Ligand-receptor interactions: detailed evaluation of occupancy-dependent affinity

The exact nature of the curvilinearity of Scatchard plots derived from hormonal and nonhormonal binding systems has not been definitively resolved. Such plots are compatible with heterogeneous receptors with different but fixed affinities and with negatively interacting binding sites resulting in occupancy-dependent affinity. In the current study we examined in detail the effect of receptor occupancy by the ligand on receptor affinity under a variety of experimental conditions. We chose the human lymphocyte-leukoagglutinin (LPHA) system, which closely mimics the IM9-insulin model. Reliable estimates of total binding capacity (728 ng/10(6) cells) essential to our report were calculated from a wide database by the least-squares model. At occupancies greater than or equal to 0.085, receptors are associated with low and fixed affinity (1.5 X 10(6) M-1), whereas at occupancies less than or equal to 0.085, affinity is high and fixed (1.8 X 10(8) M-1) or high but variable (1 X 10(7) M-1 to 1.5 X 10(6) M-1) depending on whether the binding is assumed to be noncooperative or cooperative, respectively. Calculation of receptor-ligand complex dissociation velocity over a wide range of occupancies (0.01-0.40) suggested that occupancy exerts an inversely proportional effect on affinity that is rapid and sustained. Cell activation (DNA synthesis) is initiated at receptor occupancy of approximately equal to 0.004 and is magnified as ligand binding to high affinity receptors increases up to approximately equal to 0.07 occupancy (functional sites), beyond which point further binding (to low affinity sites) becomes increasingly ineffective and cytotoxic (redundant sites). These findings suggest that occupancy influences affinity as postulated by the hypothesis of negative cooperativity. Through this effect occupancy may play a significant role in regulating ligand-induced cell responses.     

Ligand-receptor interactions: a new method for determining the binding parameters

A new experimental procedure and new plot coordinates that allow determination of the binding parameters of ligand-acceptor interaction have been proposed. Instead of titration of a constant concentration of receptors with changing concentrations of ligand, as requested by the well-known methods of Klotz and Scatchard, a series of sequential dilutions of the reacting ligand-receptor mixture is suggested. This allows the application of a new coordinate system that transforms the binding isotherms into straight lines. The case of one acceptor with two classes of receptors with different binding constants is also considered briefly, where the correspondent graphs are nonlinear. It is suggested that in some cases this approach can be a simple and convenient substitute of the broadly used methods of Klotz and Scatchard.     

Ligand-receptor and junction-mediated cell-cell interactions: comparison of the two principles

Two major forms of cell-cell interaction--namely, ligand-receptor interactions and junction-mediated interactions--have different functional implications and naturally supplement each other in the most important tissue processes. The former type of interaction may be defined as distant and mainly "intertissue"; as a rule it determines temporal coordination of cell functioning. The latter is local and preferentially "intratissue"; it determines spatial coordination of cell activity.     

SRV2, a gene required for RAS activation of adenylate cyclase in yeast

We have identified a gene, SRV2, mutations of which alleviate stress sensitivity in strains carrying an activated RAS gene. Epistasis analysis suggests that the gene affects accumulation of cAMP in the cell. Direct assays of cAMP accumulation indicate that mutations of the gene diminish the rate of in vivo production of cAMP following stimulation by an activated RAS allele. Null mutations of srv2 result in lethality, which cannot be suppressed by mutational activation of the cAMP-dependent protein kinase. The sequence of the gene indicates that it encodes an adenylate cyclase-associated protein. These results demonstrate that SRV2 protein is required for RAS-activated adenylate cyclase activity, but that it participates in other essential cellular functions as well.     

Phosphoinositide-AP-2 interactions required for targeting to plasma membrane clathrin-coated pits

The clathrin-associated AP-2 adaptor protein is a major polyphosphoinositide-binding protein in mammalian cells. A high affinity binding site has previously been localized to the NH(2)-terminal region of the AP-2 alpha subunit (Gaidarov et al. 1996. J. Biol. Chem. 271:20922-20929). Here we used deletion and site- directed mutagenesis to determine that alpha residues 21-80 comprise a discrete folding and inositide-binding domain. Further, positively charged residues located within this region are involved in binding, with a lysine triad at positions 55-57 particularly critical. Mutant peptides and protein in which these residues were changed to glutamine retained wild-type structural and functional characteristics by several criteria including circular dichroism spectra, resistance to limited proteolysis, and clathrin binding activity. When expressed in intact cells, mutated alpha subunit showed defective localization to clathrin-coated pits; at high expression levels, the appearance of endogenous AP-2 in coated pits was also blocked consistent with a dominant-negative phenotype. These results, together with recent work indicating that phosphoinositides are also critical to ligand-dependent recruitment of arrestin-receptor complexes to coated pits (Gaidarov et al. 1999. EMBO (Eur. Mol. Biol. Organ.) J. 18:871-881), suggest that phosphoinositides play a critical and general role in adaptor incorporation into plasma membrane clathrin-coated pits.     

Caspase activity is required for commitment to Fas-mediated apoptosis.

Recognition of the widespread importance of apoptosis has been one of the most significant changes in the biomedical sciences in the past decade. The molecular processes controlling and executing cell death through apoptosis are, however, still poorly understood. The ICE (Interleukin-1beta Converting Enzyme) family-recently named the caspases for cysteine aspartate-specific proteases-plays a central role in apoptosis and may well constitute part of the conserved core mechanism of the process. Potentially, these proteases may be of great significance, both in the pathology associated with failure of apoptosis and also as targets for therapeutic intervention where apoptosis occurs inappropriately, e.g. in degenerative disease and AIDS. However, this is only likely if caspase activity is required before commitment to mammalian cell death. Here, we have used both peptide inhibitors and crmA transfection to inhibit these proteases in intact cells. Our experiments show that selective inhibition of some caspases protects human T cells (Jurkat and CEM-C7) from Fas-induced apoptosis, dramatically increasing their survival (up to 320-fold) in a colony-forming assay. This suggests that dysfunction of some, but not all, caspases could indeed play a crucial part in the development of some cancers and autoimmune disease, and also that these proteases could be appropriate molecular targets for preventing apoptosis in degenerative disease.     

Ligand-receptor and receptor-receptor interactions act in concert to activate signaling in the Drosophila toll pathway

In Drosophila, the signaling pathway mediated by the Toll receptor is critical for the establishment of embryonic dorso-ventral pattern and for innate immune responses to bacterial and fungal pathogens. Toll is activated by high affinity binding of the cytokine Sp?tzle, a dimeric ligand of the cystine knot family. In vertebrates, a related family of Toll-like receptors play a critical role in innate immune responses. Despite the importance of this family of receptors, little is known about the biochemical events that lead to receptor activation and signaling. Here, we show that Sp?tzle binds to the N-terminal region of Toll and, using biophysical methods, that the binding is complex. The two binding events that cause formation of the cross-linked complex are non-equivalent: the first Toll ectodomain binds Sp?tzle with an affinity 3-fold higher than the second molecule suggesting that pathway activation involves negative cooperativity. We further show that the Toll ectodomains are able to form low affinity dimers in solution and that juxtamembrane sequences of Toll are critical for the activation or derepression of the pathway. These results, taken together, suggest a mechanism of signal transduction that requires both ligand-receptor and receptor-receptor interactions.     

MEKK2 is required for T-cell receptor signals in JNK activation and interleukin-2 gene expression

The c-Jun N-terminal kinases (JNKs) are members of the mitogen-activated protein kinase (MAPK) gene family and are essential for cell proliferation, differentiation, and apoptosis. Previously we found that activation of JNK in T-cells required costimulation of both T-cell receptor and auxiliary receptors such as CD28. In this study, we cloned a full-length human MEK kinase (MEKK) 2 cDNA from Jurkat T-cells and demonstrated that it was a major upstream MAPK kinase kinase for the JNK cascade in T-cells. The human MEKK2 cDNA encoded a polypeptide of 619 amino acids and was the human counterpart of the reported murine MEKK2. It was 94% homologous with human and murine MEKK3 at the catalytic domains and 60% homologous at the N-terminal noncatalytic region. Northern blot analysis showed that MEKK2 was ubiquitously expressed, with the highest level in peripheral blood leukocytes. In T cells, MEKK2 was found to be a strong activator of JNK but not of extracellular signal-regulated kinase MAPKs and to activate JNK-dependent AP-1 reporter gene expression. MEKK2 also synergized with anti-CD3 antibody to activate JNK in T cells, and stimulation of T cells led to induction of MEKK2 tyrosine phosphorylation. Significantly, the JNK activation induced by anti-CD3 and anti-CD28 antibodies, but not by 12-O-tetradecanoylphorbol-13-acetate and Ca(2+) ionophore A23187, was inhibited by dominant negative MEKK2 mutants. AP-1 and interleukin-2 reporter gene induction in T-cells was also inhibited by dominant negative MEKK2 mutants. Taken together, our results showed that human MEKK2 is a key signaling molecule for T-cell receptor/CD3-mediated JNK MAPK activation and interleukin-2 gene expression.     

Membrane-associated interleukin 1 is required for the activation of T cells in the anti-CD3 antibody-induced T cell response

To establish the role of the membrane-associated form of IL 1 in T cell activation, we tested the accessory function of monocytes (Mo) that were unable to secrete or release soluble IL 1. The data obtained show that strictly purified resting T cells start to proliferate in response to anti-CD3 antibody (OKT3) stimulation in the presence of paraformaldehyde (PFA)-fixed Mo or 3-day cultured Mo (macrophages). The findings that i) PFA-fixed Mo did not produce or release IL 1, ii) the accessory function of PFA-fixed Mo could be inhibited with pretreatment with anti-IL 1 antibody, iii) PFA-fixed Mo had a comitogenic effect in the murine thymocyte assay, and iv) there was a temporal difference between the capacities to function in the comitogenic assay and to produce soluble IL 1, suggest that human Mo can express membrane-associated IL 1 and that it is functionally relevant.     

Intracellular polyamine biosynthesis is required for interleukin 2 responsiveness during lymphocyte mitogenesis

The objective of the present investigation was to define a more precise role for intracellular polyamine biosynthesis with respect to specific inducible events which regulate lymphocyte mitogenesis. In this regard, we have examined the effect of polyamine depletion on interleukin 2 (IL-2) production, receptor expression, and responsiveness in Con A stimulated mononuclear leukocytes (MNL). Polyamine depletion was achieved utilizing the specific irreversible inhibitor of ornithine decarboxylase (ODC), DL-alpha-difluoromethylornithine (DFMO). Polyamine depletion of MNL augmented detectable levels of Con A-induced IL-2 activity. In contrast, the ability of polyamine depleted MNL to respond to saturating levels of IL-2 (100 U/ml) following 72 or 96 hr of Con A stimulation was reduced 100 and 81%, respectively. Nonetheless, polyamine depletion did not impair the induction of IL-2 receptor expression. High-affinity IL-2 receptor density in the polyamine depleted population was greater than control cells late in culture (96 hr). The expression of high-affinity IL-2 receptors did not correlate with an ability to respond to IL-2 in the polyamine depleted population. The results of this study demonstrate for the first time that intracellular polyamine biosynthesis is required for IL-2 responsiveness during a primary mitogenic lymphocyte response.     

Ligand-receptor interactions in the membrane of cultured cells monitored by fluorescence correlation spectroscopy

We investigated the specific binding of epidermal growth factor (EGF) to its membrane-bound receptors in cultured cells. The specificity of the binding was attested by the consistent displacement of bound rhodamine-labeled EGF (Rh-EGF) following addition of 1000-fold molar excess of unlabeled EGF. The binding specificity of EGF was further confirmed when vascular EGF was unable to displace Rh-EGF binding, demonstrating no cross-reaction. Evidence for the specific interactions was verified by an equilibrium saturation binding experiment. EGF binding to the cell membranes is saturated at nanomolar concentration. The Scatchard plots show a binding process with K(ass) of 1.5 x 10(9) M(-1). The dissociation kinetics follow a single exponential function characteristic for a relatively slow dissociation process with k(diss) = 2.9 x 10(-4) s(-1). The appearance of two binding complexes through the distribution of diffusion times may suggest that these are representatives of two different forms or subtypes of EGF receptors. This study is of pharmaceutical significance as it provides evidence that fluorescence correlation spectroscopy can be used as a rapid technique for studying ligand-receptor interactions in cell cultures. This is a step forward toward large-scale drug screening in cell cultures.     

Monocyte interleukin 2 receptor gene expression and interleukin 2 augmentation of microbicidal activity

Activation of human peripheral blood monocytes results in the expression of interleukin 2 (IL 2) receptors, which are absent on resting monocytes. In a population of purified monocytes, the appearance of IL 2 receptors occurs on the majority of cells in association with increased levels of HLA-DR. Lipopolysaccharide (LPS) induces maximum numbers of IL 2 receptors within 12 hr, whereas IFN-gamma requires 48 hr. We used cDNA encoding for the human IL 2 receptor to evaluate IL 2 receptor gene expression in resting and activated monocytes. Within 4 hr after LPS stimulation, IL 2 receptor mRNA species of 3500 and 1500 bases appear, reaching peak levels between 8 and 12 hr and declining thereafter. The LPS-activated monocyte IL 2 receptor protein is expressed on the cell surface within a few hours after the detection of IL 2 receptor mRNA. The addition of IL 2 to IL 2 receptor-positive monocytes augments their generation of reactive oxygen intermediates and their cytotoxic activity. Thus monocytes when activated undergo a series of morphologic, phenotypic, and functional changes, including the expression of IL 2 receptors, which may provide an important immunoregulatory pathway.     

Per3, a circadian gene, is required for Chk2 activation in human cells

PER3 is a member of the PERIOD genes, but does not play essential roles in the circadian clock. Depletion of Per3 by siRNA almost completely abolished activation of checkpoint kinase 2 (Chk2) after inducing DNA damage in human cells. In addition, Per3 physically interacted with ATM and Chk2. Per3 overexpression induced Chk2 activation in the absence of exogenous DNA damage, and this activation depended on ATM. Per3 overexpression also led to the inhibition of cell proliferation and apoptotic cell death. These combined results suggest that Per3 is a checkpoint protein that plays important roles in checkpoint activation, cell proliferation and apoptosis.

Structured summary

MINT-8052850: Chk2 (uniprotkb:O96017) physically interacts (MI:0915) with Per3 (uniprotkb:P56645) by anti bait coimmunoprecipitation (MI:0006)MINT-8052875: Per3 (uniprotkb:P56645) physically interacts (MI:0914) with Chk2 (uniprotkb:O96017) and ATM (uniprotkb:Q13315) by anti tag coimmunoprecipitation (MI:0007)     

Induction and regulation of human interleukin 2 gene expression: significance of protein kinase C activation

Phytohemagglutinin (PHA) and a tumor-promoting phorbol ester, 12-O-tetradecanoylphorbol-13-acetate (TPA) act synergistically to induce interleukin 2 (IL2) mRNA in human lymphocytes in vitro. The induction was inhibited by a potent inhibitor of protein kinase C (C-kinase), 1-(5-isoquinolinylsulfonyl)-2-methylpiperazine (H-7) at less than 10 microM. H-7 inhibited C-kinase activity itself in lymphocytes at the same range of the concentration but did not interfere with the translocation of C-kinase from the cytosol to the membrane fraction of the lymphocytes induced by TPA. H-7 is also known to inhibit cAMP-dependent protein kinase (A-kinase) and cGMP-dependent protein kinase (G-kinase). However, the lymphocytes cultured with dibutyryl cAMP or dibutyryl cGMP could not be activated to produce IL2 mRNA. These results show that activation of C-kinase but not A-kinase and G-kinase is necessary in signal transduction for IL2 gene expression. Prostaglandin E2, which is known to elevate intracellular cAMP level, also inhibited IL2 mRNA induction in the lymphocytes stimulated with PHA and TPA. Addition of alpha-methylornithine and methylglyoxal bis (guanyl hydrazone), which inhibit polyamine synthesis, did not affect the induction of IL2 mRNA in the lymphocytes stimulated with PHA and TPA, indicating that polyamine synthesis is not necessary for IL2 mRNA induction.     

Interleukin 1 regulation of interleukin 2 and interferon-gamma gene activation in a human leukemic HSB.2 subclone

The role of interleukin 1 (IL1) in causing IL2 and interferon-gamma (IFN-gamma) production and their associated gene activation has been studied in a human leukemic HSB.2 subclone. One of the subclones, HSB.2-C5B2 clone #28, which produced only low levels of IL2 and IFN-gamma when stimulated with phytohemagglutinin (PHA) or with a combination of phorbol-myristate-acetate (PMA) and Ca2+ ionophore, ionomycin, showed marked IL2 and IFN-gamma production in the presence of IL1. The augmentation by IL1 was observed in both dot and Northern blot analysis, indicating that IL1 regulates lymphokine genes at the pretranslational level. The kinetics of IL2 and IFN-gamma production were essentially similar for both lymphokines except that there was a faster accumulation of IFN-gamma mRNA than IL2 mRNA. In contrast to the IL2 and IFN-gamma gene activation, IL2 receptor (Tac/p55 antigen) expression was induced directly by IL1 itself as with PMA in this subclone. In these studies, IL1 action was not replaced by the drugs raising intracellular cyclic AMP (cAMP). Taken collectively, these experiments support the notion that IL1 does not trigger IL2 gene activation per se, but amplifies the preactivated lymphokine genes initiated by PMA and ionomycin, whereas IL1 can activate the IL2 receptor gene without any other known signal requirements.     

TIMP-2 is required for efficient activation of proMMP-2 in vivo

Matrix metalloproteinases (MMPs) are synthesized as latent proenzymes. A proteolytic cleavage event involving processing of the cysteine-rich N-terminal propeptide is required for their full activation. Previous in vitro studies indicated that activation of proMMP-2 can occur through formation of a trimolecular complex between MMP-14, TIMP-2, and proMMP-2 at the cell surface. Using TIMP-2-deficient mice and cells derived from them, TIMP-2 was shown to be required for efficient proMMP-2 activation both in vivo and in vitro. The requirement for TIMP-2 was not cell-autonomous as exogenously added TIMP-2 could restore activation of proMMP-2 to TIMP-2-deficient cells. Mutant mice were overtly normal, viable, and fertile on the C57BL/6 background, indicating that both TIMP-2 and activated proMMP-2 are dispensable for normal development.