Development of selective agonists and antagonists for the human vasoactive intestinal polypeptide VPAC(2) receptor.

Ro 25-1553 is a cyclic VIP derivative with a high affinity for the VPAC(2) receptor subtype. Our goal was to identify the modifications that support its selectivity for VPAC(2) receptors, and to develop a VIP or Ro 25-1553 analog behaving as a high affinity, VPAC(2) selective antagonist. The selectivity of Ro 25-1553 for the human receptor was supported mainly by the acetylation of the amino-terminus, by the introduction of a lysine residue in position 12, and by the carboxyl-terminal extension. The lactam bridge created between positions 21 and 25 contributed to the affinity of the compound for the VIP receptors but participated only marginally to its selectivity. Deletion of the first five aminoacid residues led to a low affinity antagonist with a low selectivity. Introduction of a D-Phe residue in position 2 reduced the affinity, the selectivity and the intrinsic activity, the compound being a partial agonist. Myristoylation of the amino-terminus of [K(12)]VIP(1-26) extended carboxyl-terminally with the -K-K-G-G-T sequence of Ro 25-1553 led to a high affinity, selective VPAC(2) receptor antagonist. This molecule represents the first selective human VPAC(2) receptor antagonist described to date.

Characterization of a novel VPAC(1) selective agonist and identification of the receptor domains implicated in the carboxyl-terminal peptide recognition.

Vasoactive Intestinal Polypeptide (VIP) interacts with a high affinity to two subclasses of G protein coupled receptors named VPAC(1) and VPAC(2), and has a 3 - 10 fold preference for VPAC(1) over VPAC(2) receptors. Selective ligands for each receptor subclass were recently described. [R(16)]-PACAP (1 - 23) and [L(22)]-VIP are two selective VPAC(1) agonists. Chimaeric human VPAC(2)-VPAC(1) recombinant receptors expressed in CHO cells were used to identify the receptor domains implicated in these two selective ligands recognition. The VPAC(2) preference for [R(16)]-PACAP (1 - 27) over [R(16)]-PACAP (1 - 23) did not require the receptor's NH(2)-terminus domain but involved the whole transmembrane domain. In contrast, the selectivity of [L(22)]-VIP depended only on the presence of the NH(2) terminus and EC(2) domains of the VPAC(1) receptor. The present data support the idea that in the GPCR-B family of receptors the different selective ligands require different domains for their selectivity, and that the peptides carboxyl terminal sequence (amino acids 24 - 27) folds back on the transmembrane receptor domain, close to the peptides, aminoterminus.

Different vasoactive intestinal polypeptide receptor domains are involved in the selective recognition of two VPAC(2)-selective ligands.

A vasoactive intestinal polypeptide (VIP) analog, acylated on the amino-terminal histidine by hexanoic acid (C(6)-VIP), behaved as a VPAC(2) preferring agonist in binding and functional studies on human VIP receptors, and radioiodinated C(6)-VIP was a suitable ligand for binding studies on wild-type and chimeric receptors. We evaluated the properties of C(6)-VIP, its analog AcHis(1)-VIP, and the VPAC(2)-selective agonist Ro 25-1553 on the wild-type VPAC(1) and VPAC(2) 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 VPAC(2) receptor sequence. The binding and functional profile of these chimeric receptors suggested that the high affinity of Ro 25-1553 for VPAC(2) receptors is supported by the amino-terminal extracellular domain, whereas the ability to prefer C(6)-VIP over VIP is supported by the VPAC(2) fifth transmembrane (TM5)-EC(3) 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-VPAC(1)/VPAC(2) and on the N-->EC(2)-VPAC(1)/VPAC(2) chimeric receptors. C(6)-VIP behaved as a high-affinity agonist on these constructions. The antagonists [AcHis(1),D-Phe(2),Lys(15),Arg(16), Leu(27)]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.

Vasoactive intestinal polypeptide VPAC1 and VPAC2 receptor chimeras identify domains responsible for the specificity of ligand binding and activation.

In order to identify the receptor domains responsible for the VPAC1 selectivity of the VIP1 agonist, [Lys15, Arg16, Leu27] VIP (1-7)/GRF (8-27) and VIP1 antagonist, Ac His1 [D-Phe2, Lys15, Arg16, Leu27] VIP (3-7)/GRF (8-27), we evaluated their binding and functional properties on chimeric VPAC1/VPAC2 receptors. Our results suggest that the N-terminal extracellular domain is responsible for the selectivity of the VIP1 antagonist. Selective recognition of the VIP1 agonist was supported by a larger receptor area: in addition to the N-terminal domain, the first extracellular loop, as well as additional determinants in the distal part of the VPAC1 receptor were involved. Furthermore, these additional domains were critical for an efficient receptor activation, as replacement of EC1 in VPAC1 by its counter part in the VPAC2 receptor markedly reduced the maximal response.

Vasoactive intestinal polypeptide receptor VPAC(1) subtype is predominant in rat prostate membranes.

BACKGROUND: The 28-amino-acid neuropeptide vasoactive intestinal peptide (VIP) might play an important role in the physiology of the prostate, since it stimulates glandular secretion, inhibits muscle contraction, stimulates proliferation of epithelial cells, and increases the secretion of prostate-specific antigen (PSA). This neuropeptide may act through interaction with two types of high-affinity receptors, named VPAC(1) and VPAC(2) receptors. Recently, selective agonists and antagonists for each receptor subtype were synthesized. We used them to identify the VIP receptor subclass expressed in rat prostatic tissue. METHODS: We tested the capacity of selective labeled and unlabeled agonists and antagonists of VPAC(1) and VPAC(2) receptors to bind to rat prostatic membranes and to stimulate or prevent the stimulation of adenylate cyclase activity. RESULTS: The following selective peptides were used: VPAC(1) agonist ([K(15), R(16), L(27)] VIP (1-7)/GRF (8-27)); VPAC(1) antagonist (PG 97-269); and VPAC(2) agonist (RO 25-1553). The IC(50) values of [(125)I]-VIP binding inhibition for the different peptides in rat prostatic membranes were: VIP (1.7 nM) < VPAC(1) agonist (20 nM) < VPAC(1) antagonist (40 nM) < VPAC(2) agonist (329 nM). The EC(50) values of adenylate cyclase stimulation were similar to the IC(50) values for each peptide, and the Ki values for the VPAC(1) antagonist, inhibiting the adenylate cyclase activity stimulated by VIP and the VPAC(1) agonist, were 22 and 35 nM, respectively. Comparison of binding of [(125)I]-VIP and of [(125)I]-RO 25-1553 indicates the presence of 80% of VPAC(1) and 20% VPAC(2) receptors. CONCLUSIONS: In rat prostate membranes, VPAC(1) receptors are largely predominant. Binding studies were compatible with a ratio of 80/20 of VPAC(1)/VPAC(2) receptors, whereas functionally only VPAC(1) receptors were detected.

Mutations of aromatic residues in the first transmembrane helix impair signalling by the secretin receptor.

The secretin amino terminal residues are essential for high affinity binding to the cognate receptor and for the subsequent activation of adenylate cyclase. It has been already established that two basic residues of the receptor TM 2 are involved in the interaction with aspartate 3 of the ligand. The present work investigated the hypothesis that two conserved tyrosine residues of the TM 1 (Tyrosines 124 and 128) could also participate to the positioning of the amino terminus of the ligand. Tyrosines 124 and 128 were mutated into alanine and histidine residues, and the properties of the mutant receptors, expressed in CHO cells, were compared with those of the wild-type receptor. Mutation of tyrosine 124 to Ala or His decreased the affinity of the receptor for secretin, [Glu3]secretin, [Asn3]secretin and the secretin fragment 2-27, and reduced the intrinsic activity of [Asn3]secretin. Mutation of tyrosine 128 to Ala, but not to His reduced 50-fold secretin and [Asn3]secretin affinity but only 3-fold that of [Glu3]secretin. Secretin and [Glu3]Sn were equipotent in that mutant receptor. These results suggested that tyrosine 128 of the secretin receptor interacted directly with the [Asp3] residue of secretin and thus that the amino terminal domain of secretin interacts with amino acids buried in both the TM 1 and TM 2 helices.

Properties of a recombinant human secretin receptor: a comparison with the rat and rabbit receptors.

A secretin receptor was cloned from a commercial human pancreatic complementary DNA (cDNA) bank. The amino acid sequence deduced from the nucleotide sequence differed slightly from the three different sequences previously published, suggesting a genetic polymorphism of the human receptor. The binding properties of the receptor were evaluated by testing natural secretin, related peptides, and synthetic analogs or fragments on membranes of Chinese hamster ovary (CHO) cells expressing the receptor after transfection. The second-messenger coupling was evaluated by adenylate cyclase measurement. The human secretin receptor was compared with the rat and the rabbit receptors. In the three animals species, rat and human secretin were equipotent; rabbit secretin was equipotent on human and rabbit secretin receptors and less potent on the rat receptor. Similar data were obtained for the [Arg16]-secretin analog. Deletion of histidine 1 and replacement of aspartate 3 reduced the affinity of the peptides for the three receptors; however, the reduction was more pronounced on rat than on human and rabbit secretin receptors. Finally, the low affinity of the rat and human receptors for vasoactive intestinal peptide (VIP) was identical; the rabbit receptor, however, had a 20-fold higher affinity. Thus the human secretin receptor shows properties of both rat and rabbit receptors. Evaluation of the properties of chimeric receptors will be useful to fit the ligand on the receptors.

Influence of selective VIP receptor agonists in the rat gastric fundus.

The receptor subtypes involved in the relaxant effect of vasoactive intestinal polypeptide (VIP) in the rat gastric fundus were investigated in vitro. The selective VIP2 receptor agonist [Ac-H1,E8,K12,Nle17,A19,D25,L26,K27,28,G29,30,++ +T31]VIP(cyclo21-25) (RO25-1553) induced a concentration-dependent relaxation (EC50 2.8 nM), while the selective VIP1 receptor agonist derived from growth hormone-releasing factor (GRF) [K15,R16,L27]VIP-(1-7)/GRF-(8-27) had no effect up to 1 microM. [R16] chicken secretin, a selective VIP1 receptor agonist, induced relaxation with a potency of 4.8 nM but its maximal effect was clearly lower than that of VIP, pituitary adenylate cyclase-activating peptide [PACAP-(1-27)] and RO25-1553. This effect was reproduced by porcine secretin (EC50 2.1 nM). It is concluded that the rat gastric fundus contains functional VIP2 receptors but not VIP1 receptors, and that specific secretin receptors are also present.

Interaction of lipophilic VIP derivatives with recombinant VIP1/PACAP and VIP2/PACAP receptors.

Stearyl vasoactive intestinal polypeptide has been reported to be a VIP (vasoactive intestinal polypeptide) receptor agonist of high potency with an original bioavailability and action. We synthesized three fatty acyl derivatives, myristyl-, palmityl- and stearyl-[Nle17]VIP, and tested their capacity to recognize recombinant rat- and human VIP1- and VIP2/PACAP (pituitary adenylate cyclase-activating polypeptide) receptors and to stimulate adenylate cyclase activity. The three lipophilic analogues bound with high affinity (from 0.5 to 20 nM) to both receptor subtypes but did not distinguish between them. In preparations expressing a high density of human VIP1/PACAP receptors, the three lipophilic analogues had the same efficacy as VIP and [Nle17]VIP. In preparations expressing the rat receptors, stearyl-[Nle17]VIP had a lower efficacy than the other peptides tested. In preparations expressing a low level of VIP1/PACAP receptors and in those expressing VIP2/PACAP receptors, all analogues behaved like partial agonists. The lowest efficacy was observed for stearyl-[Nle17]VIP on the VIP2/PACAP receptor subclass. Based on our results, a complex pattern of in vivo biological effects of the lipophilic VIP derivatives should be expected: these compounds might behave as full agonists, partial agonists, or antagonists of the VIP response, depending on the number and the subtype of receptor expressed.

Molecular cloning and in vitro properties of the recombinant rabbit secretin receptor.

The rabbit secretion receptor cDNA was cloned from rabbit pancreas using combined polymerase chain reaction (PCR)/rapid amplification of cDNA ends (PCR/RACE) approaches. The rabbit cDNA encoded 445 amino acids and had 80 and 85% homology with rat- and human receptor, respectively, in terms of nucleic and amino acid sequences. Several regions where the rabbit receptor sequence diverged from the rat/human receptor sequences were observed in the putative extracellular domains of the receptor. A cDNA coding for a similar sequence with a 76 bp deletion after the 5th transmembrane domain was also found; it probably encoded an inactive protein. The whole rabbit secretin receptor cDNA was subcloned in expression vector pCR3.1, then stably and transiently transfected in Chinese hamster ovary (CHO) cells. The pharmacological properties of the rat and rabbit secretin receptor studies were compared by radiolabeled secretin binding, binding inhibition, and adenylate cyclase activation (using secretin analogs and fragments). Porcine secretin was equipotent with rabbit secretin on the rabbit secretin receptor, but fivefold more potent than rabbit secretin on the rat receptor. This was due to the serine-->arginine residue replacement, in position 16 of rabbit secretin. Amino terminal modified secretin analogs (secretin (2-27), [E3]secretin, [N3]secretin) and VIP were less potent than secretin on both secretin receptors, but more potent on the rabbit than on the rat receptor. The carboxy-terminally truncated fragment (1-26) had the same reduced potency on rat and rabbit receptors. Thus, the rabbit secretin receptor had original properties, different from those of the rat receptor.

Vasoactive intestinal peptide modification at position 22 allows discrimination between receptor subtypes.

Secretin and growth hormone releasing factor (GRF) have a weak affinity for VIP (vasoactive intestinal peptide)/PACAP (pituitary adenylate cyclase activating polypeptide) receptors, but discriminate between VIP1/PACAP and VIP2/PACAP receptors. This previously allowed us to develop modified secretin and GRF derivatives as high affinity and highly selective VIP1/PACAP receptor ligands. We tested the hypothesis that the presence of a Gln residue at position 24 and a Leu residue at position 22 was responsible for their VIP1/PACAP receptor selectivity. [Gln24]VIP was not different from VIP but [Leu22]VIP had a 100-fold lower affinity for VIP2/PACAP receptors as compared to VIP1/PACAP receptors. The substitution of Tyr22 by Phe22 in VIP had no significant effect on the recognition of both receptors but [Ala22]VIP had a reduced affinity for the VIP2/PACAP receptor. This indicated that an aromatic residue at position 22 of VIP was required for a high affinity for the VIP2/PACAP receptor but not for the VIP1/PACAP receptor.

Contribution of the second transmembrane helix of the secretin receptor to the positioning of secretin.

The secretin amino-terminal residues are essential for high affinity binding to its cognate receptor and for its biological activity. Mutation of the [Asp3] residue of secretin to [Asn3] decreased the ligand's affinity for the rat wild-type receptor 100-300-fold. Receptor mutations in the transmembrane 2 domain and the beginning of the first extracellular loop allowed the identification of three residues involved in recognition of the [Asp3] residue: D174, K173 and R166. Mutation of K173 and D174 not only reduced the secretin and [Asn3]secretin affinities, but also changed the receptor's selectivity as judged by a decreased secretin and [Asn3]secretin potency ratio. The most striking effect was observed when R166 was mutated to Q, D or L. This led to receptors with a very low affinity for secretin but an up to 10-fold higher affinity than the wild-type receptor for [Asn3]secretin. This suggested that R166, highly conserved in that subgroup of receptor, is a major determinant for the recognition of the [Asp3] of the ligand.

Analogues of VIP, helodermin, and PACAP discriminate between rat and human VIP1 and VIP2 receptors.

Vasoactive intestinal polypeptide (VIP) acts through interaction with two subclasses of seven transmembrane G protein-coupled receptors named VIP1 and VIP2 receptors. These receptors have been cloned in different species, such as rat and human. Considering the different distribution of both receptor subclasses, there is considerable interest in the development of selective agonists and antagonists. The present study compares the binding properties of VIP, PACAP, GRF, secretin, and helodermin analogues on recombinant rat and human VIP1 and VIP2 receptors. On both rat and human receptors, secretin and GRF had a higher affinity for the VIP1 receptor subtypes. The amino-shortened VIP, and the carboxy terminal-shortened VIP and PACAP analogues also presented a higher affinity for the VIP1 receptor. PHI, PHV, helodermin, and helospectin were selective for the human VIP2 receptor subtypes. These results suggest that the helical structure of the carboxy terminal end is necessary for VIP2 recognition. The differences between species were the following: PHI, PHV, helodermin, and helospectin had a higher affinity for the rat VIP1 receptor than for the human VIP1 receptor. On both rat and human receptors, D-Ala4 VIP and D-Phe4 VIP had a high affinity for the VIP1 receptor and a low affinity for the VIP2 receptor. Thus, three domains of the ligand involved in VIP1/VIP2 receptor discrimination were identified: the amino acid residue in position 4 ([D-Ala4], [D-Phe4]VIP), in positions 8 and 9 (the effects of helodermin and helospectin), and the carboxy terminal end (the effects of the shortened VIP and pituitary adenylate cyclase activating polypeptide analogues).

Mutational analysis of extracellular cysteine residues of rat secretin receptor shows that disulfide bridges are essential for receptor function.

We attempted to express point-mutant secretin receptors where each of the 10 extracellular Cys residues was replaced by a Ser residue, in Chinese hamster ovary (CHO) cells. Six of the point-mutant receptors (C24-->S, C44-->S, C53-->S, C67-->S, C85-->S and C101-->S) could not be detected by binding or functional studies: the mutations resulted in functional inactivation of the receptor. In contrast, the four other point-mutant receptors (C11-->S, C186-->S, C193-->S and C263-->S) were able to bind poorly 125I-secretin, and to activate adenylate cyclase with high secretin EC50 values. These results suggest that cysteine residues 24, 44, 53, 67, 85 and 101 are necessary for receptor function, and that the two putative disulfide bridges formed by cysteine residues 11, 186, 193 and 263 are functionally relevant, but not essential for receptor expression. Secretin activated the adenylate cyclase through the quadruple mutant (C11,186,193,263-->S), the four triple mutants, and through double mutants C186,193-->S and C186,263-->S with a very high (microM) EC50 value, suggesting that, in the wild-type receptor, disulfide bridges are formed between C11-C186, and between C193-C263. Prior treatment with dithiothreitol resulted in a marked EC50 increase of the wild-type receptor and of those receptors with at least the two cysteine residues in positions 11 and 186, suggesting that the C11-C186 (but not the C193-C263) disulfide bridge was accessible to this reducing agent. Several results nevertheless indicated that, in mutant receptors, alternative disulfide bridges can be formed between cysteine 186 and cysteine 193 or 263, suggesting that these three residues are in close spatial proximity in the wild-type receptor.

The long-acting vasoactive intestinal polypeptide agonist RO 25-1553 is highly selective of the VIP2 receptor subclass.

RO 25-1553 is a synthetic VIP analogue that induced a long-lasting relaxation of tracheal and bronchial smooth muscles as well as a reduction of edema and eosinophilic mobilization during pulmonary anaphylaxis. In the present study, we tested in vitro the capacity of RO 25-1553 to occupy the different VIP/PACAP receptor subclasses and to stimulate adenylate cyclase activity. The cellular models tested expressed one single receptor subtype: Chinese hamster ovary (CHO) cells transfected with the rat recombinant PACAP I, rat VIP1, and human VIP2 receptors; SUP T1 cells expressing the human VIP2 and HCT 15 and LoVo cells expressing the human VIP1 receptor. RO 25-1553 was threefold more potent than VIP on the human VIP2 receptor, 100- and 600-fold less potent than VIP on the rat and human VIP1 receptors, respectively, and 10-fold less potent than VIP and 3000-fold less potent than PACAP on the PACAP I receptor. RO 25-1553 was a full agonist on the VIP2, the PACAP I, and the rat recombinant VIP1 receptor but a partial agonist only on the human VIP1 receptor. Thus, RO 25-1553 is a highly selective agonist ligand for the VIP2 receptor subclass.

Development of high affinity selective VIP1 receptor agonists.

The biological effects of VIP are mediated by at least two VIP receptors: the VIP1 and the VIP2 receptors that were cloned in rat, human and mice. As the mRNA coding for each receptor are located in different tissues, it is likely that each receptor modulates different functions. It is therefore of interest to obtain selective agonists for each receptor subtype. In the present work, we achieved the synthesis of two VIP1 receptor selective agonsits derived from secretin and GRF. [R16]chicken secretin had IC50 values of binding of 1,10,000, 20, and 3000 nM for the rat VIP1-, VIP2-, secretion- and PACAP receptors, respectively. This peptide, however, had a weaker affinity for the human VIP1 receptor (IC50 of 60 nM). The chimeric, substituted peptide [K15, R16, L27]VIP(1-7)/GRF(8-27) had IC50 values of binding of 1,10,000, 10,000 and 30,000 nM for the rat VIP1-, VIP2-, secretin- and PACAP receptors, respectively. Furthermore, its also showed an IC50 of 0.8 nM for the human VIP1 receptor and a low affinity for the human VIP2 receptor. It is unlikely that this GRF analogue interacted with a high affinity to the pituitary GRF receptors as it did not stimulate rat pituitary adenylate cyclase activity. The two described analogues stimulated maximally the adenylate cyclase activity on membranes expressing each receptor subtype.

In vitro properties of a high affinity selective antagonist of the VIP1 receptor.

A selective high affinity VIP1 receptor antagonist [Acetyl-His1, D-Phe2, Lys15, Arg16, Leu17] VIP(3-7)/GRF(8-27) or PG 97-269 was synthesized, by analogy with recently obtained selective VIP1 receptor agonists. The properties of the new peptide were evaluated on Chinese hamster ovary (CHO) cell membranes expressing either the rat VIP1-, rat VIP2- or the human VIP2-recombinant receptors and on LoVo cell membranes expressing exclusively the human VIP1 receptor. The IC50 values of 125I-VIP binding inhibition by PG 97-269 were 10, 2000, 2 and 3000 nM on the rat VIP1-, rat VIP2-, human VIP1- and human VIP2 receptors, respectively. PG 97-269 had a negligible affinity for the PACAP I receptor type. It did not stimulate adenylate cyclase activity, but inhibited competitively effect of VIP on the VIP1 receptor mediated stimulation of adenylate cyclase activity. The Ki values were respectively of 15 +/- 5 nM and 2 +/- 1 nM for the rat and human VIP1 receptors. Thus the described molecule in the first reported VIP antagonist with an affinity in the nM range and with a high selectivity for the VIP1 receptor subclass. It may be useful for evaluation of the physiological role of VIP in rat and human tissues.

Effect of introduction of an arginine16 in VIP, PACAP and secretin on ligand affinity for the receptors.

Rabbit secretin, which differs from all other mammalian secretins in having a Leu residue in position 6 (instead of Phe) and a basic residue (Arg) in position 16, had a lower affinity than porcine secretion on recombinant rat secretin receptors but had a greater affinity than porcine secretin on recombinant rat VIP1 and PACAP I receptors. Synthetic [L6] porcine secretin had a reduced potency on secretin and VIP1 receptors whereas [R16] porcine secretin had a similar binding profile as rabbit secretin. Thus, an arginine residue in position 16 reduced 3-fold the affinity of secretin for secretin receptors but increased 30-fold its affinity for the VIP1 and PACAP I receptors. The introduction of an arginine residue in position 16, instead of glutamine, in VIP and PACAP had a similar effect: [R16] VIP and [R16] PACAP had 3- to 10-fold higher affinities than VIP and PACAP for VIP1 and PACAP I receptors, and 3-fold lower affinities for the secretin receptors. The three [R16] peptides also had a reduced potency on the chimeric receptor consisting of the N-terminal part of the secretin receptor grafted on the VIP1 receptor, and an enhanced potency on the chimeric receptor consisting of the N-terminal part of VIP1 receptor grafted on the secretin receptor, indicating that position 16 of each ligand interacted with the N-terminal extracellular domain of the receptors.

Properties of the pituitary adenylate cyclase-activating polypeptide I and II receptors, vasoactive intestinal peptide1, and chimeric amino-terminal pituitary adenylate cyclase-activating polypeptide/vasoactive intestinal peptide1 receptors: evidence for multiple receptor states.

We analyzed the functional and binding properties of the "normal" pituitary adenylate cyclase-activating polypeptide (N-PACAP) type I, PACAP type II/vasoactive intestinal peptide (VIP)1, and chimeric N-PACAP/VIP1 receptors expressed in Chinese hamster ovary cells. The binding properties of the three receptors were investigated using three radioiodinated tracers: 125I-VIP, 125I-PACAP-27, and 125I-PACAP-29 (125I-PACAP-27-Gly28,Lys29-amide). The three tracers labeled very different receptor densities; 125I-PACAP-29 labeled more receptors than either 125I-VIP or 125I-PACAP-27 in the three cell lines. Analysis of the competition curves suggested that the three tracers labeled in a different manner three PACAP I receptor states, two PACAP II/VIP1 receptor states, and three chimeric N-PACAP/VIP1 receptor states in transfected Chinese hamster ovary cells. The previously described PACAP1A and PACAP1B receptors, which differ by their affinities for PACAP-27 and PACAP-38, actually correspond to different PACAP I receptor states. The three receptors were able to increase adenylate cyclase activity when activated by PACAP-38, PACAP-27, or VIP. In contrast with the two parent receptors, the chimeric N-PACAP/VIP1 receptor was activated by PACAP-38 at lower concentrations than PACAP-27, suggesting that the amino-terminal and core receptor domains influence each other and that the conformation of one or both domains was altered in the chimeric compared with wild-type receptors. Comparison of the binding and functional properties of three clones expressing different chimeric N-PACAP/VIP1 receptors densities indicated that 125I-PACAP-29 was necessary to correctly estimate the receptor number and that 125I-PACAP-27 or 125I-VIP labeled only a fraction of the functional receptors. We suspect (but could not demonstrate) that this might also be true for PACAP I and PACAP II/VIP1 receptors.

Addition of the (28-38) peptide sequence of PACAP to the VIP sequence modifies peptide selectivity and efficacy.

Chimeric peptides were synthesized by adding the C-terminal extension 28-38 of the pituitary adenylate cyclase activating polypeptide (PACAP) to the sequences (1-27), (2-27), (3-27) and (6-27) of VIP. The capacity of these peptides to occupy the selective PACAP- and the non-selective PACAP-VIP receptors and to stimulate adenylate cyclase activity was studied in chinese hamster ovary (CHO) cells expressing the recombinant receptors. The results were compared to those obtained with VIP and the corresponding VIP fragments. The presence of the (28-38) PACAP extension increased at least 100-fold the VIP- or VIP fragment affinities for the selective PACAP receptor but not for the non-selective PACAP-VIP receptors. Furthermore, on both receptors, the extension increased peptide intrinsic activity: VIP(3-28) was a partial agonist while VIP(3-27)/PACAP(28-38) was as potent as VIP and was apparently a full agonist; VIP(6-28) had no intrinsic activity, but VIP(6-27)/PACAP(28-38) was a partial agonist. These results suggest: (1) the presence of a specific domain for the (28-38) PACAP sequence on the selective PACAP receptor; and (2) a stabilizing effect of the (28-38) PACAP sequence on the structure of N-terminally truncated VIP.