CDB15:0001556 VIP — VIPR2

ABSTRACT: The widespread neuropeptide vasoactive intestinal peptide (VIP) has two receptors VPAC(1) and VPAC(2). Solid-phase syntheses of VIP analogs in which each amino acid has been changed to alanine (Ala scan) or glycine was achieved and each analog was tested for: (i) three-dimensional structure by ab initio molecular modeling; (ii) ability to inhibit (125)I-VIP binding (K(i)) and to stimulate adenylyl cyclase activity (EC(50)) in membranes from cell clones stably expressing human recombinant VPAC(1) or VPAC(2) receptor. The data show that substituting residues at 14 positions out of 28 in VIP resulted in a >10-fold increase of K(i) or EC(50) at the VPAC(1) receptor. Modeling of the three-dimensional structure of native VIP (central alpha-helice from Val(5) to Asn(24) with random coiled N and C terminus) and analogs shows that substitutions of His(1), Val(5), Arg(14), Lys(15), Lys(21), Leu(23), and Ile(26) decreased biological activity without altering the predicted structure, supporting that those residues directly interact with VPAC(1) receptor. The interaction of the analogs with human VPAC(2) receptor is similar to that observed with VPAC(1) receptor, with three remarkable exceptions: substitution of Thr(11) and Asn(28) by alanine increased K(i) for binding to VPAC(2) receptor; substitution of Tyr(22) by alanine increased EC(50) for stimulating adenylyl cyclase activity through interaction with the VPAC(2) receptor. By combining 3 mutations at positions 11, 22, and 28, we developed the [Ala(11,22,28)]VIP analog which constitutes the first highly selective (>1,000-fold) human VPAC(1) receptor agonist derived from VIP ever described.

ABSTRACT: The effects of a (N-stearyl, Norleucine17) vasoactive intestinal peptide hybrid ((SN)VIPhybrid) on cells stably transfected with VPAC,, VPAC2, or PAC1 receptors were investigated. (SN)VIPhybrid inhibited specific 125I-VIP binding to membranes derived from CHO cells transfected with VPAC, or VPAC2 receptors with high affinity (IC50 = 30 and 50 nM). (SN)VIPhyb inhibited specific 125I-PACAP-27 binding to membranes derived from NIH/3T3 cells transfected with PAC1 receptors with high affinity (IC50 = 65 nM). PACAP-27 caused cAMP elevation in NIH/3T3 cells transfected with PAC1 receptors and the increase cAMP caused by pituitary adenylated cyclase (PACAP) was inhibited by (SN)VIPhyb. Also, the increase in cAMP caused by VIP using CHO cells transfected with VPAC1 or VPAC2 receptors was antagonized by (SN)VIPhyb. These results indicate that (SN)VIPhyb is an antagonist for VPAC1, VPAC2, and PAC1 receptors.

ABSTRACT: VPAC/PAC receptor activation classically results in cyclic-AMP production, with limited reports evaluating calcium signalling. These studies systematically characterise intracellular cyclic-AMP ([cAMP](i)) and calcium ([Ca(2+)](i)) responses in CHO-cells expressing recombinant human (h) VPAC/PAC receptors (hVPAC(1)R, hVPAC(2)R, hPAC(1)R), using two simple, non-radioactive, HT-amenable assays. The rank order of potency (ROP) of the agonists VIP, PACAP-27 and PACAP-38 was similar in both assays for each individual receptor subtype, although potencies (EC(50)) in the [Ca(2+)](i) assay were approximately 100-fold lower. Importantly, this shift was also evident in SHSY-5Y cells endogenously expressing hPAC(1)R. Furthermore, [Ala(11,22,28)]VIP and maxadilan were selective hVPAC(1)R and hPAC(1)R agonists, respectively, and although R3P65 had no demonstrable hVPAC(2)R selectivity, these compounds exhibited comparable reductions in [Ca(2+)](i) EC(50) values. In contrast, PG97-269 and PG99-465, putatively selective hVPAC(1)R and hVPAC(2)R antagonists, respectively, were marginally less potent in [cAMP](i) studies, whereas M65 was equipotent at hPAC(1)R. Moreover, PG99-465 alone increased [cAMP](i) at all three hVPAC/PAC receptor subtypes, with full hVPAC(1)R and hPAC(1)R agonism. With equivalent agonist ROPs generated in both assays, [Ca(2+)](i) signalling provides an alternative approach to examine hVPAC/PAC receptor pharmacology. However, these studies underscore the paucity of receptor selective compounds, complexities in comparing drug potencies across assays, and the pleiotropic nature of VPAC/PAC-receptor signalling.

ABSTRACT: Ro 25-1392 [Ac-Glu8,OCH3-Tyr10,Lys12,Nle17,Ala19,A sp25,Leu26,-Lys27,28-vasoactive intestinal peptide(cyclo 21-25)] is a cyclic peptide analog of vasoactive intestinal peptide (VIP) that potently exerts cellular effects typical of VIP. The selectivity of Ro 25-1392 for type I (VIPR1) and type II (VIPR2) VIP receptors was investigated first in competitive binding studies using Chinese hamster ovary cell transfectants stably expressing recombinant human VIPR1 and VIPR2. Nonradioactive Ro 25-1392 was as potent a competitive inhibitor as VIP for the binding of 125I-VIP to VIPR2 transfectants (Ki = 9.6 +/- 1.0 and 16 +/- 1.7 nM, respectively; mean +/- S.E.M., n = 4). In contrast, Ro 25-1392 had a very low affinity for VIPR1, compared with VIP, and attained a maximum of only 40% mean inhibition of binding of 125I-VIP at 1 microM. The affinity of VIP (Ki = 3.4 +/- 1.5 nM, mean +/- S.E.M., n = 4) for binding to VIPR1 was 1000-fold greater than that of Ro 25-1392. Ro 25-1392 evoked concurrent and concentration-dependent increases in intracellular levels of calcium and cyclic AMP (EC50 = 3.0 +/- 0.4 nM, mean +/- S.E.M., n = 4) in VIPR2 transfectants, but not in VIPR1 transfectants. The VIP receptor specificity of Ro 25-1392 was confirmed by preincubation of Chinese hamster ovary transfectants with 0.1 microM Ro 25-1392 for 18 hr at 37 degrees C, to down-regulate each type of VIP receptor. Pretreatment of VIPR2 transfectants with Ro 25-1392 decreased Bmax by a mean of 58% and VIP-induced increases in the intracellular concentration of cyclic AMP by a mean of 65%. In contrast, there was no significant change in VIPR1 transfectants after pretreatment with Ro 25-1392. Ro 25-1392 thus is selectively recognized by VIPR2, with consequent initiation of cyclic AMP and Ca+2 signals and down-regulation of VIPR2. This potent analog of VIP may prove useful for investigations of VIPR2-mediated physiological effects of VIP and exploration of the roles of VIPR2 in diseases.

ABSTRACT: 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.

ABSTRACT: Vasoactive intestinal peptide (VIP1 and VIP2) receptors belong to the new class II subfamily of G protein-coupled receptors. We investigated here human VIP1 and VIP2 receptors by mutating in their extracellular domains all amino acid residues that are conserved in VIP receptors but are different in other members of their subfamily. They are present in 1) the N-terminal domain, i.e., E36, I43, S64, D132 and F138 in the VIP1 receptor and E24, I31, S53, D116 and F122 in the VIP2 receptor; 2) the second extracellular loop, i.e., T288 and S292 in the VIP1 receptor and T274 and S278 in the VIP2 receptor. These residues were changed to alanine (A), and cDNAs were transfected into Cos cells. For the VIP1 receptor, no specific 125I-VIP binding could be detected in cells transfected with the E36A mutant, whereas other mutants exhibited Kd values similar to that of the wild-type receptor, with the exception of S64A, for which a 3-fold increase of Kd was observed. For the VIP2 receptor, no specific 125I-VIP binding could be observed with the E24A mutant, whereas other mutants exhibited dissociation constants similar to that of the wild-type receptor, with the exception of I31A and T274A mutants, for which a 11- and 5-fold increase of Kd was observed, respectively. cAMP production experiments provided evidence that the E36A VIP1 receptor and the E24A VIP2 receptor mutants mediated almost no response upon VIP exposure. For the I31A and T274A mutants of the VIP2 receptor and the S64A mutant of the VIP1 receptor, the EC50 values of VIP for stimulating cAMP production were increased 35, 8 and 3 times as compared with that observed for the wild-type receptor, respectively. Immunofluorescence studies indicated that all mutants were normally expressed by Cos cells. These data provide the first evidence for differences in the structure-function relationship of VIP1 and VIP2 receptors.

ABSTRACT: 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.

ABSTRACT: 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).