Maxadilan Activates PAC1 Receptors Expressed in Xenopus laevis Melanophores

Functional interactions between ligands and their cognate receptors can be investigated using the ability of melanophores from Xenopus laevis to disperse or aggregate their pigment granules in response to alterations in the intracellular levels of second messengers. We have examined the response of long‐term lines of cultured melanophores from X. laevis to pituitary adenylate cyclase activating peptide (PACAP), a neuropeptide with vasodilatory activity, and maxadilan, a vasodilatory peptide present in the salivary gland extracts of the blood feeding sand fly. Pituitary adenylate cyclase activating peptide increased the intracellular levels of cyclic adenosine monophosphate (cAMP) and induced pigment dispersion in the pigment cells, confirming that melanophores express an endogenous PACAP receptor. Maxadilan did not induce a response in non‐transfected melanophores. When the melanophores were transfected with complementary DNA (cDNA) from the three different members of the PACAP receptor family, maxadilan induced pigment dispersion specifically and cAMP accumulation in melanophores transfected with the cDNA for PAC1 receptors but not VPAC1 or VPAC2 receptors. A melanophore line was generated that stably expresses the PAC1 receptor.     

Identification and functional properties of the pituitary adenylate cyclase activating peptide (PAC1) receptor in human benign hyperplastic prostate

Pituitary adenylate cyclase activating peptide (PACAP) is a novel neuropeptide with regulatory and trophic functions that is related to vasoactive intestinal peptide (VIP). Here we investigate the expression of specific PACAP receptors (PAC1) and common VIP/PACAP receptors (VPAC1 and VPAC2) in the human hyperplastic prostate by immunological methods. The PAC1 receptor corresponded to a 60-KDa protein whereas the already known VPAC1 and VPAC2 receptors possessed molecular masses of 58 and 68 KDa, respectively. The heterogeneity of VIP/PACAP receptors in this tissue was confirmed by radioligand binding studies using [125I]PACAP-27 by means of stoichiometric and pharmacological experiments. At least two classes of PACAP binding sites showing different affinities could be resolved, with Kd values of 0.81 and 51.4 nM, respectively. The order of potency in displacing [125I]PACAP-27 binding was PACAP-27 approximately equal to PACAP-38 > VIP. PACAP-27 and VIP stimulated similarly adenylate cyclase activity, presumably through common VIP/PACAP receptors. The PAC1 receptor was not coupled to activation of either adenylate cyclase, nitric oxide synthase, or phospholipase C. It appears to be a novel subtype of PAC1 receptor because PACAP-27 (but not PACAP-38 or VIP) led to increased phosphoinositide synthesis, an interesting feature because phosphoinositides are involved via receptor mechanisms in the regulation of cell proliferation.     

Pituitary adenylate cyclase-activating polypeptides directly stimulate sympathetic neuron neuropeptide Y release through PAC(1) receptor isoform activation of specific intracellular signaling pathways.

Pituitary adenylate cyclase-activating polypeptides (PACAP) have potent regulatory and neurotrophic activities on superior cervical ganglion (SCG) sympathetic neurons with pharmacological profiles consistent for the PACAP-selective PAC(1) receptor. Multiple PAC(1) receptor isoforms are suggested to determine differential peptide potency and receptor coupling to multiple intracellular signaling pathways. The current studies examined rat SCG PAC(1) receptor splice variant expression and coupling to intracellular signaling pathways mediating PACAP-stimulated peptide release. PAC(1) receptor mRNA was localized in over 90% of SCG neurons, which correlated with the cells expressing receptor protein. The neurons expressed the PAC(1)(short)HOP1 receptor but not VIP/PACAP-nonselective VPAC(1) receptors; low VPAC(2) receptor mRNA levels were restricted to ganglionic nonneuronal cells. PACAP27 and PACAP38 potently and efficaciously stimulated both cAMP and inositol phosphate production; inhibition of phospholipase C augmented PACAP-stimulated cAMP production, but inhibition of adenylyl cyclase did not alter stimulated inositol phosphate production. Phospholipase C inhibition blunted neuron peptide release, suggesting that the phosphatidylinositol pathway was a prominent component of the secretory response. These studies demonstrate preferential sympathetic neuron expression of PACAP-selective receptor variants contributing to regulation of autonomic function.     

Functional analysis of recombinant mutants of maxadilan with a PAC1 receptor-expressing melanophore cell line

Maxadilan, a 61-amino-acid vasodilatory peptide, was initially isolated from the salivary glands of the sand fly Lutzomyia longipalpis. Although its primary sequence has no homology to that of pituitary adenylate cyclase-activating peptide, maxadilan is an agonist for the PAC1 receptor. A total of 58 substitution and deletion mutants was engineered in an effort to determine which residues were important for receptor activation. The mutants were characterized functionally using an assay based on pigment granule translocation in PAC1-expressing Xenopus laevis melanophores. Substitution of charged residues and proline 43 could alter (but not eliminate) the agonist activity of the mutants. In contrast, we found that several multiple substitution mutants of the predicted beta-strand threonine residues became antagonists at the PAC1 receptor. The results suggest that these threonine residues are cooperatively involved in PAC1 activation.     

Role of VIP and PACAP in islet function

Vasoactive intestinal polypeptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) are two closely related neuropeptides that are expressed in islets and in islet parasympathetic nerves. Both peptides bind to their common G-protein-coupled receptors, VPAC1 and VPAC2, and PACAP, in addition to the specific receptor PAC1, all three of which are expressed in islets. VIP and PACAP stimulate insulin secretion in a glucose-dependent manner and they both also stimulate glucagon secretion. This action is achieved through increased formation of cAMP after activation of adenylate cyclase and stimulation of extracellular calcium uptake. Deletion of PAC1 receptors or VPAC2 receptors results in glucose intolerance. These peptides may be of importance in mediating prandial insulin secretion and the glucagon response to hypoglycemia. Animal studies have also suggested that activation of the receptors, in particular VPAC2 receptors, may be used as a therapeutic approach for the treatment of type 2 diabetes. This review summarizes the current knowledge of the potential role of VIP and PACAP in islet function.     

Functional characterization of structural alterations in the sequence of the vasodilatory peptide maxadilan yields a pituitary adenylate cyclase-activating peptide type 1 receptor-specific antagonist.

Maxadilan is a vasodilatory peptide derived from sand flies that is an agonist at the pituitary adenylate cyclase-activating peptide (PACAP) type 1 receptor. Surprisingly, maxadilan does not share significant sequence homology with PACAP. To examine the relationship between structure and activity of maxadilan, several amino acid substitutions and deletions were made in the peptide. These peptides were examined in vitro for binding to crude membranes derived from rabbit brain, a tissue that expresses PACAP type 1 receptors; and induction of cAMP was determined in PC12 cells, a line that expresses these receptors. The peptides were examined in vivo for their ability to induce erythema in rabbit skin. Substitution of the individual cysteines at positions 1 and 5 or deletion of this ring structure had little effect on activity. Substitution of either cysteine at position 14 or 51 eliminated activity. Deletion of the 19 amino acids between positions 24 and 42 resulted in a peptide with binding, but no functional activity. The capacity of this deletion mutant to interact with COS cells transfected with the PACAP type 1 receptor revealed that this peptide was a specific antagonist to the PACAP type 1 receptor.     

PAC1 receptors mediate pituitary adenylate cyclase-activating polypeptide- and progesterone-facilitated receptivity in female rats

Pituitary adenylate cyclase-activating polypeptide (PACAP) acts as a feed-forward, paracrine/autocrine factor in the hypothalamic ventromedial nucleus (VMN) for receptivity and sensitizes pituitary hormone release for ovulation. The present study examined receptor(s) and signaling pathway by which PACAP enhances rodent lordosis. PACAP binds to PACAP (PAC1)- and vasoactive intestinal peptide-preferring receptors (VPAC1, VPAC2). Ovariectomized rodents primed with estradiol (EB) were given PACAP or vasoactive intestinal peptide directly onto VMN cells. Only PACAP facilitated receptivity. Pretreatment with VPAC1 and VPAC2 inhibitors blocked both PACAP- and progesterone (P)-induced receptivity. Antisense (AS) oligonucleotides to PAC1 (not VPAC1 or VPAC2) inhibited the behavioral effect of PACAP and P. By real-time RT-PCR, EB, P and EB+P enhanced VMN mRNA expression of PAC1. Within the total PAC1 population, EB and EB+P induced expression of short form PAC1 and PAC1hop2 splice variants. Finally, blocking cAMP/protein kinase A signaling cascade by antagonists to cAMP activity and protein kinase A or by antisense to dopamine- and cAMP-regulated phosphoprotein of 32 kDa blocked the PACAP effect on behavior. Collectively, these findings provide evidence that progesterone receptor-dependent receptivity is, in part, dependent on PAC1 receptors for intracellular VMN signaling and delineate a novel, steroid-dependent mechanism for a feed-forward reinforcement of steroid receptor-dependent reproductive receptivity.     

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.     

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.     

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.     

The neuropeptide pituitary adenylate cyclase activating protein is a physiological activator of human monocytes

Pituitary adenylate cyclase activating protein (PACAP) and its structurally related vasointestinal peptide (VIP) bind to three G-protein-coupled receptors named VPAC1 and VPAC2 for VIP/PACAP receptors and PAC1 for PACAP preferred receptors. We report that in freshly isolated human monocytes PACAP acts as a pro-inflammatory molecule. By RT-PCR, VPAC1 mRNA was the only receptor found to be expressed; VPAC1 protein was detected by Western blotting and visualized by immunohistochemistry. Signaling pathways activated by PACAP include the extracellular regulated kinase (ERK), the stress-activated MAPK p38, the focal adhesion kinase, Pyk2 and its associated cytoskeleton protein paxillin and the phosphatidylinositol 3-kinase (PI-3K). PACAP induces a transient peak in cytoplasmic calcium associated with an increase in reactive oxygen species production and upregulation in membrane expression of the integrin CD11b as well as the complement receptor 1. Control of the different pathways and functions stimulated by PACAP were evaluated using Phospholipase C (PLC), PI-3K, ERK and p38 MAPK inhibitors and led to the conclusion that PLC and to a lesser degree PI-3K activation are upstream events occurring in VPAC1 mediated PACAP stimulation of monocytes and are in contrast to ERK and p38 mandatory for the initiation of other cellular events associated with monocytes activation.     

Functional characterization of a receptor for vasoactive-intestinal-peptide-related peptides in cultured dermal melanophores from Xenopus laevis

A receptor for vasoactive-intestinal-peptide (VIP)-related peptides was functionally characterized in a cell line derived from Xenopus melanophores using a recently described microtiter-plate-based bioassay. Activation of the melanophore VIP receptor by VIP or the peptides pituitary-adenylate-cyclase-activating polypeptide (PACAP 38), PACAP 27, and helodermin stimulated intracellular 3'-5' cyclic adenosine monophosphate (cAMP) accumulation and pigment dispersion in the cells. Helodermin, with an EC50 (concentration of peptide inducing half-maximal melanosome dispersion) of 46.5 pM, was the most potent activator of pigment dispersion, followed by PACAP 38 > VIP > PACAP 27. A similar order of potencies was observed for the peptides to induce cAMP accumulation. The responses to VIP agonists were selectively inhibited by the VIP antagonists PACAP-(6-27) and (N-Ac-Tyr(1)-D-Phe2)-growth-hormone-releasing factor[GRF](1-29)-NH2. Taken together, the results suggest that the melanophores express a VIP receptor that shares certain characteristics of, but also differs significantly from, other previously identified VIP receptors.     

Pituitary adenylate cyclase-activating polypeptide induces translocation of its G-protein-coupled receptor into caveolin-enriched membrane microdomains, leading to enhanced cyclic AMP generation and neurite outgrowth in PC12 cells

Pituitary adenylate cyclase-activating polypeptide (PACAP), a member of the secretin/glucagon/vasoactive intestinal peptide family expressed throughout the nervous system, binds to the PACAP-specific G-protein-coupled receptor family members to promote both neuronal differentiation and survival. Although the PACAP receptor is known to activate its effector protein, adenylate cyclase (AC), and thus enhance cAMP generation, the molecular mechanism utilized by the receptor to activate AC is lacking. Here, we show that PACAP induces neurite outgrowth in PC12 cells by induction of translocation of the PACAP type 1 receptor (PAC1R) into caveolin-enriched Triton X-100-insoluble microdomains, leading to stronger PAC1R-AC interaction and elevated cAMP production. Moreover, we demonstrate that translocation of PAC1R is blocked by various treatments that selectively disrupt caveolae. As a result, intracellular cAMP level is decreased and consequently the PACAP-induced neurite outgrowth retarded. In contrast, addition of exogenous ganglioside GM1 to the cells shows the opposite effects. These results therefore identify the PACAP-induced translocation of its G-protein-coupled receptor into caveolae, where both AC and the regulating G-proteins reside, as the key molecular event in activating AC and inducing cAMP-mediated differentiation of PC12 cells.     

Paradoxical antagonism of PACAP receptor signaling by VIP in Xenopus oocytes via the type-C natriuretic peptide receptor

Atrial natriuretic peptide (ANP) and the closely-related peptides BNP and CNP are highly conserved cardiovascular hormones. They bind to single transmembrane-spanning receptors, triggering receptor-intrinsic guanylyl cyclase activity. The "truncated" type-C natriuretic peptide receptor (NPR-C) has long been called a clearance receptor because it lacks the intracellular guanylyl cyclase domain, though data suggest it might negatively couple to adenylyl cyclase via G(i). Here we report the molecular cloning and characterization of the Xenopus laevis type-C natriuretic peptide receptor (XNPR-C). Analysis confirms the presence of a short intracellular C-terminus, as well as a high similarity to fish and mammalian NPR-C. Injection of XNPR-C mRNA into Xenopus oocytes resulted in expression of high affinity [(125)I]ANP binding sites that were competitively and completely displaced by natriuretic analogs and the unrelated neuropeptide vasoactive intestinal peptide (VIP). Measurement of cAMP levels in mRNA-injected oocytes revealed that XNPR-C is negatively coupled to adenylyl cyclase in a pertussis toxin-sensitive manner. When XNPR-C was co-expressed with PAC(1) receptors for pituitary adenylyl cyclase-activating polypeptide (PACAP), VIP and natriuretic peptides counteracted the cAMP induction by PACAP. These results suggest that VIP and natriuretic peptides can potentially modulate the action of PACAP in cells where these receptors are co-expressed.     

Melanophore pigment dispersion responses to agonists show two patterns of sensitivity to inhibitors of cAMP-dependent protein kinase and protein kinase C

Melanophore pigment dispersion is a sensitive bioassay for activation of the adenylyl cyclase and phospholipase C second-messenger pathways. The necessity of protein kinase activation in causing pigment dispersion was confirmed for eight agonists of endogenous melanophore receptors and for two transfected receptors. All agonists and receptors previously shown to elevate intracellular cAMP in melanophores—melanocyte stimulating hormone, light, (−) norepinephrine, 5-hydroxytrptamine, and the β₂-adrenergic receptor—were able to stimulate pigment dispersion in the presence of Ro31-8220, a potent inhibitor of protein kinase C, but were blocked in the presence of H89, an inhibitor of cAMP-dependent protein kinase. The bombesin receptor, which elevates intracellular IP₃ in melanophores, was unable to stimulate pigment dispersion in the presence of Ro31-8220 or H89. Agonists whose mechanism of activation of pigment dispersion are unknown were also tested. Endothelin 3 responses were blocked by both H89 and Ro31-8220, predicting coupling to phospholipase C. Vasoactive intestinal polypeptide, oxytocin, and calcitonin gene-related peptide β responses were blocked only by H89, predicting coupling to adenylyl cyclase. © 1996 Wiley-Liss, Inc.     

Differential expression of mRNAs for PACAP and its receptors during neural differentiation of embryonic stem cells

The expressions of mRNAs for pituitary adenylate cyclase-activating polypeptide (PACAP), vasoactive intestinal peptide (VIP), and their receptors (PAC1, VPAC1 and VPAC2) were examined in the five steps of the in vitro neuronal culture model of embryonic stem (ES) cell differentiation. mRNAs for PACAP, VIP, PAC1 receptor, and VPAC2 receptor were moderately expressed in neural stem cell-enriched cultures, while VPAC1 receptor mRNA was most prominently expressed in embryoid bodies (EBs). The expression of PAC1 receptor mRNA was further upregulated after terminal differentiation into neurons. In contrast, the expressions of PAC1 receptor and PACAP mRNAs were markedly decreased after glial differentiation. These results suggest that this in vitro neuronal culture system will be a useful model for future studies on the functional role of the PACAPergic system during different stages of neuronal development.     

Crystal structure of the PAC1R extracellular domain unifies a consensus fold for hormone recognition by class B G-protein coupled receptors

Pituitary adenylate cyclase activating polypeptide (PACAP) is a member of the PACAP/glucagon family of peptide hormones, which controls many physiological functions in the immune, nervous, endocrine, and muscular systems. It activates adenylate cyclase by binding to its receptor, PAC1R, a member of class B G-protein coupled receptors (GPCR). Crystal structures of a number of Class B GPCR extracellular domains (ECD) bound to their respective peptide hormones have revealed a consensus mechanism of hormone binding. However, the mechanism of how PACAP binds to its receptor remains controversial as an NMR structure of the PAC1R ECD/PACAP complex reveals a different topology of the ECD and a distinct mode of ligand recognition. Here we report a 1.9 Å crystal structure of the PAC1R ECD, which adopts the same fold as commonly observed for other members of Class B GPCR. Binding studies and cell-based assays with alanine-scanned peptides and mutated receptor support a model that PAC1R uses the same conserved fold of Class B GPCR ECD for PACAP binding, thus unifying the consensus mechanism of hormone binding for this family of receptors.     

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.