Receptors for vasoactive intestinal peptide and secretin on guinea pig pancreatic acini

We investigated the abilities of VIP and secretin to occupy receptors and to increase cellular cyclic AMP using dispersed acini from guinea pig pancreas. The dose-inhibition curve for inhibition of binding of 125I-VIP by VIP was broad with detectable inhibition at 0.1 nM VIP, half-maximal inhibition at 2 nM VIP and complete inhibition at 10 microM VIP. Secretin also inhibited binding of 125I-VIP was compatible with two VIP-preferring receptors with one class having a high affinity for VIP (Kd 1.1 nM) and a low affinity for secretin (Kd 5 microM) and the other class having an intermediate affinity for VIP (Kd 470 nM). The dose inhibition curve for inhibition of binding of 125I-secretin by secretin was not broad. Half-maximal inhibition occurred with 7 nM secretin or with 10 microM VIP. Computer analysis was compatible with a single secretin-preferring receptor with a high affinity for secretin (Kd 7 nM) and a low affinity for VIP (Kd 5.9 microM). Comparison of the ability of VIP to increase cyclic AMP with or without the secretin-receptor antagonist, secretin-5-27, demonstrated only occupation of the high affinity VIP-preferring or high affinity secretin-preferring receptors increase cyclic AMP. Our results demonstrate that, in contrast to previous reports, guinea pig pancreatic acini possess 3 classes of receptors that interact with VIP and secretin. The low affinity receptor seen with 125I-VIP is not the same as the secretin-preferring receptor and does not increase cellular cyclic AMP.     

Solubilization of active and stable receptors for vasoactive intestinal peptide from rat liver

Vasoactive intestinal peptide (VIP) receptors were solubilized from rat liver using the zwitterionic detergent CHAPS. Optimal conditions of solubilization were obtained with 5 mM CHAPS and 2.5 mg protein/ml. The binding of 125I-VIP to CHAPS extracts was time- and pH-dependent, saturable and reversible. The following order of potency of unlabeled VIP-related peptides for inhibiting 125I-VIP binding was observed: VIP greater than helodermin greater than peptide histidine isoleucine amide (PHI) greater than rat growth hormone releasing factor (rGRF) greater than secretin. This peptide specificity is identical to that of rat liver membrane-bound receptors. VIP binding activity in the CHAPS extract was destroyed by trypsin or dithiothreitol in accordance with the known sensitivity of membrane-bound receptors to these agents. VIP receptors in CHAPS extracts were stable for at least 5 days at 4 degrees C. Scatchard analysis of equilibrium binding data indicated the presence in CHAPS extracts of high (H) and low (L) affinity binding sites with the following characteristics: KdH = 0.27 nM and BmH = 34 fmol/mg protein; KdL = 51 nM and BmL = 1078 fmol/mg protein. The guanine nucleotide GTP inhibited 125I-VIP binding to soluble receptors and enhanced the dissociation of soluble VIP-receptor complexes, suggesting that GTP-binding proteins were functionally associated with VIP receptors in solution. Gel filtration of solubilized VIP receptors on Sephacryl S-300 revealed a single binding component with a Stokes radius of 6.1 nm. It is concluded that active VIP receptors can be extracted from liver membranes by CHAPS. The availability of this CHAPS-soluble, stable and functional receptor from a tissue which can be obtained in large amounts represents a major step toward the purification of VIP receptors.     

VIP receptors from porcine liver: high yield solubilization in a GTP-insensitive form.

Vasoactive intestinal peptide (VIP) receptors were solubilized from porcine liver membranes using CHAPS. The binding of 125I-VIP to solubilized receptors was reversible, saturable and specific. Scatchard analysis indicated the presence of one binding site with a Kd of 6.5 +/- 0.3 nM and a Bmax of 1.20 +/- 0.15 pmol/mg protein. Solubilized and membrane-bound receptors displayed the same pharmacological profile since VIP and VIP-related peptides inhibited 125I-VIP binding to both receptor preparations with the same rank order of potency e.g. VIP greater than helodermin greater than rat GRF greater than rat PHI greater than secretin greater than human GRF. GTP inhibited 125I-VIP binding to membrane-bound receptors but not to solubilized receptors supporting functional uncoupling of VIP receptor and G protein during solubilization. Affinity labeling of solubilized and membrane-bound VIP receptors with 125I-VIP revealed the presence of a single molecular component with Mr 55,000 in both cases. It is concluded that VIP receptors from porcine liver can be solubilized with a good yield, in a GTP-insentive, G protein-free form. This represents a major advance towards the purification of VIP receptors.     

Pharmacology and molecular identification of vasoactive intestinal peptide (VIP) receptors in normal and cancerous gastric mucosa in man

In human antral membranes, VIP and its natural analogs inhibited the binding of HPLC-purified 125I-VIP, according to the following order of potency: VIP greater than rh GRF greater than helodermin greater than r PHI greater than PHM greater than p PHI greater than hp GRF greater than h, p secretin. No specific binding was detected in plasma membranes purified from the human fundus. In human antral membranes, Scatchard plots were compatible with the existence of two classes of VIP receptors, the first class with high affinity and low binding capacity (Kd = 0.1 nM, Bmax = 10 fmol/mg protein) and another class with a low affinity and higher binding capacity (Kd = 12) nM, Bmax = 1,000 fmol/mg protein). The structure of the VIP receptor in purified plasma membranes prepared from human antral glands and from the HGT-1 human gastric cancer cells was subsequently probed using the cross-linking reagent DSP and 125I-VIP. In agreement with the pharmacological study and the Scatchard analysis of the binding data, SDS gel electrophoresis of the solubilized receptor identified two radiolabeled peptides Mr 67,000 and 34,000 containing disulfide bonds. According to its sensitivity to low doses of VIP and to GTP, the Mr 67,000 binding site represents the membrane domains involved in the physiologial regulation of adenylate cyclase by VIP in normal and transformed human gastric epithelia.     

A human melanoma-derived cell line (IGR39) with a very high number of vasoactive-intestinal-peptide (VIP) receptors. 1. Molecular characterization of the binding site

Using mono[125I]iodinated vasoactive intestinal peptide (125I-VIP), a very high number of specific binding sites for VIP were identified at the surface of the human melanoma cell line IGR39. The Scatchard analysis of competitive displacement experiments between native VIP and 125I-VIP was consistent with the existence of two classes of VIP-binding sites. IGR39 cells possess 0.54 x 10(6) high-affinity sites with a dissociation constant (Kd) of 0.66 nM and 1.3 x 10(6) sites of moderate affinity with a Kd of 4.7 nM. Pharmacological studies indicated that the order of potency in inhibiting 125I-VIP binding of the VIP/secretin family peptides was VIP much greater than peptide histidine methioninamide greater than human growth-hormone-releasing factor(1-44) greater than secretin. Glucagon has no effect on the binding of the labelled peptide. By means of photoaffinity labelling a polypeptide of Mr 63,000 was characterized. The labelling of this species was completely abolished by native VIP. The order of potency of VIP-related peptides in inhibiting 125I-VIP cross-linking to its receptor was the same as in the competition experiments. The glycoprotein nature of the VIP-binding sites of IGR39 cells has been investigated by affinity chromatography on wheat-germ-agglutinin-Sepharose.     

Solubilization of rat lung vasoactive intestinal peptide receptors in the active state. Characterization of the binding properties and comparison with membrane-bound receptors

We demonstrate here that rat lung membrane vasoactive intestinal peptide (VIP) receptors can be extracted in the active state using digitonin. Sepharose 4B gel filtration chromatography was utilized to demonstrate the formation of specific binding complexes between 125I-VIP and solubilized receptors. A rapid soluble receptor assay was established to separate 125I-VIP-receptor complexes from free 125I-VIP, which entailed differential precipitation of the 125I-VIP-receptor complex with polyethylene glycol and bovine gamma-globulin. Using this assay, several detergents were tested for their suitability to extract active VIP receptors, and most favorable results were obtained with digitonin, as judged by specific binding of 125I-VIP to the solubilized receptors. Time course studies indicated that the binding of 125I-VIP to digitonin extract was more rapid than to rat lung membranes. Scatchard analyses of competitive binding data indicated the presence of two classes of binding sites in the digitonin extract, as in the membrane. The values for the dissociation constants (Kd) were 200 pM for Class I and 8 nM for Class II receptors while the values for binding capacity (Bmax) were 200 and 2300 fmol/mg for Class I and II sites, respectively. Although the binding parameters of the two classes were similar to those in the membrane, the pharmacological properties were different, as evidenced by the inability of rat growth hormone releasing factor, a potent VIP agonist in the membrane, to displace specifically bound 125I-VIP from solubilized receptors. The ability to solubilize active VIP receptors represents an important step toward purification of the functional protein.     

Solubilization and hydrodynamic characterization of guanine nucleotide sensitive vasoactive intestinal peptide-receptor complexes from rat intestine

The purpose of this work was to solubilize vasoactive intestinal peptide (VIP) receptors from rat small intestinal plasma membranes and to analyze the nature and function of its molecular form(s) in a nondenaturing environment. Membranes were incubated with 3 nM 125I-VIP, washed, and treated with 1% Triton X-100. Chromatography on Sephadex G-50 showed that 60% of the extractable radioactivity was eluted with macromolecular components in the void volume. This radioactive material was dramatically reduced when 1 microM unlabeled VIP was present in the incubation medium or when membranes were pretreated with trypsin or dithiothreitol. Macromolecular components that had bound 125I-VIP were further chromatographed on Sephacryl S-300. Two peaks were observed: a major one (80%) and a minor one (20%) with Stokes radii of 5.2 and 3.1 nm, respectively. The labeling of both components was inhibited by unlabeled VIP or peptide with NH2-terminal histidine and COOH-terminal isoleucine amide (a VIP agonist). The presence of GTP (0.1 mM) in the incubation medium of membranes completely abolished the labeling of the 5.2-nm component but did not affect that of the 3.1-nm one. Moreover, GTP induced dissociation of 125I-VIP from the 5.2-nm component isolated by Sephacryl S-300 chromatography. This effect was time dependent and nucleotide specific. In contrast, GTP did not affect the stability of the 3.1-nm component. After cholera toxin catalyzed [32P]ADP-ribosylation of membranes, chromatography of solubilized material on Sephacryl S-300 showed that a peak of 32P radioactivity was coeluted with the 5.2-nm component.(ABSTRACT TRUNCATED AT 250 WORDS)     

Molecular identification of receptors for vasoactive intestinal peptide in rat intestinal epithelium by covalent cross-linking. Evidence for two classes of binding sites with different structural and functional properties

The cleavable cross-linking reagent dithiobis (succinimidyl propionate) or DTSP was shown to link 125I-labeled vasoactive intestinal peptide (125I-VIP) covalently to its receptors in rat intestinal epithelial membranes. DTSP treatment of 125I-VIP-labeled membranes inhibited the dissociation of VIP-receptor complexes in a way which was dependent on both time and concentration (ED50 = 200 microM). Polyacrylamide gel electrophoresis of membrane proteins revealed three 125I-VIP-protein complexes of Mr 76 000, 36 000 and 17 000. The labeling of those compounds was not observed when: (a) treatment of membranes by DTSP was omitted; (b) the reagent quench, ammonium acetate, was added together with DTSP; (c) DTSP-treated membranes were incubated with 2-mercaptoethanol which reduces the disulfide bond present within DTSP. Labeling of Mr-76 000 and Mr-36 000 complexes was specific in that it could be abolished by native VIP, while the labeling of the Mr-17 000 was not. Densitometric scanning of autoradiographs indicated that: (a) labeling of the Mr-76 000 complex was abolished by low VIP concentrations (0.03--10 nM), by VIP agonists with the relative potency VIP greater than a peptide having N-terminal histidine and C-terminal isoleucine amide greater than secretin, and by GTP (10(-5)--1 mM) but was unaffected by various other peptide hormones; (b) labeling of the Mr-36 000 complex was inhibited by high VIP concentrations (1--300 nM), by VIP agonists at high concentrations but was not affected by GTP and various peptide hormones. Assuming one molecule of 125I-VIP was bound per molecule of protein, two proteins with Mr-73 000 and 33 000 were identified as VIP binding sites. The Mr-73 000 protein displays many characteristics (affinity, specificity, discriminating power toward agonists, sensitivity to GTP regulation) of the high-affinity VIP receptors mediating adenylate cyclase activation. The Mr-33 000 protein displays the characteristics (affinity, specificity) of a low-affinity VIP binding site. This study thus shows the molecular characteristics of the VIP receptor and further argues for the molecular heterogeneity of VIP binding sites.     

Autoradiographic localization of vasoactive intestinal polypeptide receptors in the rat mesenteric vascular tree

By the use of combined in vitro radioreceptor binding and autoradiographic techniques, we analyzed the pharmacological properties and the anatomical localization of the vasoactive intestinal polypeptide (VIP) receptor in rat superior mesenteric artery and in medium and small mesenteric artery branches. 125I-VIP was bound by sections of rat superior mesenteric artery in a manner consistent with the labeling of specific VIP receptors, with Kd and Bmax values of 0.23 nM and 0.71 pmol/mg protein respectively. Inhibition of 125I-VIP binding with VIP and related peptides gives the following rank order of potency: VIP greater than peptide histidine methionine greater than secretin. Light microscope autoradiography reveals specific VIP binding sites within the medial layer of superior mesenteric artery and its branches. Medium and small sized vessels are richer in 125I-VIP binding sites than the larger ones.     

Solubilization and molecular characterization of active galanin receptors from rat brain.

Galanin receptors were solubilized from rat brain using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid (CHAPS). Binding of 125I-galanin to the soluble fraction was time- and temperature-dependent, saturable, and reversible. Scatchard analysis of binding data indicated that the soluble extract contained a single class of galanin binding sites with a Kd of 0.8 nM and a Bmax of 26 fmol/mg of protein. Unlabeled galanin and its fragments galanin(2-29) and galanin(1-15) antagonized the binding of 125I-galanin to CHAPS-solubilized extracts with relative potencies similar to those observed with membrane receptors. Galanin(3-29) was found inactive. Binding of 125I-galanin to CHAPS extracts was inhibited by guanine nucleotides with the following rank order of potency: GMP-P-(NH)P greater than GTP greater than GDP. Molecular analysis of the soluble galanin receptor by covalent cross-linking of 125I-galanin to CHAPS extracts using disuccinimidyl tartrate and further identification on SDS-PAGE indicated that the soluble galanin binding site behaves as a protein of Mr 54,000. After incubation of CHAPS extracts with 125I-galanin, gel filtration on Sephacryl S-300 followed by ultracentrifugation on sucrose density gradient revealed a binding component with the following hydrodynamic parameters: Stokes radius, 5 nm; s20,w, 4.5 S; Mr, 98,000; frictional ratio, 1.6. GMP-P(NH)P treatment of CHAPS extracts gave rise to a molecular form with the following characteristics: Stokes radius, 4 nm; s20,w, 3.3 S; Mr, 57,000; frictional ratio, 1.4.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solubilization of the liver vasoactive intestinal peptide receptor. Hydrodynamic characterization and evidence for an association with a functional GTP regulatory protein

Vasoactive intestinal peptide (VIP) receptors were solubilized using the nondenaturing detergent Triton X-100 after occupancy of rat liver membrane-bound receptors with 125I-VIP. Gel filtration and ultracentrifugation on sucrose density gradients revealed the existence in the soluble macromolecular fraction of two labeled components: a major (80%) heavy component and a minor (20%) light one. The two components exhibit the following hydrodynamic parameters: Stokes radius, 5.8 nm: s20,w, 5.98 s; Mr, 150,000; frictional ratio, 1.52 for the major; and Stokes radius, 3.0 nm: s20,w, 3.98 s; Mr = 52,000; frictional ratio, 1.12 for the minor component. The labeling of these components was specific in that it dramatically decreased when unlabeled VIP was added together with 125I-VIP. The pharmacological specificity was also assessed by using 10 nM histidylisoleucineamide (a VIP agonist). Many lines of evidence indicate that the light component (Mr = 52,000) is the VIP-receptor complex while the heavy component (Mr = 150,000) is a ternary complex consisting of VIP, the receptor, and a guanine nucleotide regulatory protein, probably Ns. GTP is required to dissociate 125I-VIP from the heavy component whereas it is ineffective on the light component. This effect is nucleotide specific. After cholera toxin-induced [32P]ADP ribosylation of liver membranes, a high peak of 32P radioactivity containing the alpha subunit (Mr = 42,000) of the Ns protein is coeluted with the heavy component on Sephacryl S-300. By mild urea (2 M) treatment, the heavy component is converted into the light without significant dissociation of 125I-VIP. When a Triton extract of membranes prelabeled with 125I-VIP is treated with dithiobis(succinimidyl propionate) subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis reveals a major band corresponding to Mr = 150,000. Alternatively, when prelabeled membranes are directly treated with the cross-linker, a major complex of Mr = 51,000 is observed. This may be related to different accessibility of the cross-linker to the site at which the receptor and the Ns protein interact in the two conditions. In conclusion, these data represent initial reports on the successful solubilization of functional VIP-receptor complexes and provide evidence for an interaction between liver VIP-receptor complexes and a GTP-binding protein.     

Purification of vasoactive intestinal peptide receptor from porcine liver by a newly designed one-step affinity chromatography

Vasoactive intestinal peptide (VIP) receptors were solubilized from porcine liver membrane using the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. The solubilized VIP receptor has been purified approximately 50,000-fold to apparent homogeneity by a one-step affinity chromatography using a newly designed VIP-polyacrylamide resin. The purified receptor bound 125I-VIP with a Kd of 22.3 +/- 0.7 nM and retained its peptide specificity toward VIP-related peptides. The specific activity of the purified receptor (16,400 pmol/mg of protein) was very close to the theoretical value (18,900 pmol/mg of protein) calculated assuming one binding site/protein. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified receptor revealed a single band with an Mr of 53,000 after either silver staining or radioiodination. Affinity labeling of the purified receptor with 125I-VIP using dithiobis(succinimidyl propionate) gave a single radioactive band, the labeling of which was completely inhibited by an excess of unlabeled VIP. In conclusion, an Mr 53,000 protein containing the VIP-binding site was purified to homogeneity by a one-step affinity chromatography using immobilized VIP.     

Properties of vasoactive intestinal peptide-receptor interaction in rat liver membranes

The properties of the specific receptors for vasoactive intestinal peptide (VIP) in rat liver plasma membranes have been studied by using 125I-VIP as a tracer. The binding of the peptide was a reversible, saturable and specific process, as well as time and temperature dependent. Peptide inactivation was also dependent on time and temperature and remained relatively low in the standard conditions used, as it happened in the inactivation of the binding sites. The binding data were compatible with the existence of two classes of VIP receptors: a high affinity (Kd = 4.2 x 10(-10) M) and low binding capacity (1.5 pmol VIP/mg protein) class and another one of low affinity (Kd = 1.7 x 10(-7) M) and high binding capacity (38.6 pmol VIP/mg protein). The specificity of the binding sites of VIP was established from the fact that binding of 125I-VIP was inhibited by native VIP and by 60-fold higher concentrations of secretin but not by the parent hormone glucagon, by insulin or somatostatin at concentrations as high as 10(-6) M.     

Demonstration of a functional receptor for vasoactive intestinal polypeptide on Molt 4b T lymphoblasts

Viable human T lymphoblasts derived from the "Molt 4b" cell line have been shown to possess functional plasma membrane receptors for vasoactive intestinal polypeptide (VIP). Specific binding of 125I-VIP to these lymphoblasts is rapid, reversible and linearly dependent on the number of cells present. Analysis of binding at 17 degrees C reveals a single class of high affinity binding sites over the concentration range of 10(-7) to 10(-11) M VIP (KD = 7.3 +/- 1.3 nM). The Bmax of 0.24 +/- 0.07 nM extrapolates to 15 000 +/- 4000 sites/cell. The binding of 125I-VIP to T lymphoblasts is highly specific; secretin and glucagon, peptides of similar molecular weight which show sequence homology with VIP, are unable to competitively inhibit binding of 125I-VIP to Molt 4b lymphoblasts. VIP activates adenylate cyclase in membrane preparations from Molt 4b lymphoblasts and increases cAMP in intact cells. Half maximal activation in both membrane preparations and intact cells occurs at 5 nM VIP. This demonstration of a functional receptor for VIP suggests that the Molt 4b lymphoblastic cell line may be a useful model system in which to study neuropeptide modulation of T lymphocyte function.     

High affinity peptide histidine isoleucine-preferring receptors in rat liver

Peptide Histidine Isoleucine (PHI) is generally considered a low affinity agonist for Vasoactive Intestinal Peptide (VIP) receptors. In this study, we investigated the presence and characteristics of [125I]PHI binding sites on rat liver membranes. Detergents at nonsolubilizing concentrations (1 mM CHAPS or 0.01% Tween-20) were included in the assay buffer to reduce adsorptive loss of PHI to acceptable levels and permit measurement of PHI-binding to receptors. Under these conditions, binding of PHI to liver membranes was time- and temperature-dependent, reversible and saturable. Unlabeled PHI was 9.7-fold more potent than VIP, and 357-fold more potent than secretin in displacing [125I]-PHI binding. Scatchard analysis suggested the presence of two classes of PHI receptors, with Kd 27 pM and 512 pM. The data from [125I]-PHI and [125I]-VIP binding studies suggested that one class of receptors was PHI-preferring, and the other, equally reactive with PHI and VIP. The concentration of immunoreactive PHI, measured by radioimmunoassay, in blood from the hepatic portal vein of anesthetized rats was 2-fold higher than that from the hepatic vein, suggesting uptake of circulating PHI by the liver.     

Vasoactive intestinal peptide receptor regulation of cAMP accumulation and glycogen hydrolysis in the human Ewing's sarcoma cell line WE-68

This study describes functional characteristics of receptors for vasoactive intestinal peptide (VIP) on human Ewing's sarcoma WE-68 cells. These characteristics include 125I-VIP binding capacity, cellular cAMP generation, glycogen hydrolysis, and pharmacological specificity. Binding studies with 125I-VIP showed specific, saturable, binding sites for VIP in WE-68 cells. Scatchard analysis revealed the presence of a single class of high-affinity binding sites that exhibited a dissociation constant (Kd) of 90 pM and a maximal binding capacity (Bmax) of 24 fmol/mg of protein. VIP and VIP-related peptides competed for 125I-VIP binding in the following order of potency: human (h) VIP greater than human peptide with N-terminal histidine and C-terminal methionine (PHM) greater than chicken secretin much greater than porcine secretin. Glucagon and the C-terminal fragments VIP[10-28] and VIP[16-28] and the VIP analogue (D-Phe2)VIP did not inhibit 125I-VIP binding. Addition of hVIP to WE-68 cells provoked marked stimulation of cAMP accumulation, hVIP stimulated increases in cAMP content were rapid, concentration-dependent, and potentiated by 3-isobutyl-l-methylxanthine (IBMX). Half-maximal stimulation (EC50) occurred at 150 nM hVIP. The ability of hVIP and analogues to stimulate cAMP generation paralleled their potencies in displacing 125I-VIP binding. (D-Phe2)VIP, VIP[10-28], VIP[16-28], and (p-Cl-D-Phe6, Leu17)VIP, a putative VIP receptor antagonist, affected neither basal cAMP levels nor hVIP-induced cAMP accumulation. WE-68 cell responses to hVIP were desensitized by prior exposure to hVIP. Desensitization to hVIP did not modify the cAMP response to beta-adrenergic stimulation, and beta-adrenergic agonist desensitization did not modify responses to hVIP. hVIP also induced a time- and concentration-dependent hydrolysis of 3H-glycogen newly formed from 3H-glucose in WE-68 cultures. hVIP maximally decreased 3H-glycogen content by 36% with an EC50 value of about 8 nM. The order of potency of structurally related peptides of hVIP for stimulation of glycogenolysis correlated with their order of potency for inhibition of 125I-VIP binding. IBMX potentiated the glycogenolytic action of hVIP and PHM. The simultaneous presence of the calcium channel antagonist verapamil or the calcium ionophore A 23187 did not influence the glycogenolytic and cAMP stimulatory effects of hVIP. Collectively, these data indicate that Ewing's sarcoma (WE-68) cells are endowed with genuine VIP receptors which are coupled to the formation of cAMP that probably serves a second messenger role in stimulating glycogen hydrolysis in these cells in response to VIP.(ABSTRACT TRUNCATED AT 400 WORDS)     

Guanosine nucleotides regulate B2 kinin receptor affinity of agonists but not of antagonists: discussion of a model proposing receptor precoupling to G protein

The effect of nucleotides on binding of the B2 kinin (BK) receptor agonist [3H]BK and the antagonist [3H]NPC17731 to particulate fractions of human foreskin fibroblasts was studied. At 0 degrees C, particulate fractions exhibited a single class of binding sites with a Kd of 2.3 nM for [3H]BK and a Kd of 3.8 nM for the antagonist [3H]NPC17731. Incubation with radioligands at 37 degrees C for 5 min gave a reduction of agonist, as well as antagonist, binding that was between 0-40% depending on the preparation, even in the absence of guanosine nucleotides. As shown by Scatchard analysis, this reduction in specific binding was due to a shift in the affinity of at least a fraction of the receptors. The presence at 37 degrees C of the guanine nucleotides GTP, GDP and their poorly hydrolyzable analogs left [3H]NPC17731 binding unaffected, but reduced the receptor affinity for [3H]BK to a Kd of about 15 nM. The maximal number of receptors, however, was unchanged. This affinity change was strongly dependent on the presence of bivalent cations, in particular Mg2+. It was reversed by incubation at 0 degrees C. The rank order of the guanosine nucleotides for [3H]BK binding reduction was GTP[gammaS] = Gpp[NH]p > GTP = GDP > GDP[betaS]. GMP, ATP, ADP and AMP showed no influence on agonist binding. A model for the interaction of the B2 kinin receptor with G proteins is discussed.     

Hydrodynamic characterization of vasoactive intestinal peptide receptors extracted from rat lung membranes in Triton X-100 and n-octyl-beta-D-glucopyranoside

Rat lung membrane vasoactive intestinal peptide (VIP) receptors were covalently labeled with 125I-VIP, extracted in Triton X-100 and n-octyl-beta-D-glucopyranoside, and analyzed by gel filtration and sucrose density gradient sedimentation. The fractions were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography, and the identity of the 125I-VIP.receptor complex was demonstrated by its co-migration with the covalently labeled 55-kDa receptor unit identified previously. Furthermore, the radioactivity in the peak corresponding to the 125I-VIP.receptor complex was displaced in the presence of unlabeled VIP in a dose-dependent manner. The following hydrodynamic properties were determined for VIP receptors in each detergent solution: in Triton X-100, Stokes radius of 6.1 +/- 0.4 nm, sedimentation coefficient (S20,w) of 7.35 +/- 0.45 S, and partial specific volume (v) of 0.809 +/- 0.015 ml/g; in n-octyl-beta-D-glucopyranoside, Stokes radius of 5.6 +/- 0.00 nm, S20,w of 10.87 +/- 0.22 S, and partial specific volume of 0.783 +/- 0.020 ml/g. The apparent molecular weight of the 125I-VIP.receptor.detergent complex was calculated as 270,000 +/- 36,000 in Triton X-100 and 320,000 +/- 32,000 in n-octyl-beta-D-glucopyranoside. The amount of detergent bound to the receptor was estimated by using the two sets of hydrodynamic data and the significantly different partial specific volumes of the two detergents. Thus, the molecular weight of the receptor alone was calculated as 54,600 daltons, indicating that approximately 3.9 g of Triton X-100 and 4.9 g of n-octyl-beta-D-glucopyranoside were bound per g of receptor. This species contained the 55-kDa binding unit and appeared to be glycosylated as evidenced by its specific binding to wheat germ agglutinin-Sepharose. These results indicate that the rat lung VIP receptor is a glycoprotein with a single polypeptide chain of 55 kDa. The large amount of detergent bound suggests that the receptor is extensively embedded in the membrane.     

Solubilization of the active vasoactive intestinal peptide receptor from human colonic adenocarcinoma cells

The non-ionic detergent n-octyl-beta-D-glucopyranoside was used to solubilize the VIP (vasoactive intestinal peptide) receptor from human colonic adenocarcinoma cell line HT29-D4. The binding of monoiodinated 125I-VIP to the solubilized receptor was specific, time-dependent, and reversible. Scatchard analysis of data obtained from competitive displacement of monoiodinated 125I-VIP by native VIP suggested the presence of two classes of VIP binding sites with Kd values of 0.32 and 46.7 nM. The binding capacities of these two classes were 1.7 x 10(10) and 30.2 x 10(10) sites/mg of proteins, respectively. The solubilized receptor retained the specificity of the human VIP receptor towards the peptides of the VIP/secretin/glucagon family. The order of potency in inhibiting monoiodinated 125I-VIP binding was VIP (IC50 = 1.0 x 10(-9) M) much greater than peptide histidine methionine amide (IC50 = 10(-7) M) greater than growth hormone-releasing factor (IC50 = 3 x 10(-7) M) greater than secretin (IC50 greater than 10(-6) M); glucagon had no effect on VIP binding. The reducing agent dithiothreitol inhibited in a dose-dependent manner the binding of 125I-VIP. Covalent cross-linking experiments between the solubilized receptor and 125I-VIP showed that after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography two major and one minor polypeptides of Mr 67,000, 72,000, and 83,000 were specifically labeled. When analyzed by gel filtration, the n-octyl-beta-D-glucopyranoside-solubilized 125I-VIP-receptor complex was resolved into two major peaks with molecular mass in the range of 60-70 and 270-300 kDa. Thus, the soluble form of the VIP receptor was probably a multimeric complex in which disulfide bonds may play an important role to hold the receptor in an active configuration.     

Hypothalamic binding sites for pituitary adenylate cyclase activating polypeptide: characterization and molecular identification.

The goal of these experiments was to identify and characterize binding sites in the rat hypothalamus for the peptide, pituitary adenylate cyclase activating polypeptide (PACAP). The 27 amino acid form of PACAP (PACAP27) was used as the radiolabeled ligand in these experiments. Binding of [125I]PACAP27 to hypothalamic membrane preparations was rapid, reversible on addition of unlabeled peptide, and at least partially regulated by GTP. Nonhydrolyzable GTP analogs, guanosine-5'-O-(3-thiotriphosphate) (GTP gamma S), guanosine-5'-(2-thiodiphosphate) (GDP beta S), and guanylylimidophosphate (GppNHp) also displaced [125I]PACAP27 binding to hypothalamic membrane preparations in a dose-dependent manner. The order of potency for the three analogs was GTP gamma S greater than GDP beta S greater than GppNHp. Both forms of the peptide, PACAP27 and PACAP38, were highly potent in displacing bound [125I]PACAP27, whereas VIP or PACAP(1-23) were unable to displace binding at concentrations of up to 500 nM. Scatchard analysis of the PACAP27 and PACAP38 displacement curves revealed that the fit of both curves was consistent with a single class of high-affinity binding sites, although the site exhibited a greater affinity for PACAP38 compared with PACAP27 (PACAP27 Kd = 1452 +/- 59 pM; PACAP38 Kd = 175 +/- 13 pM; Bmax 23.2 +/- 1.1 pmol/mg protein). The possibility of the existence of a class of binding sites with extremely low affinity cannot be discounted. After covalent cross-linking of [125I]PACAP27 with its receptor, the molecular weights of the complexes were estimated by electrophoresis and autoradiography. A major band of 60 Kd was evident when membranes were incubated with VIP or PACAP(1-23). Previous incubation with unlabeled PACAP27 or PACAP38 eliminated visualization of this band. These results suggest that a specific, high-affinity binding site for PACAP27 is present in rat hypothalamus, and that this site shows a greater affinity for PACAP38 compared with PACAP27. The molecular weight of the peptide-receptor complex is 60,000 kDa, and therefore the receptor itself has an apparent molecular weight 57,000.