Characterization of the carboxyl-terminal domain of the rat glucose-dependent insulinotropic polypeptide (GIP) receptor. A role for serines 426 and 427 in regulating the rate of internalization.

Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone involved in the regulation of insulin secretion. In non-insulin-dependent diabetes mellitus insulin responses to GIP are blunted, possibly due to altered signal transduction or reduced receptor number. Site-directed mutagenesis was used to construct truncated GIP receptors to study the importance of the carboxyl-terminal tail (CT) in binding, signaling, and receptor internalization. Receptors truncated at amino acids 425, 418, and 405, expressed in COS-7 or CHO-K1 cells, exhibited similar binding to wild type receptors. GIP-dependent cAMP production with the 405 mutant was decreased in COS-7 cells. Maximal cAMP production in CHO-K1 cells was reduced with all truncated forms. Binding was undetectable with a receptor truncated at amino acid 400; increasing tail length by adding 5 alanines restored binding and signaling. Mutants produced by alanine scanning of residues 394-401, adjacent to transmembrane domain 7, were all functional. CT truncation by 30 or more amino acids, mutation of serines 426/427, singly or combined, or complete CT serine knockout all reduced receptor internalization rate. The majority of the GIP receptor CT is therefore not required for signaling, a minimum chain length of approximately 405 amino acids is needed for receptor expression, and serines 426 and 427 are important for regulating rate of receptor internalization.     

VIP receptors on canine submucosal synaptosomes

The present study characterized [¹²⁵I]VIP binding to synaptosomes from the submucosa of canine small intestine. Studies of saturation, competition binding, and kinetic studies revealed high- and low-affinity binding sites. Studies with GTP-γ-S and cholera toxin suggested that the receptor was coupled to a G-protein, possibly G_s. Competition with VIP analogs suggested that the N-terminal end of the molecule played the major role in determining affinity and that this receptor was for VIP, not PACAP. Cross-linking VIP to the receptor revealed a single peptide (M_r 60,000). We suggest that VIP may act to modulate mediator release from enteric nerve endings.     

VIP provides cellular protection through a specific splice variant of the PACAP receptor: a new neuroprotection target

Vasoactive intestinal peptide (VIP) was known to provide neuroprotection. Three VIP receptors have been cloned: VPAC1, VPAC2 and PAC1. A specific splice variant of PAC1 in the third cytoplasmatic loop, hop2, was implicated in VIP-related neuroprotection. We aimed to clone the hop2 splice variant, examine its affinity to VIP and investigate whether it mediates the VIP-related neuroprotective activity. The PAC1 cDNA was cloned from rat cerebral astrocytes. Using genetic manipulation the hop2 splice variant was obtained, then inserted into an expression vector and transfected into COS-7 cells that were used for binding assays. Results showed that VIP bound the cloned hop2 splice variant. Stearyl-neurotensin(6-11) VIP(7-28) (SNH), an antagonist for VIP, was also found to bind hop2. In addition, VIP protected COS-7 cells expressing hop2 from oxidative stress. Parallel assays demonstrated that VIP increased cAMP accumulation in COS-7 cells expressing hop2. These results support the hypothesis that hop2 mediates the cytoprotective effects attributed to VIP.     

Modifications of the binding properties of the human VIP receptor of IGR39 cells by sulfhydryl reagents.

The effects of specific sulfhydryl reagents, N-ethylmaleimide (NEM), p-chloromercuribenzoic acid (PCMB) and 5-5'-dithiobis(2-nitrobenzoic acid) (DTNB), were tested on the vasoactive intestinal peptide (VIP) receptor binding capacity of the human superficial melanoma-derived IGR39 cells. On intact cell monolayers NEM and PCMB inhibit the specific [125I]VIP binding in a time and dose-dependent manner while DTNB has no effect at any concentration tested. Inhibitory effects of NEM and PCMB on high and low affinity VIP receptor are not identical. With NEM-treated cells, only low affinity sites remained accessible to the ligand. Their affinity constant is not modified. With PCMB-treated cells, the binding capacity of high affinity sites is reduced by 56% while the binding capacity of low affinity sites is not significantly affected. For both types of binding sites, the affinity constants remain in the same range of that of untreated cells. On cells made permeable by lysophosphatidylcholine, DTNB is able to inhibit the specific [125I]VIP binding in a time and dose-dependent manner. The three sulfhydryl reagents stabilize the preformed [125I]VIP receptor complex whose dissociation in the presence of native VIP is significantly reduced. Labeling of free SH groups with tritiated NEM after preincubation of cells with DTNB and VIP made possible the characterization of reacting SH groups which probably belong to the receptor. Taken together, these data allow us to define three classes of sulfhydryl groups. In addition, it is shown that high and low affinity sites have different sensibility to sulfhydryl reagents.     

Characterization of functional VIP/PACAP receptors in the human erythroleukemic HEL cell line

R. LEMA-KISOKA, N. HAYEZ, I. LANGER, P. ROBBERECHT, E. SARIBAN AND C. DELPORTE. Characterization of functional VIP/PACAP receptors in the human erythroleukemic HEL cell line. PEPTIDES. The presence of VIP/PACAP receptors was investigated on the human erythroleukemic cell line HEL. Specific binding of [¹²⁵I]-PACAP or [¹²⁵I]-VIP on HEL cells or membranes was very low and did not allow to perform competition curves. At 37°C PACAP transiently increased cAMP levels in the presence of the non-specific phosphodiesterase inhibitor IBMX, suggesting rapid desensitization. Kinetic studies revealed that optimal conditions to measure the EC₅₀ of PACAP(1–27) were 10 min at 20°C. Under those conditions, PACAP-related peptides increased cAMP levels with EC₅₀ in agreement with the pharmacological profile of the VPAC₁ receptor subtype: PACAP = VIP > [K¹⁵, R^16, L²⁷]VIP(1–7)/GRF(8–27) = [R¹⁶]ChSn (two VPAC₁ agonists) HELODERMIN = secretin. RO 25–1553, a selective activator of VPAC₂ receptor was inactive at 1 μM. Dose-response curves of VPAC₁ agonist molecules (PACAP, VIP, [K¹⁵, R¹⁶, L²⁷]VIP(1–7)/GRF(8–27), [R¹⁶]ChSn) were shifted to the right by the VPAC₁ receptor antagonist [AcHis¹, D-Phe², Lys¹⁵, Leu¹⁷]VIP(3–7)/GRF(8–27), with a K_i of 3 ± 1 nM (n = 3). The presence of VPAC₁ receptor mRNA was confirmed by RT-PCR. Preincubation with PACAP or PMA showed that VPAC₁ receptors underwent homologous and heterologous desensitization.

This study provides the first evidence for the expression of functional VPAC₁ receptors undergoing rapid desensitization in HEL cells.     

Novel analogs of VIP with multiple C-terminal domains

The effect of multiplication of the N-terminal domain of vasoactive intestinal peptide (VIP) on the binding activity of the peptide was recently evaluated. A VIP analog with multiple N-terminal domains was found to be slightly more potent as compared to [Nle(17)]VIP towards VIP receptor type 1 (VPAC1)-related cAMP production. Here, the effect of multiplication of the C-terminal domain of VIP was evaluated with the aim of possibly amplifying peptide-receptor (VPAC1) binding and activation. Several VIP analogs were designed and synthesized, each carrying multiplication of the C-terminal domain that was obtained by either a simple linear tandem extension or by a unique branching methodology. Results show that despite significant alterations in the C-terminal domain of VIP that is considered essential to induce potent receptor binding, few peptides demonstrated only slight reduction in receptor binding and activation in comparison to [Nle(17)]VIP. Furthermore, a specific branched VIP analog with multiple C-terminal domains was equipotent to [Nle(17)]VIP in the cAMP production assay. Therefore, it is concluded that the association between the VIP ligand to the VIP receptor could be tolerable to size increases in the C-terminal region of the VIP ligand and multiplication of the C-terminal does not increase activity.     

Functional vasoactive intestinal polypeptide (VIP) receptors in human neuroblastoma subclones that contain VIP precursor mRNA and release VIP-like substances

Three phenotypically distinct subclones (SH-SY-5Y, SH-EP, SH-IN) of the human neuroblastoma cell line SK-N-SH were found to possess vasoactive intestinal polypeptide (VIP) precursor mRNA, release immunoreactive VIP, and express high-affinity VIP receptors coupled to adenylate cyclase. The apparent molecular mass for the receptor polypeptide, as determined by covalent cross-linking of 125I-VIP, was 49 kDa. After 2 days in culture, a concentration of immunoreactive VIP equivalent to the binding affinity of VIP to its receptor was found in the medium in two of these clones (SH-IN and SH-EP). Conditioned medium from SH-IN cells competitively displaced 125I-VIP binding and increased cAMP levels in SH-EP cells, indicating that all of the necessary components for a potential autocrine action of VIP exist in SK-N-SH cells. After numerous cell passages, the SH-EP subclone converted to a distinct phenotype in which VIP precursor mRNA and VIP immunoreactivity in the cell and medium were no longer detectable. In correlation, the VIP receptor number increased, and the EC50 for VIP stimulation of cAMP production shifted to a lower concentration. This points to the possibility that the continuous presence of endogenous VIP in earlier passage SH-EP cells causes a modification in VIP receptor number and cell responsiveness to VIP.     

Epidermal growth factor receptor-dependent and -independent cell-signaling pathways originating from the urokinase receptor

Urokinase-type plasminogen activator (uPA) and vitronectin activate cell-signaling pathways by binding to the uPA receptor (uPAR). Because uPAR is glycosylphosphatidylinositol-anchored, the signaling receptor is most likely a uPAR-containing multiprotein complex. This complex may be heterogeneous within a single cell and among different cell types. The goal of this study was to elucidate the role of the EGF receptor (EGFR) as a component of the uPAR-signaling machinery. uPA activated extracellular signal-regulated kinase (ERK) in COS-7 cells and in COS-7 cells that overexpress uPAR, and this response was blocked by the EGFR inhibitor, tyrphostin AG1478, implicating the EGFR in the pathway that links uPAR to ERK. By contrast, Rac1 activation, which occurred as a result of uPAR overexpression, was EGFR-independent. COS-7 cell migration was stimulated, in an additive manner, by uPAR-dependent pathways leading to ERK and Rac1. AG1478 inhibited only the ERK-dependent component of the response. CHO-K1 cells do not express EGFR; however, these cells demonstrated ERK activation in response to uPA, indicating the presence of an EGFR-independent alternative pathway. As anticipated, this response was insensitive to AG1478. When CHO-K1 cells were transfected to express EGFR or a kinase-inactive mutant of EGFR, ERK activation in response to uPA was unchanged; however, the EGFR-expressing cells acquired sensitivity to AG1478. We conclude that the EGFR may function as a transducer of the signal from uPAR to ERK, but not Rac1. In the absence of EGFR, an alternative pathway links uPAR to ERK; however, this pathway is apparently silenced by EGFR expression.     

(N-stearyl, norleucine17) VIP hybrid inhibits the growth of pancreatic cancer cell lines

The effects vasoactive intestinal peptide (VIP) antagonists were investigated on pancreatic cancer cell lines. (N-Stearyl, Norleucine17) VIP hybrid ((SN)VIPhyb) inhibited 125I-VIP binding to human Capan-2 cells with an IC50 value of 0.01 microM whereas VIP hybrid had an IC50 value of 0.2 microM. By RT-PCR and Northern blot, VPAC1 receptor mRNA was detected in CAPAN-2 cells. One microM (SN)VIPhyb and 10 microM VIPhyb inhibited the ability of 30 nM VIP to elevate cyclic AMP and increase c-fos mRNA. (SN)VIPhyb, 1 microM inhibited the clonal growth of CAPAN-2 cells in vitro. In vivo, (SN)VIPhyb (10 microg/day s.c.) inhibited CAPAN-2 xenograft growth in nude mice. These results indicate that (SN)VIPhyb is a pancreatic cancer VPAC receptor antagonist.     

The orphan receptor cDNA RDC4 encodes a 5-HT1D serotonin receptor.

The cDNA of RDC4, a putative receptor of the G protein-coupled receptor family, has been cloned by PCR methodology. The primary structure of this receptor showed homology with the serotonin 5-HT1A receptor. In this work, RDC4 mRNA has been injected in Y1 adrenal cells and Xenopus oocytes and RDC4 cDNA has been transfected transiently in cos-7 cells. In all these systems serotonin elicited a rise in cyclic AMP levels. Binding studies on membranes of the transfected cos-7 cells using [3H]-LSD showed a pattern of drug affinities consistent with the known properties of a 5-HT1D receptor. RDC4 therefore codes for a 5-HT1D receptor which in the studied systems is positively coupled to adenylate cyclase.     

Gonadotropin-releasing hormone receptor initiates multiple signaling pathways by exclusively coupling to G(q/11) proteins

The agonist-bound gonadotropin-releasing hormone (GnRH) receptor engages several distinct signaling cascades, and it has recently been proposed that coupling of a single type of receptor to multiple G proteins (G(q), G(s), and G(i)) is responsible for this behavior. GnRH-dependent signaling was studied in gonadotropic alphaT3-1 cells endogenously expressing the murine receptor and in CHO-K1 (CHO#3) and COS-7 cells transfected with the human GnRH receptor cDNA. In all cell systems studied, GnRH-induced phospholipase C activation and Ca(2+) mobilization was pertussis toxin-insensitive, as was GnRH-mediated extracellular signal-regulated kinase activation. Whereas the G(i)-coupled m2 muscarinic receptor interacted with a chimeric G(s) protein (G(s)i5) containing the C-terminal five amino acids of Galpha(i2), the human GnRH receptor was unable to activate the G protein chimera. GnRH challenge of alphaT3-1, CHO#3 and of GnRH receptor-expressing COS-7 cells did not result in agonist-dependent cAMP formation. GnRH challenge of CHO#3 cells expressing a cAMP-responsive element-driven firefly luciferase did not result in increased reporter gene expression. However, coexpression of the human GnRH receptor and adenylyl cyclase I in COS-7 cells led to clearly discernible GnRH-dependent cAMP formation subsequent to GnRH-elicited rises in [Ca(2+)](i). In alphaT3-1 and CHO#3 cell membranes, addition of [alpha-(32)P]GTP azidoanilide resulted in GnRH receptor-dependent labeling of Galpha(q/11) but not of Galpha(i), Galpha(s) or Galpha(12/13) proteins. Thus, the murine and human GnRH receptors exclusively couple to G proteins of the G(q/11) family. Multiple GnRH-dependent signaling pathways are therefore initiated downstream of the receptor/G protein interface and are not indicative of a multiple G protein coupling potential of the GnRH receptor.     

Vasoactive intestinal peptide (VIP) receptors in the canine gastrointestinal tract

Vasoactive intestinal peptide (VIP) is a putative neurotransmitter in both the brain and peripheral tissues. To define possible target tissues of VIP we have used quantitative receptor autoradiography to localize and quantify the distribution of ¹²⁵I-VIP receptor binding sites in the canine gastrointestinal tract. While the distribution of VIP binding sites was different for each segment examined, specific VIP binding sites were localized to the mucosa, the muscularis mucosa, the smooth muscle of submucosal arterioles, lymph nodules, and the circular and longitudinal smooth muscle of the muscularis externa. These results identify putative target tissues of VIP action in the canine gastrointestinal tract. In correlation with physiological data, VIP sites appear to be involved in the regulation of a variety of gastrointestinal functions including epithelial ion transport, gastric secretion, hemodynamic regulation, immune response, esophageal, gastric and intestinal motility.     

Modulation of the expression of the VIP receptor by serum factors on the human melanoma cell line IGR39.

IGR39 cells, isolated from a human superficial melanoma, display at their surface high and low affinity receptors for the vasoactive intestinal peptide (VIP). When grown in DME medium supplemented with 10% fetal calf serum, cells display 1.6 x 10(5) high affinity (Kd 0.74 nM) and 5.6 x 10(5) low affinity (Kd 55 nM) VIP binding sites per cell. When cultured in a chemically defined medium containing EGF, transferrin, and selenium, IGR39 cells display many neurite-like extensions. Following these morphological changes, the specific [125I]VIP binding is increased four- to fivefold after 6 days in culture. This phenomenon is reversible and is the result of an increased number of VIP binding sites available at the cell surface, without modification of their affinities. The molecular mass of the binding sites is also unchanged whatever cell culture conditions. Increase in [125I]VIP binding is inversely correlated to the serum concentration in the culture medium. When added to the chemically defined medium, sera from various origins as well as some serum substitutes reduce [125I]VIP binding to the same extent as that of the serum. The total cAMP production by VIP-stimulated IGR39 cells is enhanced by a factor of six to seven when cells are cultured in serum-free medium, in good correlation with the increase of VIP binding capacity. These data suggest that factor(s) present in fetal calf serum inhibit(s) the expression of VIP receptor, thus demonstrating the importance of a strict control of cell culture conditions for in vitro studies.     

Expression and regulation of the orphan receptor RDC1 and its putative ligand in human dendritic and B cells

Based on phylogenetic analysis and chromosomal mapping, the orphan receptor RDC1 was proposed to be a chemokine receptor. In this study we examined the expression of RDC1 on leukocytes by measuring mRNA levels and receptor expression using a new specific mAb. Both mRNA and protein levels were high in monocytes and B cells, relatively low on immature dendritic cells (DC), and up-regulated during final stages of maturation. Strikingly, in mature plasmacytoid DC the mRNA was up-regulated, but did not correlate with protein surface expression. We indeed report that CpG-activated plasmacytoid DC produce a putative ligand for RDC1, which selectively down-regulates RDC1, but not CXCR4 on primary human B cells. RDC1 expression was found to be tightly regulated during B cell development and differentiation. In blood-derived switch memory B cells, the expression of RDC1 appeared to correlate with the ability to differentiate into plasma cells upon activation, suggesting that RDC1 is a marker for memory B cells, which are competent to become Ab-secreting cells.     

The neuropeptide VIP regulates the expression of osteoclastogenic factors in osteoblasts

Osteoclast formation is controlled by stromal cells/osteoblasts expressing macrophage colony-stimulating factor (M-CSF) and receptor activator of NF-κB ligand (RANKL), crucial for osteoclast progenitor cell proliferation, survival and differentiation, and osteoprotegerin (OPG) that inhibits the interaction between RANKL and its receptor RANK. Recent data have strongly indicated that the nervous system plays an important role in bone biology. In the present study, the effects of the neuropeptide vasoactive intestinal peptide (VIP), present in peptidergic skeletal nerve fibers, on the expression of RANKL, OPG, and M-CSF in osteoblasts and stromal cells have been investigated. VIP and pituitary adenylate cyclase-activating polypeptide 38 (PACAP-38), but not secretin, stimulated rankl mRNA expression in mouse calvarial osteoblasts. In contrast, VIP inhibited the mRNA expressions of opg and m-csf, effects shared by PACAP-38, but not by secretin. VIP did not affect rankl, opg, or m-csf mRNA expression in mouse bone marrow stromal cells (BMSCs). The effects by VIP on the mRNA expression of rankl, opg, and m-csf were all potentiated by the cyclic AMP phosphodiesterase inhibitor rolipram. In addition, VIP robustly enhanced the phosphorylation of ERK and the stimulatory effect by VIP on rankl mRNA was inhibited by the MEK1/2 inhibitor PD98059. These observations demonstrate that activation of VPAC(2) receptors in osteoblasts enhances the RANKL/OPG ratio by mechanisms mediated by cyclic AMP and ERK pathways suggesting an important role for VIP in bone remodeling.     

Novel glycosylated VIP analogs: synthesis, biological activity, and metabolic stability.

Vasoactive intestinal peptide (VIP) is a prominent neuropeptide, exhibiting a wide spectrum of biological activities in mammals. However, the clinical applications of VIP are mainly hampered because of its rapid degradation in vivo. Peptide glycosylation, a procedure frequently used to increase peptide resistance to proteolytic degradation and consequently increase peptide metabolic stability, has not been performed yet on VIP. The presence of three N-glycosylation sites on VIP receptor type 1 (VPAC1) was previously demonstrated. Therefore, glycosylation of the VIP ligand could potentially increase its receptor affinity because of glyco-glyco interactions between the ligand and the receptor. In order to enhance VIP's metabolic stability and to increase its ligand-receptor binding/activation, eight glycosylated VIP derivatives were successfully synthesized by the solid-phase procedure. Each VIP analog was monoglycosylated by a monosaccharide addition to one amino-acid residue along the sequence. Glycosylation did not affect the alpha-helical structure shown by the native VIP in organic environment. Few glycosylated VIP analogs displayed highly potent VPAC1 receptor binding and cAMP-induced activation; only 4-6 fold lower in comparison to the native VIP. Furthermore, the peptide analog glycosylated on Thr11 ([11Glyc]VIP) showed a significantly enhanced stability toward trypsin enzymatic degradation in comparison to VIP. Analysis of the degradation products of [11Glyc]VIP showed that differently from VIP, incubation of the peptide [11Glyc]VIP with trypsin resulted in no cleavage at the Arg12-Leu13 peptide bond, suggesting that VIP glycosylation may lead to enhanced metabolic stability.     

TH2 lymphocytes secrete functional VIP upon antigen stimulation

In addition to the peptidergic innervation, immune cells may also represent a source for VIP in the lymphoid organs. Previous studies reported increased VIP mRNA and protein expression in mitogen-stimulated B and T lymphocytes. To determine whether specific T cell subsets are responsible for VIP production, we derived TH1 and TH2 effector cell lines from T-cell receptor transgenic mice. TH1 and TH2 cells were stimulated with the specific (pigeon cytochrome C peptide) or nonspecific (ovalbumin) antigen presented by MHC class II compatible antigen-presenting cells. Upon stimulation with the specific antigen, TH2, but not TH1 cells express VIP mRNA and intracelllular VIP protein, as determined by Northern blots and FACS analysis. Supernatants harvested from antigen-stimulated TH2 cells contain secreted VIP, as determined by Elisa, and induce cAMP in HEK293 cells transfected with the specific VIP/PACAP receptor VPAC1. These results confirm that TH2, but not TH1 cells, express and secrete functional VIP following specific antigen stimulation. The release of VIP within the lymphoid microenvironment following antigenic stimulation provides a physiological basis for the immunoregulatory effects of VIP on neighboring immune cells, such as downregulation of macrophage activation, effects on lymphocyte migration, on antigen-induced T cell apoptosis, and on T cell differentiation.     

A fragment of vasoactive intestinal peptide, VIP(10-28), is an antagonist of VIP in the colon carcinoma cell line, HT29

The 19 amino acid carboxyl terminus fragment of vasoactive intestinal peptide (VIP), VIP(10-28), inhibits [125I]VIP binding in intact HT29 colonic adenocarcinoma cells and in membranes from these cells. However, VIP(10-28) alone has no effect on adenylate cyclase activity (membranes) or cyclic AMP synthesis (intact cells) in HT29 cells although VIP receptor agonists are markedly stimulatory. The indicated antagonist character of VIP(10-28) was confirmed by rightward parallel shifts of VIP dose response curves in the presence of VIP(10-28) in adenylate cyclase and cyclic AMP synthesis experiments. The equilibrium dissociation constant values for VIP(10-28) from these experiments agree with values from inhibition binding studies. The lack of effect of VIP(10-28) on forskolin dose response curves in HT29 adenylate cyclase assays indicates the specificity of the VIP(10-28) antagonism, thus suggesting that VIP(10-28) may be a useful tool in studying VIP receptor regulation and other aspects of the mechanisms of VIP action. The potential regulatory role of a proteolytically generated fragment of VIP acting antagonistically at VIP receptors is discussed.