Peptide T from human immunodeficiency virus does not interact with VIP receptor-effector system in immunocompetent cells of rat and mouse

Human immunodeficiency virus (HIV) infection is initiated by attachment of the virus to specific target cells. An octapeptide sequence contained within the envelope of HIV, peptide T, mediates the viral binding. Since there is an appreciable structural similarity between peptide T and an eight amino acid sequence of VIP, it is interesting to investigate the interaction of peptide T with the VIP receptor-effector system of immunocompetent cells from both rat and mouse. In this paper, we show the lack of interaction between peptide T and VIP receptor-effector system in peripheral blood lymphocytes, spleen lymphocytes and macrophages of rat and in macrophages of mouse. These results do not support the hypothesis that HIV through peptide T may interact with the VIP receptor-effector system present in immunocompetent cells.     

Peptide T does not affect induction of pineal N-acetyltransferase by vasoactive intestinal peptide

It has been suggested that the HIV virus binds to VIP recognition sites which can be blocked by the octapeptide, peptide T. Stimulation of VIP receptors on pinealocytes activates adenylate cyclase and increases the activity of the enzyme serotonin N-acetyltransferase (NAT). We examined whether peptide T or D-Ala peptide T amide affected this induction. We found no evidence for peptide T interference with NAT induction and conclude that if peptide T inhibits attachment of HIV virus to VIP receptors, it does so at regions other than that occupied by VIP in stimulating adenylate cyclase and NAT.     

CD4 receptor binding peptides that block HIV infectivity cause human monocyte chemotaxis. Relationship to vasoactive intestinal polypeptide

The octapeptide Ala-Ser-Thr-Thr-Thr-Asn-Tyr-Thr (peptide T) and two structural analogs are potent agonists of human monocyte chemotaxis, evincing identical rank potency orders as was previously shown for their inhibition of human immunodeficiency virus (HIV) envelope binding and T cell infectivity. Chemotactic activity could be inhibited by anti-CD4 monoclonal antibodies (Mabs), but not other mononuclear cell Mabs. The core peptide required for chemotactic activity is a pentapeptide related to the sequence Thr-Thr-Asn-Tyr-Thr. Homologous pentapeptides, identified by computer search, were detected in several other non-HIV-related viruses as well as the neuropeptide vasoactive intestinal polypeptide (VIP). The CD4 molecule, therefore, appears to be a recognition molecule for a small signal peptide ligand whose active sequence is a homolog of peptide T and which may be the neuropeptide VIP.     

Identification of high affinity receptors for vasoactive intestinal peptide on human lymphocytes of B cell lineage

Specific, high affinity receptors for vasoactive intestinal peptide (VIP) have been identified on a human pre-B cell line, Nalm 6, and on a human plasma cell line, Dakiki. The single class of high affinity sites exhibited a KD of 12.6 +/- 2.9 nM for VIP in Nalm 6 cells and 9.1 +/- 2.7 nM in Dakiki plasma cells. The homologous peptides, peptide histidine methionine (PHM), growth hormone releasing factor (GHRF), and secretin were all less effective than VIP in competitively inhibiting binding of 125I-VIP to Nalm 6 and Dakiki plasma membranes. The putative receptor was characterized as a 47-kDa protein using covalent cross-linking techniques and VIP stimulated adenylate cyclase in pre-B cells. Human lymphocytes of B cell lineage thus appear to express functional VIP receptors homologous to the receptor identified in T lymphoblasts, brain, pituitary, and intestine.     

The rat liver vasoactive intestinal peptide binding site. Molecular characterization by covalent cross-linking and evidence for differences from the intestinal receptor.

To identify the molecular components of the vasoactive intestinal peptide (VIP) binding sites in the liver, 125I-labelled VIP was covalently linked to liver membranes by using the cleavable cross-linker dithiobis(succinimidylpropionate). Purified rat liver plasma membranes were incubated with 125I-VIP, washed and treated with 1 mM-cross-linker. Polyacrylamide-gel electrophoresis of membrane proteins followed by autoradiography revealed a major 125I-VIP-protein complex of Mr 51 000. A minor Mr 89 000 complex was also observed. An identical pattern of protein labelling was obtained using crude membranes from rat liver. Labelling of the Mr 51 000 and 89 000 species was specific in that it could be abolished by native VIP, but was unaffected by 1 microM-glucagon and cholecystokinin octapeptide. Densitometric scanning of autoradiographs indicated that the labelling of the two species was abolished by similar low VIP concentrations (0.1-100 nM). It was also reduced by two VIP agonists, peptide histidine isoleucine amide and secretin, with a potency that is 1/7 and 1/200 that of native VIP, respectively. The guanine nucleotide GTP in the concentration range between 10(-7) and 10(-3) M reduces the labelling of the major Mr 51 000 protein and that of the minor Mr 89 000 protein, but with a slightly higher potency. Assuming one molecule of 125I-VIP was bound per molecule of protein, a major Mr 48 000 protein and a minor Mr 86 000 protein were identified as components of the high-affinity VIP binding sites in liver. This contrasts markedly with the pattern of labelling of rat intestinal epithelial membranes, where a Mr 73 000 protein was identified as a high-affinity VIP receptor and a Mr 33 000 protein as a low-affinity VIP binding site [Laburthe, Bréant & Rouyer-Fessard (1984) Eur. J. Biochem. 139, 181-187], suggesting structural differences between VIP binding sites in rat liver and intestinal epithelium.     

Interaction of a bovine thymic peptide extract with vasoactive intestinal peptide (VIP) receptors

Bovine t hymic peptide extract (1–100 g/ml) is shown to completely inhibit the binding of [¹²⁵I]VIP to rat blood mononuclear cells, lymphoid cells of spleen, and liver plasma membranes. In the three models, the bovine thymic peptide extract inhibits [¹²⁵I]VIP binding with a potency that is 4000–7000 times lower than that of the native VIP, on a weight basis. In rat liver plasma membranes, the bovine thymic peptide extract stimulates adenylate cyclase with a maximal efficiency that is similar to that of VIP. At maximal doses, VIP and thymic peptide extract do not exert an additive effect on adenylate cyclase, suggesting that the activation of the enzyme by the bovine thymic peptide extract occurs through VIP receptors. Finally, no VIP-like immunoreactivity was detected in the thymic peptide extract using an antiserum raised against mammalian VIP. All these data suggest the presence in the bovine thymic peptide extract of a new substance which behaves as a VIP agonist in rat.     

Vasoactive intestinal peptide modulates Langerhans cell immune function

Epidermal nerves lie in close proximity to Langerhans cells (LC) and are capable of releasing peptides that modulate LC function, including calcitonin gene-related peptide and pituitary adenylate cyclase-activating polypeptide. The neuropeptide vasoactive intestinal peptide (VIP) has also been found in cutaneous nerves and mRNA, for the VIP receptor vasoactive intestinal peptide receptor type 1, and vasoactive intestinal peptide receptor type 2 have been found in murine LC and the LC-like cell line XS106. We examined the effects of VIP on LC function and cutaneous immunity. VIP inhibited elicitation of a delayed-type hypersensitivity response in previously immunized mice by epidermal cells enriched for LC content pulsed with Ag in vitro. VIP also inhibited the ability of unseparated epidermal cells to present Ag to a T cell clone and hybridoma and the ability of highly enriched LCs to present to the T cell clone. Inhibition of presentation to the hybridoma was observed with an antigenic peptide that does not require processing, suggesting that VIP is active at a step independent of Ag processing. To elucidate the mechanism(s) by which VIP may mediate these effects, we determined the effects of VIP on LC cytokine production using the XS106 cell line as a surrogate for LC. VIP augmented the production of the IL-10 in LPS-stimulated XS106 cells while down-regulating IL-12 and IL-1beta production. Thus, VIP, like pituitary adenylate cyclase-activating polypeptide and calcitonin gene-related peptide, down-regulates LC function and the associated immune response.     

Pharmacological characterization of the novel helodermin/VIP receptor present in human SUP-T1 lymphoma cell membranes

[Acetyl-His1]VIP stimulated adenylate cyclase with higher potency than VIP in membranes from human SUP-T1 lymphoblasts and was used as an efficient radioiodinated ligand with low non-specific binding to evaluate the relationship between receptor occupancy and adenylate cyclase activation and the possible interference of peptide T (an epitope derived from HIV envelope protein gp120). Various peptides inhibited [125I-acetyl-His1]VIP binding and activated the enzyme, their order of potency being: helodermin greater than [acetyl-His1]VIP greater than VIP = PHI = [Phe1]VIP greater than [D-Phe2]VIP = [D-Ala4]VIP = [D-Phe4]PHI greater than or equal to [D-Phe4]VIP greater than [D-His1]VIP giving further support for the existence of a novel subtype of helodermin/VIP receptors. [D-Ala1]peptide T and VIP-(10-28) did not recognize the binding site and did not inhibit, even at high concentration, VIP - or VIP analogue - stimulated adenylate cyclase activities.     

Selective gene expression and activation-dependent regulation of vasoactive intestinal peptide receptor type 1 and type 2 in human T cells

Vasoactive intestinal peptide (VIP) has potent antiproliferative and anti-inflammatory functions in the immune system. Two structurally distinct G-protein-associated receptors, VIP receptor type 1 (VPAC1) and VIP receptor type 2 (VPAC2), mediate the biological effects of VIP. The regulation of VIP receptor gene expression and the distribution of these receptors in different compartments of the human immune systems are unknown. This study reports, for the first time, a quantitative analysis of VPAC1 and VPAC2 mRNA expression in resting and activated T cells as well as in resting monocytes. Purified human peripheral blood CD4(+) T cells and CD8(+) T cells were stimulated via the TCR/CD3 receptor complex. Using the novel fluorometric-based kinetic (real-time) RT-PCR, we determined that VPAC1 is constitutively expressed in resting T cells and monocytes; the levels of expression were significantly higher in monocytes and CD4(+) T cells than in CD8(+) T cells. VPAC1 mRNA expression is significantly higher relative to VPAC2 in resting CD4(+) T cells and CD8(+) T cells. VPAC2 is expressed at very low levels in resting T cells but is not detectable in resting monocytes. In vitro stimulation of Th cells with soluble anti-CD3 plus PMA induced a T cell activation-dependent down-regulation of VPAC1. VPAC1 is down-regulated under conditions of optimal T cell stimulation. Our results suggest that selective VIP effects on T cell function may be mediated via selective expression of VPAC1 and VPAC2 on T cells and monocytes. Furthermore, down-regulation of VPAC1 in CD4(+) T cell subpopulations is highly correlated with T cell activation.     

Vasoactive intestinal peptide receptors on AR42J rat pancreatic acinar cells.

VIP receptors on AR42J rat pancreatic cells were analyzed by competition binding, affinity labeling and by N-glycanase digestion analyses. These studies revealed the presence of specific, high affinity (Kd approximately 1 nM) VIP receptors with a mass of 67 kDa or 59 kDa under reducing or non-reducing conditions, respectively. N-glycanase digestion of affinity labeled membranes generated a core receptor protein of approximately 44 kDa and evidence for at least two N-linked glycans on the mature receptor. The receptor lacked O-linked oligosaccharides but contained terminal sialic acid residues on its N-linked glycan(s) based on digestions with O-glycanase and neuraminidase. The similarity of the AR42J VIP receptor to the recently cloned cDNA for human VIP receptors makes this cell line an attractive model for further analysis of VIP receptor signal transduction events.     

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.     

Functional expression and tissue distribution of a novel receptor for vasoactive intestinal polypeptide.

Vasoactive intestinal polypeptide (VIP), a 28 amino acid peptide hormone, plays many physiological roles in the peripheral and central nerve systems. A functional cDNA clone of the VIP receptor was isolated from a rat lung cDNA library by cross-hybridization with the secretin receptor cDNA. VIP bound the cloned VIP receptor expressed in mouse COP cells and stimulated adenylate cyclase through the cloned receptor. The rat VIP receptor consists of 459 amino acids with a calculated Mr of 52,054 and contains seven transmembrane segments. It is structurally related to the secretin, calcitonin, and parathyroid hormone receptors, suggesting that they constitute a new subfamily of the Gs protein-coupled receptors. VIP receptor mRNA was detected in various rat tissues including liver, lung, intestines, and brain. In situ hybridization revealed that VIP receptor mRNA is widely distributed in neuronal cells of the adult rat brain, with a relatively high expression in the cerebral cortex and hippocampus.     

Effect of bilateral adrenalectomy on VIP receptor/effector system in rat intestinal epithelial cells

The number of vasoactive intestinal peptide (VIP) receptors and the efficiency of VIP in the stimulation of cyclic AMP accumulation in rat jejunal epithelial cells increased after bilateral adrenalectomy. However, this condition increased neither receptor affinity nor VIP potency. In addition, jejunal VIP levels followed a parallel increase. These changes reversed to control conditions after glucocorticoid replacement with dexamethasone indicating that adrenalectomy modifies the intestinal VIP receptor/effector system and suggest a relationship between corticosteroids and VIP in the functions of intestinal epithelium.     

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.     

Vasoactive intestinal peptide receptor on liver plasma membranes: Solubilization and cross-linking

The hepatic receptor for VIP was solubilized from rat liver plasma membranes with 1.4% digitonin and shown to conserve its ability to bind to the ligand. This solubilized receptor demonstrated the high affinity and specificity for VIP (K_D1 nM, binding preference: VIP > PHI > secretin > thymosin ₁) which were observed with the nonsolubilized VIP receptor on intact liver plasma membranes. ¹²⁵I-VIP was next cross-linked to either the solubilized or nonsolubilized receptor using disuccinimido suberate or disuccinimido dithiobis(propionate), and the resulting complexes analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. A broad autoradiographic band which demonstrated a high affinity for VIP was identified at Mr 56,000 (53,000 in the absence of the reducing agent dithiothreitol) for both the solubilized and nonsolubilized receptors. We have thus been able to solubilize from rat liver plasma membranes a receptor with high affinity and specificity for VIP, and confirmed its structural similarity with the native VIP receptor in nonsolubilized membranes using cross-linking techniques.     

A strong homology exists between the active T-cell binding gp120 octapeptide of human immunodeficiency virus and the subtilisin cleavage peptide of bovine ribonuclease A

A homology has been found between an octapeptide involved in attachment of the human immunodeficiency virus to helper/inducer T cells and an octapeptide segment of bovine pancreatic ribonuclease A. This segment (residues 19-26) contains the sites for subtilisin cleavage of this enzyme into the S-peptide and S-protein. From the X-ray crystal structure of ribonuclease, this sequence is known to be exposed to solvent and interacts little with the rest of the protein. A structure for the human immunodeficiency virus attachment peptide can be deduced from this homology, as a well-defined structure has been determined for this sequence in ribonuclease. This can be readily accomplished using previously developed computer methods based upon conformational energy calculations. The calculated structure for human immunodeficiency virus peptide is identical to the ribonuclease segment (19-26) in backbone conformation. It is stabilized by internal interactions of nonpolar residues, and by exposure of polar hydroxyl groups. The results suggest that the T-cell human immunodeficiency virus receptor may be hydrophilic in nature and that conservation of the sequence in two presumably functionally unrelated proteins is related to the need for conservation of exposed structure.     

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.     

Adenylate cyclase stimulation by VIP in rat and human parotid membranes

Adenylate cyclase activity was stimulated by vasoactive intestinal peptide (VIP) in rat parotid membranes, in the presence of 100 microM guanosine triphosphate (GTP). The threshold concentration of VIP was 300 nM and the activity doubled at the maximal VIP concentration tested (30 microM). The relative potency of peptides of the VIP family was: VIP greater than peptide histidine isoleucinamide (PHI) greater than secretin. The beta-adrenergic agent isoproterenol was a more efficient activator of rat parotid adenylate cyclase and its stimulatory effect, like that of VIP, depended on the presence of GTP. The effects of VIP and isoproterenol were both potentiated by 10 microM forskolin. By comparison with rat parotid preparations, membranes from a human parotid gland responded similarly to the VIP family of peptides (VIP greater than PHI greater than secretin). In both rat and human parotid membranes, two proteins (Mr 44 kDa and 53 kDa) of the alpha-subunit of Ns (the guanyl nucleotide-binding stimulatory protein) were labelled by ADP-ribosylation, in the presence of cholera toxin. Taken together, these results indicate that VIP receptors, when coupled to Ns, were able to activate the adenylate cyclase system in rat and human parotid membranes.     

Vasoactive intestinal peptide effects on GH3 pituitary tumor cells: high affinity binding, affinity labeling, and adenylate cyclase stimulation. Comparison with peptide histidine isoleucine and growth hormone-releasing factor

The vasoactive intestinal polypeptide (VIP) receptor was characterized on the GH3 rat pituitary tumor cell line using competitive binding studies with peptides having sequence homology with VIP. Further studies investigated receptor coupling to the adenylate cyclase complex by measurement of cAMP levels. Finally, the molecular weight of the receptor was estimated by affinity labeling techniques. Studies using 125I-VIP and unlabeled competing peptides revealed a single class of high affinity binding sites with a dissociation constant (KD) of 17 +/- 2 nM (mean +/- S.E.M.) for VIP, 275 +/- 46 nM for peptide histidine isoleucine (PHI), and 1380 +/- 800 nM for human pancreatic growth hormone releasing factor (GHRF). VIP and PHI each stimulated intracellular cAMP accumulation in a dose-dependent manner; both peptides demonstrated synergism with forskolin. In contrast, GHRF neither stimulated accumulation of cAMP nor demonstrated synergism with forskolin. VIP plus PHI (1 microM each) caused no significant increase in cAMP over either VIP or PHI alone, implying that the two peptides act through the same receptor. Covalent crosslinking of 125I-VIP to its binding site using either disuccinimidyl suberate (DSS) or ethylene glycol bis(succinimidyl succinate) (EGS) was followed by SDS-PAGE and autoradiography. The result is consistent with an Mr 47 000 VIP-binding subunit comprising or being associated with the VIP receptor of GH3 pituitary tumor cells.