25-1553 recognition. This is not the case for AcHis1-VIP and C6-VIP, which had an identical potency (lower than that of VIP) on both VPAC1 and N-VPAC2/VPAC1 receptors. These results supported the hypothesis that the amino-terminal sequence of the ligands interacted with the core of the receptor, whereas the remainder of the molecule, probably starting around amino acid 9 (the first mutated residue in Ro 25-1553) interacts with the extracellular amino-terminal domain of the receptor. These conclusions are in line with several observations on closely related receptors. 1) The amino-terminal domain of the secretin receptor is essential for the high-affinity interaction of secretin with its receptor (Vilardaga et al., 1995), and recognizes amino acids 8 to 15 (Gourlet et al., 1996b) and 22 (Dong et al., 1999) of secretin; the amino-terminal portions of the PACAP receptor (Van Rampelbergh et al., 1996; Hashimoto et al., 1997) and of the parathyroid hormone/parathyroid hormone-related peptide receptor (Zhou et al., 1997) also participate in the selective recognition of their respective ligands. 2) The amino terminus of secretin, and particularly the aspartate residue in position 3, interacts with several receptor residues located in the first and second TM domains (Vilardaga et al., 1996; Di Paolo et al., 1998,1999).
We should very much like to identify the receptor region that recognizes the amino-terminal VIP histidine because this interaction is very important for receptor activation. Unfortunately, this histidine residue is likely also involved in stabilizing the active VIP conformation: conceiving VIP analogs that retain the right conformation but interact differentially with the receptor histidine anchoring site is by no means a trivial problem. We were therefore extremely interested by the observation that C6-VIP is a full agonist, with a greater affinity than VIP for VPAC2 receptor and only slightly lower affinity than VIP for VPAC1 receptor. The VPAC receptor region that is responsible for the differential VIP/C6-VIP recognition must be very close to the histidine anchoring site.
VIP was more potent than C6-VIP on the N→EC2-VPAC2/VPAC1chimeric receptor but less potent than VIP on the N→EC3-VPAC2/VPAC1chimeric receptor and on the VPAC2 receptor: the hexanoyl group probably recognized a binding site situated somewhere between TM5 and EC3, and the amino-terminal histidine binding site (which is essential for VPAC1 receptor activation) should be sought in the same region or in its immediate vicinity.
To further support this hypothesis, we attempted to express and study the binding and functional properties of the “mirror image” chimeras. Our results confirmed that the VPAC2TM7 sequence is not sufficient to support preferential C6-VIP > VIP recognition (Fig. 3, C and F). We were, unfortunately, unable to detect the expression of one of these chimeras cloned in CHO cells (N→EC2-VPAC1/VPAC2) by functional or binding studies, and VIP had a surprisingly low affinity and efficacy on the two remaining chimeras (Fig. 2, A, B, D, and E). We subsequently observed that C6-VIP had a higher affinity and efficacy than VIP on these chimeras (Table 1). Taken together, these results suggested that they could not be fully activated by the natural agonist, VIP, and that acylation with a 6-carbon acyl chain was sufficient to rescue the ability of the peptides to stabilize the active receptor conformation.
As pointed out recently by Colquhoun (1999), it is not possible to evaluate separately the affinity of agonists for the resting inactive receptors and their effect on receptor activation. If we assume that the agonist initially recognizes inactive receptors and then induces a conformational change to the active receptor conformation:
Removal of the amino-terminal VIP amino acids, acylation of the His1 by long-chain fatty acids, or replacement of amino acids 2, 4, or 6 by d-amino acids markedly reduced not only the affinities of the peptides but also their intrinsic efficacies at VPAC1 and VPAC2 receptors: in the case of VIP related peptides, K 1 probably reflects the peptide anchoring through the central and carboxyl-terminal amino acids, andK 2, additional interactions between the amino-terminal VIP amino acids and the receptor in its active conformation.
To test whether the two VPAC1/VPAC2 chimeric receptors had a normal conformation in the resting state, we measured their affinities (K 1) for two antagonists: the VPAC1 antagonist ([AcHis1,d-Phe2,Lys15,Arg16,Leu27]VIP(3-7)/GRF(8-27)] and VIP(5-28). The selectivity of the VPAC1antagonist is conferred mainly by its the carboxyl-terminal (8-27) region, that is, a peptide region thought to interact with the extracellular amino-terminal receptor domain. Our results, indicating that it had a high affinity for the N-VPAC1/VPAC2 chimeric receptor and low affinity for the N-VPAC2/VPAC1 chimeric receptor, further supported this hypothesis. They also suggested that the resting conformations of the amino-terminal domains of the VPAC1/VPAC2 chimeric receptors were normal or almost normal, because the antagonist affinities (K 1) were only slightly lower than those of the VPAC1 receptor. In contrast, the ability of the TM chimeric receptors domain to recognize the amino-terminal VIP sequence and to change conformation in response to agonist recognition (K 2) was reduced, so we cannot exclude the hypothesis that the chimeric receptor TM domain had an altered conformation or was misoriented with respect to the amino-terminal domain. Merely acylating the amino-terminal histidine residue with an hexanoyl group (equivalent in size to a Leu or an Ile) was sufficient to “rescue” the affinity of the agonists and their ability to activate the receptor (K 2), suggesting that the energy input necessary to activate the receptor increased only slightly in the chimeric receptors compared with VPAC1 and VPAC2 receptors.
To conclude, our results suggested that 1) that the selectivity of the selective VPAC2 agonist, Ro 25-1553, and of the selective VPAC1 antagonist was supported by the extracellular amino-terminal receptor domain. The ability of the receptor to recognize preferentially either VIP or the C6-VIP analog, in contrast, depended on the sequence of the carboxyl-terminal TM5 to EC3receptor domain. 2) The low affinity of the chimeric N-VPAC1/VPAC2 and N→EC1-VPAC2/VPAC1receptors for VIP and AcHis1-VIP reflected the reduced ability of these peptides to activate the chimeras rather than their inability to recognize the resting receptor conformation. 3) Acylation of the amino-terminal VIP histidine by a hexanoyl chain restored the ability of the peptides to activate these chimeric receptors. 4) Radioiodinated C6-VIP was a good ligand for VPAC1, VPAC2, and chimeric receptor identification.
Footnotes
- Received June 14, 1999.
- Accepted September 7, 1999.
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Send reprint requests to: Dr. Magali Waelbroeck, Department of Biochemistry and Nutrition, Faculty of Medicine, UniversitéLibre de Bruxelles, Bât. G/E, CP 611, 808 Route de Lennik, 1070 Brussels, Belgium. E-mail: mawadbr{at}ulb.ac.be
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This work was supported by grants from the European Community (PAVE project), the Communauté Française de Belgique (ARC), and an Interuniversity Poles of Attraction, Primer Minister Office, Federal Government, Belgium. M.G.J. was a recipient of a Marie Curie Research Training Grant (European Commission).
Abbreviations
- VIP
- vasoactive intestinal polypeptide
- PACAP
- pituitary adenylate cyclase-activating polypeptide
- GRF
- growth hormone-releasing peptide
- C6-VIP
- hexanoyl-VIP
- CHO
- Chinese hamster ovary
- TM
- transmembrane
- AcHis1
- acetyl-His1
- The American Society for Pharmacology and Experimental Therapeutics