VIP and PACAP receptors coupled to adenylyl cyclase in human lung cancer: a study in biopsy specimens

Vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are important neuropeptides in the control of lung physiology. Both of these commonly bind to specific G protein coupled receptors named VPAC(1)-R and VPAC(2)-R, and PAC(1)-R (with higher affinity for PACAP). VIP and PACAP have been implicated in the control of cell proliferation and tumor growth. This study examined the presence of VIP and PACAP receptors in human lung cancer samples, as well as the functionality of adenylyl cyclase (AC) stimulated by both peptides. Results from RT-PCR and immunoblot experiments showed the expression of VPAC(1)-, VPAC(2)- and PAC(1)-R in lung cancer samples. Immunohistochemical studies showed the expression of VPAC(1) and VPAC(2) receptors. These receptors were positively coupled to AC, but the enzyme activity was impaired as compared to normal lung. There were no changes in Galpha(s) or Galpha(i) levels. Present results contribute to a better knowledge of VIP/PACAP actions in lung cancer and support the interest for the development of VIP/PACAP analogues with therapeutic roles.     

A structure–function study of PACAP using conformationally restricted analogs: Identification of PAC1 receptor-selective PACAP agonists

Pituitary adenylate cyclase-activating polypeptide (PACAP) has widespread physiological/pathophysiological actions and there is increased interest for its use therapeutically, especially in the CNS (neuroprotection). Unfortunately, no selective PACAP-analogs exist for PACAP-preferring PAC1-receptors, primarily because of its high sequence identity to VIP and particularly, because of the inability of structure–function studies to separate the pharmacophore of PAC1-R from VPAC1-R, which has high affinity for PACAP and VIP. The present study attempted to develop PAC1-R-selective agonists primarily by making conformationally restricted PACAP-analogs in positions important for receptor-selectivity/affinity. Forty-six PACAP-related-analogs were synthesized with substitutions in positions 1–4, 14–17, 20–22, 28, 34, 38 and receptor-selectivity determined in PAC1-R,VPAC1-R,VPAC2-R-transfected or native cells from binding or cAMP-generation experiments. Fifteen PACAP-analogs had 6–78-fold higher affinities for PAC1-R than VPAC1-R and 13 were agonists. Although binding-affinities correlated significantly with agonist potency, the degree of receptor-spareness varied markedly for the different PACAP-analogs, resulting in selective potencies for activating the PAC1 receptor over the VPAC1 receptor from 0- to 103-fold. In addition, a number of PACAP-analogs were identified that had high selectivity for PAC1-R over VPAC2-R as well as PACAP-analogs that could prove more useful therapeutically because of substitutions known to extend their half-lives (substitutions at potential sites of proteolysis and attachment of long-chain fatty acids). This study provides for the first time a separation of the pharmacophores for PAC1-R and VPAC1-R, resulting in PACAP-related analogs that are PAC1-R-preferring. Some of these analogs, or their modifications, could prove useful as therapeutic agents for various diseases.     

Structural motifs of pituitary adenylate cyclase-activating polypeptide (PACAP) defining PAC1-receptor selectivity

Pituitary adenylate cyclase-activating polypeptide (PACAP) interacts with three types of PACAP/VIP-receptors. The PAC1-receptor accepts PACAP as a high affinity ligand but not vasoactive intestinal peptide (VIP) similarly binding to VPAC1- and VPAC2-receptors. To identify those amino acids not present in VIP defining PAC1-receptor selectivity of PACAP, radio receptor binding assays on AR4-2J cells were performed. It could be shown that PACAP(1-27) exhibited a distinct and much higher susceptibility to VIP-amino acid substitutions, compared to PACAP(1-38). Positions 4 and 5 seem to be most important for receptor binding of PACAP(1-27), whereas position 13 was identified to be crucial for maximal affinity of PACAP(1-38). PACAP(29-38) extension analogues of VIP revealed a stabilizing effect of the C-terminus of PACAP(1-38) on the optimal peptide conformation. The substitution analogues were also checked for their capacity to stimulate IP3 and cAMP formation in AR4-2J cells. Compared to PACAP(1-27) and PACAP(1-38), most analogues revealed potencies reduced congruously to their lower binding affinities. However, one of the analogues, PACAP(1-27) substituted in position 5, may represent a weak antagonist since this peptide was less potent in inducing second messengers than in label displacement. Our findings indicate that PACAP(1-27) and PACAP(1-38) differ in terms of their requirement of the amino acids in positions 4, 5, 9, 11 and 13 for maximal interaction with the PAC1-receptor.     

Cytosolic Ca2+ responses to sub-picomolar and nanomolar PACAP in pancreatic beta-cells are mediated by VPAC2 and PAC1 receptors

Pituitary adenylate cyclase-activating polypeptide (PACAP) potentiates glucose-induced insulin release and increases cytosolic Ca2+ concentration ([Ca2+]i) in islet beta-cells in a concentration-dependent manner with two peaks at 10(-13) and 10(-9) M. PAC1 receptor (PAC1-R) and VPAC2 receptor (VPAC2-R) are expressed in pancreatic beta-cells and thought to be involved in insulin release. We aimed to determine the receptor types involved in the [Ca2+]i responses to 10(-13) and 10(-9) M PACAP. We measured [Ca2+]i in beta-cells and examined comparative effects of PAC1-R-selective agonist maxadilan, its antagonist M65, VPAC2-R-selective agonist Ro25-1553, and native ligands of PACAP and VIP. In the presence of 8.3 mM glucose, maxadilan, Ro25-1553, PACAP, and VIP at 10(-13) and 10(-9) M all increased [Ca2+]i. PACAP and maxadilan elicited greater effects at 10(-9) M than at 10(-13) M both in the incidence and amplitude of [Ca2+]i responses. For VIP and Ro25-1553, in contrast, the effects at 10(-9) and 10(-13) M were comparable. Furthermore, the amplitude of [Ca2+]i responses to 10(-9) M PACAP, but not 10(-13) M PACAP, was suppressed by M65. The results suggest that VPAC2-R and PAC1-R contribute equally to [Ca2+]i responses to sub-picomolar concentrations of PACAP, while PAC1-R has greater contribution to [Ca2+]i responses to nanomolar concentrations of this peptide.     

Identification of a VIP-specific receptor in guinea pig tenia coli

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) interact with VPAC(2) receptors in rabbit and guinea pig (GP) gastric muscle but with functionally distinct VIP and PACAP receptors in GP tenia coli. This study examined whether selectivity for VIP was determined by two residues (40, 41) in the extracellular domain that differ in the VIP receptors of GP gastric and tenial muscle. A mutant rat VPAC(2) receptor (L40F, L41F), and two chimeric receptors in which the NH(2)-terminal domain of rat VPAC(2) receptor was replaced with that of GP gastric (chimeric-G) or tenia coli (chimeric-T) VIP receptors, were constructed and expressed in COS-1 cells. VIP and PACAP bound with equal affinity to wild-type and mutant rat VPAC(2) receptors and to chimeric-G receptor (IC(50): VIP 0.3 +/- 0.1 to 1.5 +/- 0.4 nM, PACAP 0.4 +/- 0.1 to 2.5 +/- 0.1 nM) and stimulated cAMP with equal potency (EC(50): VIP 13 +/- 5 to 48 +/- 8 nM, PACAP 8 +/- 3 to 31 +/- 14 nM). VIP bound with high affinity also to chimeric-T receptor (IC(50): 0.5 +/- 0.1 nM) and stimulated cAMP with high potency (EC(50): 3 +/- 1 nM). In contrast, PACAP exhibited >1,000-fold less affinity for binding or potency for stimulating cAMP. We conclude that GP tenia coli express a VIP-specific receptor and that selectivity is determined by a pair of extracellular phenylalanine residues.     

Molecular cloning and functional expression of a VIP-specific receptor

Three receptors for VIP and pituitary adenylate cyclase-activating peptide (PACAP) have been cloned and characterized: PAC(1), with high affinity for PACAP, and VPAC(1) and VPAC(2) with equally high affinity for VIP and PACAP. The existence of a VIP-specific receptor (VIP(s)) in guinea pig (GP) teniae coli smooth muscle was previously surmised on the basis of functional studies, and its existence was confirmed by cloning of a partial NH(2)-terminal sequence. Here we report the cloning of the full-length cDNAs of two receptors, a VPAC(2) receptor from GP gastric smooth muscle and VIP(s) from GP teniae coli smooth muscle. The cDNA sequence of the VIP(s) encodes a 437-amino acid protein (M(r) 49,560) that possesses 87% similarity to VPAC(2) receptors in rat and mouse and differs from the VPAC(2) receptor in GP gastric smooth muscle by only two amino-acid residues, F(40)F(41) in lieu of L(40)L(41). In COS-1 cells transfected with the GP teniae coli smooth muscle receptor, only VIP bound with high affinity (IC(50) 1.4 nM) and stimulated cAMP formation with high potency (EC(50) 1 nM). In contrast, in COS-1 cells transfected with the GP gastric smooth muscle receptor, both VIP and PACAP bound with equally high affinity (IC(50) 2.3 nM) and stimulated cAMP with equally high potency (EC(50) 1.5 nM). We conclude that the receptor cloned from GP teniae coli smooth muscle is a VIP(s) distinct from VPAC(1) and VPAC(2) receptors. The ligand specificity in this species is determined by a pair of adjacent phenylalanine residues (L(40)L(41)) in the NH(2)-terminal ligand-binding domain.     

VIP and PACAP stimulate TSH release from the bullfrog pituitary

We have recently shown that corticotropin-releasing hormone (CRH) is a major thyrotropin (TSH)-releasing factor in amphibians, but we have also found that, besides CRH, other hypothalamic substances stimulate TSH secretion in frog. In order to characterize novel TSH secretagogues, we have investigated the effect of frog (Rana ridibunda) vasoactive intestinal polypeptide (VIP) (fVIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) (fPACAP38 and PACAP27) on TSH release from bullfrog (Rana catesbeiana) pituitary cells in primary culture. Incubation of pituitary cells for 24h with graded concentrations of fVIP, fPACAP38 and PACAP27 (10(-9) to 10(-6)M) induced a dose-dependent stimulation of TSH release with minimum effective doses of 10(-9)M for fVIP and 10(-8)M for fPACAP38 and PACAP27. The PAC1-R/VPAC2-R antagonist PACAP(6-38) (10(-7) and 10(-6)M) dose-dependently suppressed the stimulatory effects of fVIP and fPACAP38 (10(-7)M each). Likewise, this antagonist (10(-6) and 10(-5)M) dose-dependently attenuated the stimulatory effect of PACAP27 (10(-7)M). On the other hand, the VPAC1-R/VPAC2-R antagonist [d-pCl-Phe(6), Leu(17)]VIP (10(-6) and 10(-5)M) dose-dependently inhibited the stimulatory effect of fVIP (10(-9)M) and PACAP27 (10(-8)M), but did not affect the response to fPACAP38 (10(-8)M). These data indicate that, in amphibians, the activity of thyrotrophs can be regulated by VIP and PACAP acting likely through VPAC2-R and PAC1-R.     

Ontogeny of PAC1-R and VPAC1-R in the frog, Rana esculenta

The distribution of pituitary adenylate cyclase-activating polypeptide (PACAP) and PACAP receptors in the brain of amphibians has been previously described. In the present study, we have investigated the ontogeny of the selective PACAP receptor, PAC1-R, and the PACAP-vasoactive intestinal polypeptide (VIP) mutual receptor, VPAC1-R, in frog embryos by whole-mount in situ hybridization histochemistry. At stage 20, expression of PAC1-R and/or VPAC1-R mRNAs was detected in the brain, the auditory vesicles, the external gills, the buds of the lateral lines and the coelomatic cavity. At stage 25, PAC1-R and/or VPAC1-R mRNAs were observed in the buds of the orbital lateral line, the pancreas and heart. At stage 30, PAC1-R and VPAC1-R mRNAs were widely distributed in the telencephalon and diencephalon as well as in the bud of the lateral line, the heart and the pancreas. The anatomical distribution of PAC1-R and VPAC1-R mRNAs, although similar, did not totally overlap, indicating that PACAP and VIP may exert differential effects in frog during development.     

Expression of functional PACAP/VIP receptors in human prostate cancer and healthy tissue

Vasoactive intestinal peptide (VIP) is involved in prostate cell proliferation and function. VIP and pituitary adenylate cyclase-activating peptide (PACAP) are similarly recognized by VPAC(1)/VPAC(2) receptors whereas PACAP binds with higher affinity than VIP to PAC(1) receptor. Here we systematically studied the presence and distribution of functional PAC(1), VPAC(1) and VPAC(2) receptors in human normal and malignant prostate tissue. Functional PACAP/VIP receptors were detected in normal and malignant prostate by adenylyl cyclase stimulation with PACAP-27/38 and VIP. RT-PCR experiments showed PAC(1) (various isoforms due to alternative splicing), VPAC(1) and VPAC(2) receptor expression at the mRNA level, whereas Western blots found the three receptor protein classes in normal and pathological conditions. No conclusive differences could be established when comparing control and cancer tissue samples. Immunohistochemistry showed a weaker immunostaining in tumoral than in normal epithelial cells for the three receptor subtypes. In conclusion, we demonstrate the expression of functional PAC(1), VPAC(1) and VPAC(2) receptors in human prostate as well as its maintenance after malignant transformation.     

Identification of key residues that cause differential gallbladder response to PACAP and VIP in the guinea pig

Pituitary adenylate cyclase-activating polypeptide (PACAP) and vasoactive intestinal polypeptide (VIP) have opposite actions on the gallbladder; PACAP induces contraction, whereas VIP induces relaxation. Here, we have attempted to identify key residues responsible for their interactions with PACAP (PAC1) and VIP (VPAC) receptors in the guinea pig gallbladder. We synthesized PACAP-27/VIP hybrid peptides and compared their actions on isolated guinea pig gallbladder smooth muscle strips using isotonic transducers. [Ala4]- and [Val5]PACAP-27 were more potent than PACAP-27 in stimulating the gallbladder. In contrast, [Ala4, Val5]- and [Ala4, Val5, Asn9]PACAP-27 induced relaxation similarly to VIP. [Asn9]-, [Thr11]-, or [Leu13]PACAP-27 had 20-70% contractile activity of PACAP-27, whereas [Asn24,Ser25,Ile26]PACAP-27 showed no change in the activity. All VIP analogs, including [Gly4,Ile5,Ser9]VIP, induced relaxation. In the presence of a PAC1 receptor antagonist, PACAP(6-38), the contractile response to PACAP-27 was inhibited and relaxation became evident. RT-PCR analysis revealed abundant expressions of PAC1 receptor, "hop" splice variant, and VPAC1 and VPAC2 receptor mRNAs in the guinea pig gallbladder. In conclusion, PACAP-27 induces contraction of the gallbladder via PAC1/hop receptors. Gly4 and Ile5 are the key NH2-terminal residues of PACAP-27 that distinguish PAC1/hop receptors from VPAC1/VPAC2 receptors. However, both the NH2-terminal and alpha-helical regions of PACAP-27 are required for initiating gallbladder contraction.     

Role of PACAP and VIP in astroglial functions

Astrocytes represent at least 50% of the volume of the human brain. Besides their roles in various supportive functions, astrocytes are involved in the regulation of stem cell proliferation, synaptic plasticity and neuroprotection. Astrocytes also influence neuronal physiology by responding to neurotransmitters and neuropeptides and by releasing regulatory factors termed gliotransmitters. In particular, astrocytes express the PACAP-specific receptor PAC1-R and the PACAP/VIP mutual receptors VPAC1-R and VPAC2-R during development and/or in the adult. There is now clear evidence that PACAP and VIP modulate a number of astrocyte activities such as proliferation, plasticity, glycogen production, and biosynthesis of neurotrophic factors and gliotransmitters.     

Development of High Affinity Selective VIP1 Receptor Agonists

Gourlet, P., A. Vandermeers, P. Vertongen, J. Rathe, P. De Neef, J. Cnudde, M. Waelbroeck and P. Robberecht. Development of high affinity selective VIP₁ receptor agonists. Peptides 18(10) 1539–1545, 1997.—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 agonists 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-, secretin- 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.     

Differential regulation of activity-dependent neuroprotective protein in rat astrocytes by VIP and PACAP

Activity-dependent neuroprotective protein (ADNP) was shown to be a vasoactive intestinal peptide (VIP) responsive gene in astrocytes derived from the cerebral cortex of newborn rats. The present study was set out to identify VIP receptors that are associated with increases in ADNP expression in developing astrocytes. Using VIP analogues specific for the VPAC1 and the VPAC2 receptors, it was discovered that VIP induced changes in ADNP expression in astrocytes via the VPAC2 receptor. The constitutive synthesis of ADNP and VPAC2 was shown to be age-dependent and increased as the astrocyte culture developed. Pituitary adenylate cyclase-activating polypeptide (PACAP) also induced changes in ADNP expression. The apparent changes induced by VIP and PACAP on ADNP expression were developmentally dependent, and while stimulating expression in young astrocytes, an inhibition was demonstrated in older cultures. In conclusion, VIP, PACAP and the VPAC2 receptor may all contribute to the regulation of ADNP gene expression in the developing astrocyte.     

Peptide agonist docking in the N-terminal ectodomain of a class II G protein-coupled receptor, the VPAC1 receptor. Photoaffinity, NMR, and molecular modeling

The neuropeptide vasoactive intestinal peptide (VIP) strongly impacts on human pathophysiology and does so through interaction with class II G protein-coupled receptors named VIP pituitary adenylate cyclase-activating peptide (PACAP) receptors (VPACs). The molecular nature of VIP binding to receptors remains elusive. In this work, we have docked VIP in the human VPAC1 receptor by the following approach. (i) VIP probes containing photolabile residues in positions 6, 22, and 24 of VIP were used to photolabel the receptor. After receptor cleavage and Edman sequencing of labeled receptor fragments, it was shown that Phe6, Tyr22, and Asn24 of VIP are in contact with Asp107, Gly116, and Cys122 in the N-terminal ectodomain (N-ted) of the receptor, respectively. (ii) The structure of VIP was determined by NMR showing a central alpha helix, a disordered N-terminal His1-Phe6 segment and a 3(10) Ser25-Asn28 helix termination. (iii) A three-dimensional model of the N-ted of hVPAC1 was constructed by using the NMR structure of the N-ted of corticotropin-releasing factor receptor 2beta as a template. As expected, the fold is identified as a short consensus repeat with two antiparallel beta sheets and is stabilized by three disulfide bonds. (iv) Taking into account the constraints provided by photoaffinity, VIP was docked into the hVPAC1 receptor N-ted. The 6-28 fragment of VIP nicely lies in the N-ted C-terminal part, but the N terminus region of VIP is free for interacting with the receptor transmembrane region. The data provide a structural rationale to the proposed two-step activation mechanism of VPAC receptor and more generally of class II G protein-coupled receptors.     

Expression of pituitary adenylate cyclase-activating polypeptide (PACAP) and the PACAP-selective receptor in cultured rat astrocytes, human brain tumors, and in response to acute intracranial injury

Pituitary adenylate cyclase-activating polypeptide (PACAP) is a bioactive peptide with diverse activities in the nervous system. In addition to its more classic role as a neurotransmitter, PACAP functions as a neurotrophic factor. PACAP exerts these activities by binding to PACAP-selective (PAC1) or nonselective (VPAC1, VPAC2) receptors (-R). Glial cells also exhibit PACAP binding, which is associated with the increased proliferation of astrocytes. The present report demonstrates a distinct spatiotemporal regulation of PACAP, PAC1-R, VPAC1-R, and VPAC2-R expression in primary cultured rat astrocytes. To determine the role of PACAP and PAC1-R expression on glial proliferation, two in vivo models were examined--human brain tumors of glial origin and the reactive gliosis induced by a penetrating stab wound to the mature rat brain. Relative to normal human brain, PAC1-R expression is significantly upregulated in glioma, particularly oligodendrogliomas. While similar polymerase chain reaction (PCR) analysis does not detect PACAP expression, in situ hybridization studies reveal PACAP expression in a limited number of cells within the tumor. In sharp contrast, neither PACAP nor PAC1-R expression are upregulated consequent to injury. These results suggest a distinct role for PACAP and PAC1-R in glioma development and nervous system response to injury.     

VIP receptor antagonists inhibit mammary carcinogenesis in C3(1)SV40T antigen mice

The effects of a vasoactive intestinal peptide (VIP) receptor antagonist on mammary carcinogenesis were investigated using the C3(1)SV40T antigen (ag) mice. Ten microg/day VIPhybrid (VIPhyb) administered daily subcutaneously increased significantly the survival of C3(1)SV40Tag mice. At 5.2 months, VIPhyb significantly reduced the mammary tumor burden in C3(1)SV40Tag mice relative to control animals. 125I-VIP bound with high affinity to mouse mammary tumor homogenate. Because (Lys15, Arg16, Leu27)VIP1-7GRF8-27 (VPAC1 selective) but not Ro25-1553 (VPAC2 selective) inhibited specific 125I-VIP binding to mammary tumor membranes with high affinity, VPAC1 receptors predominate. By RT-PCR, VPAC1 receptor mRNA was detected in mammary tumors. By Western blot, a major 60 Kdalton band was detected in mammary tumor extracts using VPAC1 receptor antisera. By immunocytochemistry, VPAC1-R immunostaining was detected in the cytosol and plasma membrane but not the nucleus of fixed mammary tumor tissue. Using laser capture microdissected tumor cells and surface enhanced laser desorption/ionization (SELDI) techniques on mammary tumor cells, the proteomic profile was altered in mice treated with VIPhyb. Because VPAC1 receptor antagonists increase the survival and reduce the tumor burden in C3(1)SV40Tag mice, they may function as chemopreventive agents in mammary cancer.     

Rat hypothalamic GRF elicits its biologic action in GH3 cells by interaction with VIP- preferring receptor site(s)

GH3 cells can be used effectively to study the in vitro mechanism of action of GRF. In these cells, there is a time and concentration-dependent release of cAMP into the medium. Rat hypothalamic GRF, (rGRF) is 7 to 10 fold more active than human hypothalamic GRF (hGRF). VIP, a peptide which is structurally homologous to GRF, stimulates cAMP efflux in GH3 cells, with a higher affinity than hGRF or rGRF. We propose that in contradistinction to the normal rat pituitary, the stimulation of cAMP release by GRF in GH3 cells occurs via activation of VIP-preferring receptors and that GRF (rGRF in particular) behaves as a partial VIP agonist.     

Generation of highly selective VPAC2 receptor agonists by high throughput mutagenesis of vasoactive intestinal peptide and pituitary adenylate cyclase-activating peptide

Pituitary adenylate cyclase-activating peptide (PACAP) has a specific receptor PAC1 and shares two receptors VPAC1 and VPAC2 with vasoactive intestinal peptide (VIP). VPAC2 activation enhances glucose-induced insulin release while VPAC1 activation elevates glucose output. To generate a large pool of VPAC2 selective agonists for the treatment of type 2 diabetes, structure-activity relationship studies were performed on PACAP, VIP, and a VPAC2 selective VIP analog. Chemical modifications on this analog that prevent recombinant expression were sequentially removed to show that a recombinant peptide would retain VPAC2 selectivity. An efficient recombinant expression system was then developed to produce and screen hundreds of mutant peptides. The 11 mutations found on the VIP analog were systematically replaced with VIP or PACAP sequences. Three of these mutations, V19A, L27K, and N28K, were sufficient to provide most of the VPAC2 selectivity. C-terminal extension with the KRY sequence from PACAP38 led to potent VPAC2 agonists with improved selectivity (100-1000-fold). Saturation mutagenesis at positions 19, 27, 29, and 30 of VIP and charge-scanning mutagenesis of PACAP27 generated additional VPAC2 selective agonists. We have generated the first set of recombinant VPAC2 selective agonists described, which exhibit activity profiles that suggest therapeutic utility in the treatment of diabetes.     

Signaling involved in pituitary adenylate cyclase-activating polypeptide-stimulated ADNP expression

Activity-dependent neurotrophic protein (ADNP) was discovered as a novel response gene for VIP and has neuroprotective potential. When the VIP paralog, PACAP38 was added to mouse neuron-glia co-cultures, it induced ADNP mRNA expression in a bimodal fashion at subpico- and nanomolar concentrations with greater response at subpicomolar level. The response was attenuated by a PAC1-R antagonist at both concentrations and by a VPAC1-R antagonist at nanomolar concentration only. An IP3/PLC inhibitor attenuated the response at both concentrations of PACAP38, but a MAPK inhibitor had no effect. A PKA inhibitor suppressed the response at nanomolar concentration only. These findings suggest that ADNP expression is mediated through multiple receptors and signaling pathways that are regulated by different concentrations of PACAP.