Binding of cholecystokinin-8 (CCK-8) peptide derivatives to CCKA and CCKB receptors.

The structural requirements for the selective binding of cholecystokinin-8 (CCK-8)-related peptides to peripheral (CCKA) receptors are not sufficiently understood. In this study, the interaction of a series of newly shortened analogues of CCK-8 with both receptor subtypes was analyzed by displacement studies using [3H]-CCK-8 and 125I-Bolton-Hunter (BH)-CCK-8 as radioligands. The pentapeptide derivative of CCK-8, succinyl-Tyr (SO3H)-Met-Gly-Trp-Met-phenethylamide, was found to bind selectively with high affinity to the CCKA receptor. The replacement of Met28 and/or Met31 by norleucine and of L-Trp30 by its D-analogue had no significant effect on the binding properties of the peptide. Further C-terminal shortening resulted in a drastic loss of affinity and selectivity of the CCK receptor binding.

Phosphatidylinositol (PI) 3-kinase and PI 4-kinase binding to the CD4-p56lck complex: the p56lck SH3 domain binds to PI 3-kinase but not PI 4-kinase.

CD4 serves as a receptor for major histocompatibility complex class II antigens and as a receptor for the human immunodeficiency virus type 1 (HIV-1) viral coat protein gp120. It is coupled to the protein-tyrosine kinase p56lck, an interaction necessary for an optimal response of certain T cells to antigen. In addition to the protein-tyrosine kinase domain, p56lck possesses Src homology 2 and 3 (SH2 and SH3) domains as well as a unique N-terminal region. The mechanism by which p56lck generates intracellular signals is unclear, although it has the potential to interact with various downstream molecules. One such downstream target is the lipid kinase phosphatidylinositol 3-kinase (PI 3-kinase), which has been found to bind to activated pp60src and receptor-tyrosine kinases. In this study, we verified that PI 3-kinase associates with the CD4:p56lck complex as judged by the presence of PI 3-phosphate generated from anti-CD4 immunoprecipitates and detected by high-pressure liquid chromatographic analysis. However, surprisingly, CD4-p56lck was also found to associate with another lipid kinase, phosphatidylinositol 4-kinase (PI 4-kinase). The level of associated PI 4-kinase was generally higher than PI 3-kinase activity. HIV-1 gp120 and antibody-mediated cross-linking induced a 5- to 10-fold increase in the level of CD4-associated PI 4- and PI 3-kinases. The use of glutathione S-transferase fusion proteins carrying Lck-SH2, Lck-SH3, and Lck-SH2/SH3 domains showed PI 3-kinase binding to the SH3 domain of p56lck, an interaction facilitated by the presence of an adjacent SH2 domain. PI 4-kinase bound to neither the SH2 nor the SH3 domain of p56lck. CD4-p56lck contributes PI 3- and PI 4-kinase to the activation process of T cells and may play a role in HIV-1-induced immune defects.

Regulation of CD4-p56lck-associated phosphatidylinositol 3-kinase (PI 3-kinase) and phosphatidylinositol 4-kinase (PI 4-kinase).

CD4 serves as a receptor for MHC class II antigens and as a receptor for the human immunodeficiency virus (HIV-1) viral coat protein gp120. It is coupled to the protein-tyrosine kinase p56lck, an interaction necessary for an optimal response of certain T cells to antigen. Although anti-CD4 crosslinking may increase lck activity, the effects of HIV-1 gp120 have been controversial. Activated protein-tyrosine kinases are known to associate with certain intracellular proteins possessing src-homology regions (SH-2 domains) such as phosphatidylinositol 3-kinase (PI 3-kinase). In this paper, we demonstrate that the CD4:p56lck complex associates with significant amounts of phosphatidylinositol (PI) kinase activity. High pressure liquid chromatographic (HPLC) analysis of the reaction products demonstrated the presence of phosphatidylinositol 3-phosphate (PI 3-P) and phosphatidylinositol 4-phosphate (PI 4-P), thus indicating that PI 3 and PI 4 kinases associate with CD4-p56lck. The p85 subunit of PI 3-kinase was also detected in anti-CD4 immunoprecipitates by immunoblotting with anti-p85 antiserum. Significantly, p56lck binding to CD4 appears to be necessary for the detection of lipid kinase activity associated with p56lck. Also, anti-HIV gp120 and anti-CD4 crosslinking induced a 10-15-fold increase in levels of both PI 3- and PI 4-kinase activity in anti-CD4 precipitates. Stimulation of CD4-p56lck-linked PI kinases by crosslinked HIV-1 gp120 may play a role in HIV-1-induced immune defects.

Functional and immunologic characterization of human immunodeficiency virus type 1 envelope glycoproteins containing deletions of the major variable regions.

Deletions of the major variable regions (V1/V2, V3, and V4) of the human immunodeficiency virus type 1 (HIV-1) gp120 exterior envelope glycoprotein were created to study the role of these regions in function and antigenicity. Deletion of the V4 region disrupted processing of the envelope glycoprotein precursor. In contrast, the deletion of the V1/V2 and/or V3 regions yielded processed exterior envelope glycoproteins that retained the ability to interact with the gp41 transmembrane glycoprotein and the CD4 receptor. Shedding of the gp120 exterior glycoprotein by soluble CD4 was observed for the mutant with the V3 deletion but did not occur for the V1/V2-deleted mutant. None of the deletion mutants formed syncytia or supported virus entry. Importantly, the affinity of neutralizing antibodies directed against the CD4-binding region for the multimeric envelope glycoprotein complex was increased dramatically by the removal of both the V1/V2 and V3 structures. These results indicate that, in addition to playing essential roles in the induction of membrane fusion, the major variable regions mask conserved neutralization epitopes of the HIV-1 gp120 glycoprotein from antibodies. These results explain the temporal pattern associated with generation of HIV-1-neutralizing antibodies following infection and suggest stratagems for eliciting improved immune responses to conserved gp120 epitopes.

Effects of amino acid changes in the extracellular domain of the human immunodeficiency virus type 1 gp41 envelope glycoprotein.

Changes were introduced into conserved amino acids within the ectodomain of the human immunodeficiency virus type 1 (HIV-1) gp41 transmembrane envelope glycoprotein. The effect of these changes on the structure and function of the HIV-1 envelope glycoproteins was examined. The gp41 glycoprotein contains an amino-terminal fusion peptide (residues 512 to 527) and a disulfide loop near the middle of the extracellular domain (residues 598 to 604). Mutations affecting the hydrophobic sequences between these two regions resulted in two phenotypes. Some changes in amino acids 528 to 562 resulted in a loss of the noncovalent association between gp41 and the gp120 exterior glycoprotein. Amino acid changes in other parts of the gp41 glycoprotein (residues 608 and 628) also resulted in subunit dissociation. Some changes affecting amino acids 568 to 596 resulted in envelope glycoproteins partially or completely defective in mediating membrane fusion. Syncytium formation was more sensitive than virus entry to these changes. Changes in several amino acids from 647 to 675 resulted in higher-than-wild-type syncytium-forming ability. One of these amino acid changes affecting tryptophan 666 resulted in escape from neutralization by an anti-gp41 human monoclonal antibody, 2F5. These results contribute to an understanding of the functional regions of the HIV-1 gp41 ectodomain.

Antiviral effects of different CD4-immunoglobulin constructs against HIV-1 and SIV: immunological characterization, pharmacokinetic data and in vivo experiments.

The CD4 cell surface antigen belongs to the immunoglobulin superfamily and is the primary receptor for the human immunodeficiency virus 1 (HIV-1). The high affinity interaction between HIV-1 and CD4 is mediated by the viral envelope glycoprotein gp120. Recombinant soluble CD4 (rsCD4) has been shown in vitro to be an effective inhibitor of HIV-1 and HIV-2 propagation in lymphoid cells. A variety of antibody-like molecules were constructed, consisting of different parts of the extracellular domain of CD4 fused to immunoglobulin constant regions. The fusion proteins were expressed in mammalian cell lines and purified via affinity chromatography. The specificity and anti-viral effects of the different CD4-immunoglobulin constructs against HIV were analysed by different immunological tests, i.e., immunofluorescence, neutralisation and in vitro assays. In pharmacokinetic studies, differences were found in serum half-life between the four- and two-domain CD4 constructs in cynomolgus monkeys and between glycosylated and deglycosylated CD4-Fc constructs in rabbits. In two in vivo experiments using the four-domain CD4-Fc in SIV-infected macaques, no beneficial effects were observed.

Ganglioside-induced CD4 endocytosis occurs independent of serine phosphorylation and is accompanied by dissociation of P56lck.

Gangliosides induce a selective and complete modulation of CD4 from the surface of T cells. CD4 down-modulation occurs by CD4 endocytosis. This process is independent of serine phosphorylation of the cytoplasmic tail of CD4 and does not require the association between the tyrosine protein kinase p56lck and the cytoplasmic tail of CD4. Ganglioside-induced CD4 endocytosis is accompanied by the loss of p56lck activity associated with CD4. Sequential immunoprecipitation analysis using an anti-CD4 antibody and an anti-p56lck antiserum showed that this is caused by the dissociation of the enzyme from the cytoplasmic tail of CD4. The kinetics of p56lck dissociation after ganglioside treatment is identical to that of CD4 endocytosis, suggesting that p56lck is displaced in the process of endosome formation. The results indicate that CD4 endocytosis alone can cause the dissociation of the p56lck complex without the requirement for CD4 phosphorylation.

Lack of correlation between soluble CD4-induced shedding of the human immunodeficiency virus type 1 exterior envelope glycoprotein and subsequent membrane fusion events.

The noncovalent association of the gp120 and gp41 envelope glycoproteins of human immunodeficiency virus type 1 (HIV-1) is disrupted by soluble CD4 binding, resulting in shedding of the gp120 exterior envelope glycoprotein. This observation has led to the speculation that interaction of gp120 with the CD4 receptor triggers shedding of the exterior envelope glycoprotein, allowing exposure of gp41 domains necessary for membrane fusion steps involved in virus entry or syncytium formation. To test this hypothesis, a set of HIV-1 envelope glycoprotein mutants were used to examine the relationship of soluble CD4-induced shedding of the gp120 glycoprotein to envelope glycoprotein function in syncytium formation and virus entry. All mutants with a threefold or greater reduction in CD4-binding ability exhibited marked decreases in gp120 shedding in response to soluble CD4, even though several of these mutants exhibited significant levels of envelope glycoprotein function. Conversely, most fusion-defective mutants with wild-type gp120-CD4 binding affinity, including those with changes in the V3 loop, efficiently shed gp120 following soluble CD4 binding. Thus, soluble CD4-induced shedding of gp120 is not a generally useful marker for conformational changes in the HIV-1 envelope glycoproteins necessary for the virus entry or syncytium formation processes. Some gp120 mutants, despite being expressed on the cell surface and capable of efficiently binding soluble CD4, exhibited decreased gp120 shedding. These mutants were still sensitive to neutralization by soluble CD4, indicating that, for envelope glycoproteins exhibiting high affinity for soluble CD4, competitive inhibition may be more important than gp120 shedding for the antiviral effect.

Effects of CD4 synthetic peptides on HIV type I envelope glycoprotein function.

Benzylated derivatives of a peptide (CD4(81-92)) representing the CDR3-like region of CD4 were previously found to inhibit gp120 binding, HIV-1 infectivity, and syncytium formation. These results have been interpreted to indicate a role for the corresponding CD4 region in these processes. The peptide (TbYICbEbVEDQKAcEE) is the prototype of a series of similar CD4(81-92) derivatives. We report that this peptide noncompetitively inhibits binding to CD4 of both gp120 and a mAb (MAX.16H5), both of which recognize the CDR2-like region of CD4. The binding of an antibody (Leu 3a) that is directed against a different area of the D1 domain of CD4 was also inhibited. The peptide derivative inhibited both HIV-1- and HTLV-1-mediated syncytium formation in the same concentration range. Nonbenzylated cyclic and linear peptides representing the CDR3-like region of CD4 (CD4(84-101)) had only minor effects on gp120 binding which were not sequence specific. The results of this study suggest that the effects of benzylated CD4(81-92) derivatives on HIV-1 binding or fusion should not be used to reach conclusions about the function of the corresponding CD4 region.

CCK-8-related C-terminal tetrapeptides: affinities for central CCKB and peripheral CCKA receptors.

We investigated the binding affinity of new tetrapeptides derived from the C-terminal sequence of CCK8 to central CCKB and peripheral CCKA receptors. Compound 1 (Boc-Trp-Met-Asp-Phe-NH2) showed high affinity for central CCKB receptors (Ki 4.2 x 10(-8) M, pancreas/cortex ratio = 283). Compounds 2 (Suc-Trp-Met-Asp-Phe-NH2) and 3 (Suc-Trp-Leu-Asp-Phe-NH2) also exhibited high affinity (Ki 2.7 x 10(-8) M and 5.6 x 10(-8) M, respectively) but their CCKB selectivity was nearly 50 times higher (Ki ratio greater than 14,000). Replacement of Met or Leu by other amino acids resulted in less effective tetrapeptides.

[3H]naloxone as an opioid receptor label: analysis of binding site heterogeneity and use for determination of opioid affinities of casomorphin analogues.

The nonselective antagonist [3H]naloxone was used to identify opioid receptors in rat brain membranes. The multiple naloxone binding sites were related to different opioid receptors by means of selective opioid ligands as well as various beta-casomorphin analogues. Analysis of binding site heterogeneity was performed using several computer curve fitting methods. The results indicate that structurally modified casomorphin peptides are able to discriminate between mu 1- and mu 2-binding sites. The affinities to the mu-sites obtained with [3H]naloxone as label are in a good agreement with those from experiments with the mu-selective radioligand [3H]DAGO. The mu 1-site affinities of these casomorphin derivatives are well correlated with their antinociceptive potencies. This finding suggests the mediation of the analgesic activity via the high-affinity mu 1-subtype.

Substance P and Arg-Pro-Lys-Pro-NH-C12-H25-induced mediator release from different mast cell subtypes of rat and guinea-pig.

Histamine was released from mast cells in isolated perfused heart and kidney of the rat, but not from mast cells in guinea-pig tissues, by a substance P (SP) analogue (SP(1-4)-NH-C12H25), SP(1-4)-C12 for abbreviation. This peptide also released histamine from peritoneal mast cells and basophil leucocytes of the rat. Substance P itself was compared with SP(1-4)-C12 and some structurally related peptides and showed weaker activity. SP(1-4)-C12 also released leukotrienes C4, D4, E4 and thromboxane B2 from rat heart. However, there was little effect on heart rate and force of contraction and no effect on perfusion pressure (vascular resistance) of either rat heart or kidney. The findings demonstrate the structural requirements for histamine release by SP (a possible mediator of 'neurogenic' inflammation), the metabolic energy-dependence of the release process and the functional heterogeneity and interspecies differences in mast cell populations.

Modulation of the adoptive immune response in mice by histamine.

The immunomodulatory actions of histamine in mice were examined by a combined in vitro/in vivo approach. Spleen cells from mice were incubated between 2 and 24 hours with histamine (10(-12)-10(-3) M) under conditions which prevent a change of the free histamine concentration. The cells were subsequently transferred to sublethally irradiated syngenic mice in order to measure the adoptive IgM response. Only stimulatory effects of histamine were found at NMRI mice. However, both stimulatory and inhibitory actions were observed at different histamine concentrations if mice of the strains AB or XVII were used. The graft versus host reaction was measured after transfer of histamine treated spleen cells (strain XVII) to neonatal F1 (XVII X B10.LP) hybrid mice and revealed both suppressive and stimulatory effects of histamine at different concentrations. A maximal expression of the immunomodulatory effects of histamine was found after 8 hours of preincubation with the donor cells. The action of selective histamine antagonists and cell separation experiments indicated that the effect of histamine on the adoptive IgM response was mediated by H2-receptors on spleen T-cells. Summarizing, the results indicate that low histamine concentrations elicit bidirectional immunomodulatory effects in mice which vary considerably among different strains.

Histamine induced murine suppressor and potentiating factors.

Mouse spleen cells were incubated for 24 hours in the presence of histamine (10(-13)-10(-3) M). Aliquots of the histamine free supernatants were intravenously injected into either syngenic or allogenic mice which were subsequently analysed by the Jerne plaque assay in respect of their specific IgM response against red blood cells from sheep. Depending on the histamine concentration during the preincubation and the mouse strain, the effects of the supernatants were found to be suppressive or potentiating. Both the histamine induced suppressor and potentiating factor are generated by T-cells and can be precipitated by (NH4)2SO4. These precipitates were fractionated by gel filtration which revealed a molecular weight of both factors in the range of 20 to 50 kDa. Both lymphokines were found to be sensitive to trypsin, low pH, and heat treatment but were not affected by neuraminidase. They do only interfere with the early events of the IgM response within 24 hours after the administration of the antigen. The conclusion is that the bidirectional modulation of the humoral immune response by histamine in mice is partially due to the induction of these lymphokines by a different efficacy of the histamine receptor activation at different T-cell subsets.

Structural requirements for mast cell triggering by substance P-like peptides.

Experiments were based on the hypothesis that the histamine releasing action of substance P and some other neuropeptides is not due to the interaction of the neuropeptide with a specific membrane receptor. Rather, this may be a property of many amphiphilic compounds having a minimal number of positives charges and a hydrophobic chain which is not necessarily a peptide. According to this hypothesis, 18 substance P derivatives and fragments were synthesized and tested on rat and hamster peritoneal mast cells, among them five compounds which contain a non-peptide chain instead of the C-terminal substance P heptapeptide. The results are in accord with the above hypothesis.

Histamine release induced by Arg-Pro-Lys-Pro(CH2)11CH3 from rat peritoneal mast cells.

The substance Arg-Pro-Lys-Pro-(CH2)11CH3 [SP1-4C12] was synthesized by forming a peptide bond between Arg-Pro-Lys-Pro, the N-terminal sequence of substance P and dodecylamine. The aim was to examine the roles of the N- and C-terminal sequences of substance P in stimulating histamine release from mast cells of the rat peritoneal cavity. SP1-4 C12 induces concentration-dependent histamine release in the range 8 to 200 nM. SP1-4C12 was 50 times more potent than substance P and 300 times more potent than dodecylamine. Unlike dodecylamine itself, SP1-4C12 induced noncytolytic histamine release which was inhibited by benzalkonium chloride and by the substance P antagonist [D-Pro4,D-Trp7,9,10]SP4-11. Histamine release induced by SP1-4C12 was inhibited at temperatures below 16 degrees C and did not require the presence of extracellular calcium ions. It is suggested that substance P and some other basic histamine liberators initiate histamine secretion by a mechanism that involves the insertion of a hydrophobic region into the membrane lipid which is necessary to present positively charged moieties to a receptor site involved in activating the secretory mechanism.

Mast cell activation--a receptor-independent mode of substance P action?

Substance P is a representative of a group of amphiphilic neuropeptides which act as mast cell secretagogues. Our experiments with some new substance P derivatives suggest that these effects are dependent on two structural elements: (i) a hydrophobic chain which is not essentially a peptide, and (ii) a hydrophilic part with two positively charged amino acids. The mast cell triggering effect is unlikely to be mediated by a selective substance P receptor, but has strong similarities to the mode of action of polycations.

Electrophoretic characterization of muscarinic receptors under denaturating and nondenaturating conditions: computer-assisted Ferguson plot analysis.

The physical properties of the covalently labeled [( 3H]propylbenzilycholine mustard) muscarinic acetylcholine receptor from rat brain were studied by sodium dodecyl sulfate-polyacrylamide electrophoresis and computer-assisted Ferguson plot analysis. No proteolytic degradation or dimerization of the ligand binding subunit was found. No clues for different molecular weight forms or anomalous migration of the muscarinic receptor were detected. The weighted regression analysis of Ferguson plots gave an apparent molecular mass of 64-65 kDa. A new method for the electrophoretic separation of native (quinuclidinyl[3H]benzilate labeled) muscarinic receptor-detergent complexes was used for the comparison of wheat germ agglutinin binding, and not lectin binding receptors which were obtained by selective solubilization from porcine striatum. For this purpose, the computer-assisted Ferguson plot analysis is particularly suitable, since it renders possible the statistical assessment of both size and charge differences. Both receptor-detergent complexes were found to differ; statistically significant in their net charge but not in their size. The data support the view that muscarinic receptors from different sources may differ considerably in their glycosylation and that the receptor from porcine striatum can reversibly associate with a low-molecular-mass component which contains sialic acid.

Muscarinic receptor-detergent complexes with different biochemical properties: selective solubilization, lectin affinity chromatography and ligand binding studies.

Muscarinic receptors were solubilized by nonionic, zwitterionic and ionic detergents from porcine striatum. A mixture of digitonin and gitonin (3:2) was found to be most suitable in respect to receptor yield and stability. The solubilization of muscarinic receptors by this detergent appears to be dependent on the existence of free detergent micelles. Consequently, the receptor solubilization was studied at different protein-to-detergent ratios. Based on these experiments, a double extraction procedure was developed in which the receptor is solubilized subsequent to the solubilization of other membrane proteins. After elimination of the detergent excess, the binding of the receptor-detergent complex to six immobilized lectins was studied. In accordance with previous reports, we found a considerable portion of the digitonin/gitonin solubilized receptors (one step extraction procedure) specifically bound to wheat germ agglutinin via sialic acid residues. Muscarinic receptors solubilized by a double extraction procedure (either from porcine striatum or rat brain) did not bind to the lectin. This is not owing to selective extraction or partial denaturation, and indicates that considerable portions of the glycan residues are not covalently bound to the receptor polypeptide. A GTP-insensitive heterogenous agonist binding was found only at the non-wheat germ agglutinin binding receptors. The data analysis was performed by the affinity spectra method.

Monoclonal antibodies against rat mast cells differentiate between subtypes.

Four monoclonal mouse anti rat mast cell antibodies were selected which detect an antigenic determinant occurring on connective tissue mast cells of the rat. A strong antigen density was found on peritoneal mast cells whereas pleural and mesenteric mast cells exhibit considerably smaller amounts of the antigen. It does not occur on lung mast cells and basophils, thus permitting a mast cell subtype differentiation according to the expression of a surface antigen. The monoclonal antibodies do not react with IgE or IgE Fc-receptor determinants and do not interfere with the histamine secretion from peritoneal mast cells.