Galanin receptors in a hamster pancreatic beta-cell tumor: identification and molecular characterization.

High affinity binding sites for galanin are identified and characterized in membranes from a hamster pancreatic beta-cell tumor. Using the radioiodinated peptide [125I] galanin, interaction of the peptide with pancreatic membranes is shown to be saturable, reversible, and time, temperature, membrane protein concentration, pH, and ionic strength dependent. In optimized equilibrium conditions of binding (90 min at 10 C), native galanin competitively inhibits the binding of [125I]galanin in a dose-dependent manner (from 10(-11)-10(-8) M); half-maximal inhibition is induced by 1 nM peptide. Scatchard analysis indicates the existence of a single population of sites of high affinity (Kd = 1.5 nM) and low capacity (44 fmol/mg protein). The monophasic dissociation process confirms the homogeneity of galanin-binding sites. Galanin-binding sites are highly specific, since apart from native galanin, none of the numerous biologically active peptides tested competes with [125I] galanin for binding to pancreatic membranes. The cross-linking of [125I]galanin to beta-cell membranes is performed using the chemical bifunctional reagent ethylene glycol bis-(succinimidyl succinate). After sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis in the presence or absence of dithiothreitol, one single band of 57,000 mol wt is observed, which may be corresponding to the [125I]galanin-receptor complex. Indeed, labeling of this 57,000 mol wt component is abolished only by native galanin but is unaffected by various other digestive peptides. Assuming one molecule of [125I]galanin is bound per molecule of protein, a 54,000 mol wt protein is identified as the pancreatic galanin receptor. In conclusion, our results indicate for the first time the identification of galanin receptors. Their presence in pancreatic beta-cells suggests a direct role of galanin in regulating endocrine beta-cell function.

Interaction of peptide YY with rat intestinal epithelial plasma membranes: binding of the radioiodinated peptide.

High affinity binding sites for peptide YY (PYY) have been identified and characterized in plasma membranes prepared from rat jejunal epithelium by studying the kinetics, stoichiometry, and chemical specificity of the interaction of 125I-labeled PYY with membranes. Binding of [125I]PYY was rapid, saturable, reversible, specific, and depended on temperature, pH, and ionic strength. In optimized steady state conditions of binding (2 h of incubation at 15 C), the degradation of both [125I] PYY and binding sites did not exceed 20%. The concentration dependence of PYY binding, determined by adding increasing concentrations of [125I]PYY, indicated that specific binding saturated at 2-3 nM peptide. Scatchard analysis revealed a single class of binding sites with a dissociation constant (Kd) of 434 +/- (SE) 56 pM and a binding capacity of 336 +/- 41 fmol/mg protein (n = 11). Identical results were obtained when increasing concentrations of unlabeled PYY were added to a fixed concentration of [125I]PYY, indicating that the radioiodinated peptide has the same apparent affinity as native PYY. Peptides structurally unrelated to PYY, such as members of the vasoactive intestinal peptide family, insulin, or cholecystokinin octapeptide, were unable to compete with [125I]PYY for binding to membranes. Rat, human, and avian pancreatic polypeptides, which display, respectively, 42%, 47%, and 53% homology with PYY, did inhibit [125I]PYY binding but with an approximate or equal to 100,000-fold lower potency than PYY, indicating the strict structural requirement for recognition by PYY binding sites. In contrast, natural or synthetic neuropeptide Y, which has 25 out of 36 amino acids in common with PYY, retained a high affinity for PYY binding sites [only 4.7 +/- 1.2 (n = 5) times lower than that of PYY]. Specific [125I]PYY binding was particularly high in the upper small intestine and could not be detected in stomach, large intestine, or liver. These findings indicate that rat small intestinal epithelium expresses specific binding sites for the candidate gut hormone PYY that also binds the neuropeptide Y with high affinity, suggesting that the two peptides may regulate the function of small intestinal epithelium, through interaction with a common receptor site.

Peptide-YY and neuropeptide-Y inhibit vasoactive intestinal peptide-stimulated adenosine 3',5'-monophosphate production in rat small intestine: structural requirements of peptides for interacting with peptide-YY-preferring receptors.

Previous binding studies indicated that peptide-YY (PYY) and neuropeptide-Y (NPY) shared a common PYY-preferring receptor site in rat small intestinal epithelium. We showed here that PYY and NPY inhibited vasoactive intestinal peptide (VIP)-stimulated cAMP production in epithelial cells isolated from rat small intestine and examined their structure-activity relationship. Inhibition of VIP-stimulated cAMP by PYY or NPY is time and dose dependent; half-maximal effects were observed for 10 and 107 nM, respectively. In contrast, the structurally related peptide, pancreatic polypeptide, was only active at 1 microM. PYY or NPY reduced the efficacy of VIP by about 50% without altering its potency. Both peptides also suppressed prostaglandin E1-, prostaglandin E2-, and forskolin-stimulated cAMP production and reduced basal cAMP levels. Their inhibitory effects were observed throughout the small intestine, including duodenum, jejunum, and ileum, but not in large intestine. PYY or NPY and epinephrine (through alpha 2-adrenergic receptors) did not exert additive inhibitory effects on intestinal cAMP production. Several fragments of PYY and NPY were used to characterize their structural requirement for inhibiting VIP-stimulated cAMP production and competing with [125I]PYY for binding to intestinal membranes. A highly significant correlation was observed between IC50 values measured in the two assays. No partial sequence of PYY retained the full activity of intact PYY, but the C-terminal portion of PYY was shown to be much more important than the N-terminal portion. Deletion of 21 amino acids from the N-terminus [PYY-(22-36)] only resulted in a 4- to 5-fold decrease in potency compared to that of PYY-(1-36). In contrast, PYY-(27-36) exhibited a drastic loss of potency. The N-terminal fragments PYY-(1-22) and PYY-(1-28) also had very low potencies. Similar results were obtained with NPY fragments. These results provide the first insight on the negative coupling of PYY-preferring receptors with the cAMP production system in small intestine and evidence of the crucial role of the C-terminal portion of PYY in interaction with these receptors.

Interaction of GRF with VIP receptors and stimulation of adenylate cyclase in rat and human intestinal epithelial membranes. Comparison with PHI and secretin.

GRF (10(-8) - 10(-5) M) is shown to inhibit competitively the binding of [125I]VIP to human and rat intestinal epithelial membranes. The affinity of GRF for VIP receptor is 700-800-times lower than that of VIP in both species. The order of affinity of different peptides is VIP greater than PHI greater than secretin greater than GRF in rat, and VIP greater than GRF greater than PHI greater than secretin in man. The important species specificity of VIP receptors in recognizing PHI and secretin does not occur in the case of GRF. GRF stimulates adenylate cyclase through its interaction with VIP receptors in rat and human membranes. However, while GRF behaves as a VIP agonist in human tissue, it is a partial agonist/antagonist of VIP in the rat.

Interaction of Gila monster venom with VIP receptors in intestinal epithelium of human. A comparison with rat.

Gila monster venom (1-300 micrograms/ml) is shown to inhibit completely the binding of [125I]VIP to human and rat intestinal epithelial cell membranes. In both models, the venom inhibits [125I]VIP binding and stimulates adenylate cyclase with a maximal efficiency that is similar to that of VIP and a potency that is 10000-50000 times lower than that of the peptide, on a weight basis. At maximal doses, VIP and Gila monster venom do not exert an additive effect on adenylate cyclase, suggesting that the activation of the enzyme by the venom occurs through VIP receptors. As is the case for VIP, adenylate cyclase activation by Gila monster venom requires the presence of GTP in the incubation medium. Finally, no VIP-like immunoreactivity was detected in the venom using an antiserum raised against mammalian VIP. All these data suggest the presence in the venom of the Gila monster, of a new substance which behaves as a VIP agonist in human as well as rat intestine.

Phorbol ester induces loss of VIP stimulation of adenylate cyclase and VIP-binding sites in HT29 cells.

Treatment of HT29 cells with the tumor promoting phorbol ester PMA resulted in an attenuation of VIP-stimulated cAMP production in intact cells and VIP-stimulated adenylate cyclase activity in cell membranes. PMA did not decrease the ability of cholera toxin and forskolin to elevate cAMP levels in intact cells. Fluoride-stimulated adenylate cyclase activity in HT29 cells homogenates was not affected by PMA. The maximal VIP binding capacity of homogenates prepared from HT29 cells treated with PMA was decreased by 50%. It is concluded that protein kinase C regulates VIP receptor function possibly through phosphorylation of the VIP receptor.

Insulin-like growth factor II/mannose-6-phosphate receptors are transiently increased in the rat distal intestinal epithelium after resection.

The levels of insulin-like growth factor II/mannose-6-phosphate (IGF-II/Man-6-P) receptor and the insulin-like growth factor I (IGF-I) receptor were measured in the intestinal epithelium after 50% resection of the small intestine. Controls were either pair-fed to match the reduced food intake of the resected group or fed ad libitum. [125I]IGF-II binding was transiently increased 2-fold in the distal segment of the small intestine 3 days after resection compared with the pair-fed control. Receptor levels increased from 2.60 nmol/mg protein (pair-fed) to 4.63 nmol/mg protein (resected; p less than 0.001) with no significant change in affinity of IGF-II binding (Kd = 11.2 vs. 9.8 nmol/l). The increase in IGF-II/Man-6-P receptors coincided with increased activity of thymidine kinase in the distal intestinal segment after the resection. [125I]IGF-I binding remained unchanged after the resection. However, the decreased food intake of the pair-fed and resected groups caused a 2-fold increase in the amount of IGF-I receptors (0.18 nmol/mg protein; p less than 0.001) compared with the control fed ad libitum (0.08 nmol/mg protein). IGF-II/Man-6-P receptors were only moderately increased during restricted food intake (2.60 vs. 1.78 nmol/mg protein; p less than 0.005). These results suggest that the IGF-II/Man-6-P receptor may play a role in the adaptive regenerative response of the intestinal epithelium.

Cloning and functional characterization of the human VIP1/PACAP receptor promoter.

The 5'-flanking region (1.5 kb) of the gene coding for the human VIP1/PACAP receptor was isolated, sequenced, and characterized. Transient expression of constructs containing sequentially deleted 5'-flanking sequences of the VIP1/PACAP receptor fused to a luciferase reporter gene showed that this sequence was active as a promoter in the intestinal cancer cell line, HT-29, expressing endogenous VIP1/PACAP receptor. The shortest DNA fragment with significant promoter activity encompassed the region from -205 to +76 bp. Deletion of a CCAAT-box sequence in the construction corresponding to -173 to +76 bp dramatically reduced the promoter activity. The promoter -205 to +76 bp has a housekeeping gene structure without TATA-box. It contains GC-rich regions characterized by potential Sp1 and AP2 sites and some potential regulatory elements, such as CRE and ATF, and a CCAAT-box sequence (-182 to -178) crucial for gene transcription.

Stable expression of the recombinant human VIP1 receptor in clonal Chinese hamster ovary cells: pharmacological, functional and molecular properties.

We stably transfected Chinese hamster ovary (CHO) cells with the recombinant human vasoactive intestinal peptide (VIP)1 receptor. A clone referred to as Clone 15 was isolated and studied for receptor properties. The following data were obtained: (1) one class of binding site was identified by Scatchard analysis of [125I]VIP binding to cell membranes with a Kd of 0.41 nM and a Bmax of 1.62 pmol/mg protein; (2) the constant Ki for the inhibition of [125I]VIP binding by VIP and related peptides was: VIP (0.9 nM) = pituitary adenylate cyclase-activating peptide (PACAP)-27 (1.3 nM) < PACAP-38 (6.8 nM) < helodermin (46.0 nM) < human growth hormone-releasing factor (GRF) (0.6 microM) < peptide histidine methionineamide (2.0 microM) < secretin (> 10 microM); (3) cross-linking experiments using [125I]VIP identified a single M(r) 67000 recombinant receptor; (4) VIP stimulated cAMP production in Clone 15 cells with an EC50 of 0.20 nM; (5) some previously described VIP receptor antagonists including [4-Cl-D-Phe6, Leu17]VIP, [Ac-Tyr1,D-Phe2]GRF-(1-29) amide and VIP-(10-28) inhibited [125I]VIP binding with a Ki of 0.7, 1.6 and 2.5 microM, respectively. They failed to stimulate cAMP production in Clone 15 cells and inhibited, at least partially, the VIP (0.3 nM)-evoked cAMP production; (6) exposure of Clone 15 cells to 10 nM VIP for 24 h resulted in a sharp decrease in Bmax in Clone 15 cells (0.43 vs. 1.62 pmol/mg protein in control cells) and in the potency and efficacy of VIP in stimulating cAMP. Moreover, immunofluorescence studies using confocal microscopy indicated that the receptor was internalized and sequestered in vesicular structures within the cells. It is concluded that Clone 15 cells provide a valuable tool to further characterize various functional and pharmacological aspects of the human VIP1 receptor.

Interaction of PHM, PHI and 24-glutamine PHI with human VIP receptors from colonic epithelium: comparison with rat intestinal receptors.

PHM, the human counterpart of porcine Peptide Histidine Isoleucine amide (PHI), is shown to be a VIP agonist with low potency on human VIP receptors located in colonic epithelial cell membranes. Its potency is identical to that of PHI but by 3 orders of magnitude lower than that of VIP itself in inhibiting 125I-VIP binding and in stimulating adenylate cyclase activity. This contrasts markedly with the behaviour of PHI on rat VIP receptors located in intestinal epithelial cell membranes where PHI is a potent agonist with a potency that is 1/5 that of VIP. In another connection, we show that 24-glutamine PHI has the same affinity as 24-glutamic acid PHI (the natural peptide) for rat or human VIP receptors. These results indicate that while PHI may exert some physiological function through its interaction with VIP receptors in rodents, its human counterpart PHM is a very poor agonist of VIP in human. Furthermore, they show that the drastic change in position 24 of PHI (neutral versus acid residue) does not affect the activity of PHI, at least on VIP receptors.

Ac-Tyr1hGRF discriminates between VIP receptors from rat liver and intestinal epithelium.

The vasoactive intestinal peptide (VIP) stimulates adenylate cyclase activity in rat liver and intestinal epithelium with low and high efficacy, respectively. The human growth hormone releasing factor (hGRF) derivative with acetylated N-terminus e.g. Ac-Tyr1hGRF binds to VIP receptors in both tissues with a similar affinity. However, Ac-Tyr1hGRF is a partial VIP agonist with high intrinsic activity in liver (50% that of VIP) whereas it behaves as a VIP antagonist in intestine. These results further argue for a possible heterogeneity of VIP receptor-coupled adenylate cyclase among tissues on a pharmacological basis.

Receptors for insulin-like growth factors I and II in rat gastrointestinal epithelium.

Distinct receptors for insulin-like growth factors (IGFs) have been characterized in rat intestinal epithelium using 125I-labeled IGF-I and 125I-labeled IGF-II. In jejunal epithelial plasma membranes, IGF-I receptors were observed with a dissociation constant (Kd) of 7.2 nM and a binding capacity of 0.56 pmol/mg protein. Distinct IGF-II receptors were also found with a Kd of 9.5 nM and a binding capacity of 2.61 pmol/mg protein. For IGF-I receptors the following order of affinity was observed: IGF-I greater than IGF-II greater than insulin greater than proinsulin. IGF-II receptors recognize IGF-II with a 20-fold higher affinity than IGF-I and display no cross-reactivity with insulin and proinsulin. Affinity labeling of intestinal membranes also discriminates between the two types of receptors, revealing a radioligand-receptor complex of relative molecular weight (Mr) 130,000 using 125I-IGF-I and 250,000 for 125I-IGF-II under reducing conditions. Separation of proliferative crypt cells from mature villus cells in the small intestine makes it possible to show that a gradient of IGF receptors is present along the crypt-villus axis. 125I-IGF-I and 125I-IGF-II binding is 4.0- and 1.8-fold higher in crypt cells than in villus cells, respectively. Specific 125I-IGF binding is detectable throughout the gastrointestinal tract. The level of IGF binding is similar in stomach, small intestine, and cecum, but higher values are observed in colon.

Expression of two types of receptor for insulinlike growth factors in human colonic epithelium.

The presence of receptors for insulinlike growth factor I and II in human colonic epithelium is demonstrated. Scatchard analysis of binding data obtained with 125I-insulinlike growth factor I showed an insulinlike growth factor I receptor with a dissociation constant of 8.6 nM and a binding capacity of 0.8 pmol/mg membrane protein. Distinct insulinlike growth factor II receptors labeled with 125I-insulinlike growth factor II were also found with a dissociation constant of 6.9 nM and a binding capacity of 4.7 pmol/mg membrane protein. Two sets of observations make it possible to discriminate between the two types of insulinlike growth factor receptors. (a) Unlabeled insulinlike growth factor I was 3 times more potent than insulinlike growth factor II in inhibiting [125I] insulinlike growth factor I binding. Conversely, unlabeled insulinlike growth factor II was 10 times more potent than insulinlike growth factor I in competing with 125I-insulinlike growth factor II for binding to membranes. Insulin and proinsulin did not compete with either of the tracers. (b) Affinity labeling of membranes followed by sodium dodecylsulfate polyacrylamide gel electrophoresis under reducing conditions, revealed a radio-ligand-receptor complex of molecular weight 130,000 and 250,000 using 125I-insulinlike growth factor I and 125I-insulinlike growth factor II, respectively. These observations indicate that adult human colonic epithelium is abundantly equipped with two sets of receptors that recognize preferentially either insulinlike growth factor I or insulinlike growth factor II.

Functional and immunological evidence for stable association of solubilized vasoactive-intestinal-peptide receptor and stimulatory guanine-nucleotide-binding protein from rat liver.

We have reported the solubilization of complexes between vasoactive intestinal peptide (VIP) and its receptor from rat liver in a GTP-sensitive form of Mr 150,000 [Couvineau, A., Amiranoff, B. & Laburthe, M. (1986) J. Biol. Chem. 261, 14482-14489]. In the present study, we demonstrate a stable association of solubilized VIP receptor and stimulatory guanine nucleotide-binding protein (Gs protein), taking advantage of the ability of the glycoproteic VIP receptor (Mr 48,000), and the inability of the Gs protein, to adsorb to wheat germ agglutinin (WGA). 125I-VIP-receptor complexes solubilized in Triton X-100 were adsorbed on WGA-Sepharose, extensively washed and the radioactivity retained was eluted with 1 mM GTP showing that: (a) radioactivity corresponds to free 125I-VIP and (b) alpha s (Mr 42,000) and beta (Mr 35,000) subunits of Gs protein are detectable in the GTP eluate by immunoblotting using antisera against these subunits. Such an effect of GTP implied that a stable ternary complex consisting of VIP, receptor and Gs protein had been adsorbed to WGA-Sepharose. When Triton-solubilized 125I-VIP-receptor complexes were adsorbed on WGA-Sepharose, then retained material was specifically eluted with 0.3 M N-acetylglucosamine, analysis of the sugar eluate showed the following results. (a) GTP induces the dissociation of 125I-VIP-receptor complexes of Mr 150,000 contained in the eluate indicating that 125I-VIP-receptor-G protein complexes had been adsorbed to the WGA column. (b) The Mr-42,000 alpha s subunit can be specifically ADP-ribosylated by cholera toxin. (c) Immunoblotting using antisera against the alpha s and beta subunits of Gs protein, reveals Mr-42,000 and Mr-35,000 components corresponding to alpha s and beta subunits, respectively. (d) Affinity cross-linking using dithiobis(succinimidyl-propionate) of 125-I-VIP-receptor complexes eluted from the WGA column reveals a major band corresponding to Mr 150,000. Immunoblotting using antisera against the beta-subunit shows the presence of the beta subunit (Mr 35,000) in this Mr-150,000 component. In conclusion, these data provide functional and immunochemical evidence for the physical association of solubilized VIP-receptor complexes with alpha s and beta subunits of Gs protein.

Distribution of common peptide YY-neuropeptide Y receptor along rat intestinal villus-crypt axis.

Rat small intestinal epithelium is equipped with peptide YY (PYY)-preferring receptors, which also recognize neuropeptide Y (NPY) with high affinity. We therefore examined the distribution of PYY-NPY receptors along the villus-crypt axis after separation of mature villus cells from proliferative crypt cells. Specific 125I-labeled PYY binding was nine times higher in crypt cells than in villus cells. This was not due to differential degradation of PYY or PYY binding sites by the two cell populations. Rather, Scatchard analysis of equilibrium binding data showed that binding capacity (Bmax) of receptors increased from villus to crypt. Bmax were 166 +/- 36 and 21 +/- 3 fmol/mg protein, and dissociation constants (Kd) were 0.10 +/- 0.02 and 0.05 +/- 0.02 nM in crude membranes prepared from crypt and villus cells, respectively. For all cell populations, NPY and rat pancreatic polypeptide were 8- and 1,800-fold less potent than PYY in inhibiting 125I-PYY binding, respectively. Therefore, receptors appear to be PYY preferring along the entire villus-crypt axis. Both peptides (at the maximally active concentration of 1 microM) reduced vasoactive intestinal peptide (VIP)-stimulated adenosine 3',5'-cyclic monophosphate (cAMP) by 50% in crypt cells. PYY was four to six times more potent than NPY in agreement with the expression of a PYY-preferring receptor. By contrast, neither PYY nor NPY altered VIP-stimulated cAMP levels in villus cells. These results indicate that PYY-preferring receptors, negatively coupled to the cAMP production system, are preferentially expressed in crypt cells where intestinal ionic secretion is believed to take place.