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.

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).

The pituitary adenylate cyclase activating polypeptide (PACAP I) and VIP (PACAP II VIP1) receptors stimulate inositol phosphate synthesis in transfected CHO cells through interaction with different G proteins.

The PACAP receptor (PACAP I receptor, selective for PACAP) and the PACAP II VIP1 receptor (recognizing PACAP and VIP with the same high affinity) were stably expressed in Chinese Hamster Ovary (CHO) cells. Cell lines expressing different receptor densities, as measured by binding saturation curves, were selected. Inositol phosphate production was stimulated dose dependently in all the cell lines by PACAP and VIP, and the order of potency of the agonists was identical to that of high affinity receptor occupancy. The stimulatory effect of a saturating peptide concentration was proportional to the total receptor density. At similar receptor densities, however, the PACAP receptor mediated stimulation was higher than the VIP receptor-mediated stimulation. Pretreatment of the cells with pertussis toxin for 8 h had no effect on receptor densities, did not alter the PACAP stimulated inositol phosphate synthesis by the cells expressing the PACAP I receptor but markedly inhibited the response of the cells expressing the PACAP II VIP1 receptor. Thus, the present results indicate that the two G(s)-coupled PACAP I and PACAP II VIP1 receptors may stimulate IP production. The maximal stimulation depended on the number of receptor expressed; the PACAP I and PACAP II VIP1 receptors probably activated the phospholipase C through G proteins of the G(q), and of the G(i)/G(o) families, respectively.

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.

Detection of c-Ki-ras gene codon 12 mutations from pancreatic duct brushings in the diagnosis of pancreatic tumours.

Differential diagnosis of pancreatic cancer and chronic pancreatitis is sometimes difficult and cytological examination of brushings or aspirated material collected during endoscopic retrograde cholangiopancreatography (ERCP) remains disappointing. As point mutations in codon 12 of the c-Ki-ras 2 gene are found in most pancreatic adenocarcinoma and not in chronic pancreatitis, this study analysed prospectively the presence of these mutations in brushing samples collected during ERCP in 45 patients (26 males, 19 females) showing a dominant stricture of the main pancreatic duct at pancreatography: 24 with pancreatic adenocarcinoma, 16 with chronic pancreatitis, and five intraductal mucin hypersecreting neoplasms. Twenty of 45 patients presented equivocal ERCP findings that did not permit a definite diagnosis. Ki-ras mutations at codon 12 were detected using a rapid and sensitive method based on polymerase chain reaction mediated restriction fragment length polymorphism analysis and confirmed by direct sequencing of polymerase chain reaction products. Results were compared with those provided by routine brush cytology. A definitive diagnosis was established for each patient. Mutations were detected in 20 of 24 patients with pancreatic adenocarcinoma (83%), but in none of the chronic pancreatitis patients and intraductal mucin hypersecreting neoplasms, irrespective of their location. By contrast, only 13 of 24 pancreatic adenocarcinoma (54%) were detected by conventional cytological examination, which yielded four false negative and seven non-contributive results. Sensitivity, specificity, and accuracy of molecular biological and cytological methods were 83%-76%, 100-83%, and 90%-58%, respectively. Notably the mutations could be detected in six patients with small tumour size (< or = 2 cm). In conclusion, Ki-ras analysis performed on pancreatic brushing samples is an efficient procedure, more accurate than cytology in the diagnosis of pancreatic adenocarcinoma, and highly specific in the differentiation between neoplastic and chronic inflammatory ductal changes, especially in patients showing inconclusive ERCP findings.

Expression of pituitary adenylate cyclase activating polypeptide and receptors in human brain tumors.

The capacity of brain tumor samples to synthesize pituitary adenylate cyclase activating polypeptide (PACAP) was evaluated by the reverse transcriptase-polymerase chain reaction technique (RT-PCR). The expression of PACAP receptors was assessed by a combination of RT-PCR techniques, conventional binding techniques, and also by the ability of PACAP to stimulate adenylate cyclase activity. A weak PACAP mRNA and PACAP receptor mRNA expression was detected in only 3 of 16 meningiomas. A weak PACAP-stimulated adenylate cyclase activity (+20%) was detected in 10 of the 16 samples but binding of labeled PACAP was never observed. In the 16 gliomas studied (including two oligodendrogliomas and two ependymomas), PACAP mRNA was identified in 13 samples and PACAP receptor mRNA in 15 samples. PACAP receptors were identified in all the samples by binding studies and/or by PACAP stimulation of the adenylate cyclase activity. PACAP mRNA was never detected in pituitary adenomas (three prolactinomas, two mixed PRL-GH-producing tumors, three GH-secreting tumors, three gonadotrophinomas, one ACTH-producing tumor, two nonsecreting tumors) whereas PACAP receptor mRNA was highly expressed in all the tumors except prolactinomas, where it was at the limit of detection, confirming the binding and adenylate cyclase activation results. Thus, it is unlikely that the neuropeptide PACAP could influence meningioma's cell growth; PACAP secreted from extratumoral areas may influence pituitary tumors and PACAP could participate to gliomas development.

Molecular cloning and functional characterization of a human VIP receptor from SUP-T1 lymphoblasts.

We have cloned and sequenced a cDNA isolated from a human SUP-T1 lymphoblast cell line library. It encoded a 457 amino acids protein having 87% identity with the rat PACAP type II, VIP2 receptor. Chinese hamster ovary (CHO) cells stably transfected with cloned cDNA expressed a specific binding of 125I[Acetyl-His1]PACAP-27. This binding was inhibited by GTP, and by the peptides helodermin, VIP, PACAP-27 and PACAP-38 that also stimulated adenylate cyclase activity. The order of potency was PACAP-38 > VIP > or = helodermin > or = PACAP-27. Comparison of the results in two cell lines expressing different receptor densities suggested that helodermin and PACAP-38 had a higher intrinsic activity than VIP and PACAP-27.

Calmodulin, calbindin-D28K and calretinin in rat and chicken pineal glands: immunocytochemical and immunoblotting analysis.

In pineal gland, melatonin is synthesized in pinealocytes. Pharmacological studies using calmodulin antagonists suggested that melatonin synthesis was regulated through calmodulin. However, immunohistochemical studies showed that calmodulin could only be detected in pineal glial cells, and not in pinealocytes. To further investigate this discrepancy, we have tried to detect calmodulin not seen by immunohistochemical methods. We have used rat and chicken pineal homogenate supernatants and Triton X-100-treated pellets denatured by sodium dodecyl sulfate, subjected to electrophoresis and immunoblotting using anti-calmodulin antibodies. Two different IgG (#465 and #860) purified from anti-calmodulin sera were used. In rat pineal homogenate supernatants, calmodulin could be detected by immunoblotting using both antibodies. Some calmodulin could also be detected in the Triton-treated pellet fractions, but no additional cross-reacting bands were detected. However, in both chicken pineal homogenate supernatants and Triton-extracted pellets, in addition to a calmodulin immunoreactive band, two other proteins with approximate molecular masses (M(r)) of 56 kDa and 60 kDa were detected using anti-calmodulin #465. For comparison, similar immunoblot experiments were performed for detection of calbindin-D28K and calretinin, two other calcium binding proteins expressed in different pineal cell populations. Interestingly, Triton extraction of chicken pineal pellets revealed additional bands cross-reacting with each antibody. Anti-calbindin-D28K cross-reacted strongly with a M(r) = 68 kDa protein and weakly with a M(r) = 56 kDa protein. Anti-calretinin cross-reacted strongly with a M(r) = 93 kDa protein and weakly with a M(r) = 56 kDa protein.

Calretinin in rat ovary: an in situ hybridization and immunohistochemical study.

Calretinin is a cytosolic calcium-binding protein of the calmodulin superfamily, with high homology with calbindin D28k. The only cells in which calretinin has been described so far are neurons, in the central nervous system and in retina. In the present work, we describe the expression of the calretinin gene in the interstitial cells of rat ovary. Immunohistochemistry, using a calretinin-specific antibody, allowed to detect the protein from 19 days after birth. Western blot from ovary homogenates confirmed the labelling of a 29 kDa band, the size of calretinin. In situ hybridization confirmed immunochemical data; calretinin transcripts were clearly shown in the same cell population. This represents the first description of calretinin outside the nervous system. Its function in ovary remains to be determined.