A model for the interaction of the GM-CSF, IL-3 and IL-5 receptors with their ligands

The high affinity receptors for GM-CSF, IL-3 and IL-5 are heterodimers consisting of a ligand-specific alpha chain and a common beta chain. These proteins are members of a family of proteins known as the "cytokine receptor family" which is characterized by the presence of a 200-residue ligand-binding module. The GM-CSF, IL-3 and IL-5 receptor alpha chains constitute a distinct subgroup and share features not found in other members of the cytokine receptor family, features which we propose to be important for their interaction with the common beta chain and for their binding of the structurally-related ligands. The growth hormone receptor is a well-characterized member of the cytokine receptor family. Based on the structure of the complex between growth hormone and its receptor, we have proposed sites of contact between the GM-CSF, IL-3 and IL-5 receptors and their cognate ligands.     

Molecular models of two competitive inhibitors, IL-2delta2 and IL-2delta3, generated by alternative splicing of human interleukin-2

Molecular models of IL-2delta2 and IL-2delta3, two alternative splice variants of human IL-2 without exon 2 and 3, respectively, are described. These alternative splice variants attract particular interest as potential competitive inhibitors of the cytokine. Tertiary structure of IL-2 consists of four-helix bundle including helices A, B, C and D and a beta-pleated sheet. Exon 2 encodes the A-B loop (Asn30-Lys49 residues) linking helices A and B running in one direction. Rotation of the helix A around putative centre during the construction of IL-2delta2 model have not produced any significant changes in the hydrophobic core of IL-2 molecule. However, a large hole was formed on the surface of IL-2delta2 molecule instead of A-B loop in IL-2 fold. A high affinity IL-2 receptor is formed by combination of alpha, beta, and gamma(c) chains. Comparison of the model of the receptor bound IL-2 with the model of IL-2delta2 has shown that their beta-chain binding sites have minimum differences as distinct from alpha and gamma(c) chain-binding sites. Exon 3 encodes Ala50-Lys97 fragment which forms helices B and C with their short connecting loop. Model IL-2delta3 consists of helices A and D and long linking loop. This loop was composed of A-B and C-D loops which run in opposite directions in IL-2 structure and contain beta-strands making a beta-pleated sheet. Conformation of the linking loop relatively to helices A and D was stabilized by creation of a disulphide bond between cysteines 105 and 125. In addition, the hydrophobic residues of beta-sheet interact with the hydrophobic surface of A-D helical complex and close the latter from contacts with solution. Comparison of the model of IL-2 bound to receptor with IL-2delta3 model has shown that absence of helices B and C in IL-2delta3 model results in insignificant conformational changes only in residues interacting with gamma(c) chain of the receptor. The beta/gamma(c) heterodimer is an intermediate affinity receptor of IL-2. Most likely, both IL-2delta2 and IL-2delta3 are naturally occurring IL-2 antagonists since they keep the ability of binding with an intermediate affinity receptor of this cytokine and fail to engage the alpha chain of its high affinity receptor.     

Key amino acids in the gamma subunit of the gamma-aminobutyric acidA receptor that determine ligand binding and modulation at the benzodiazepine site

Pharmacological analyses of gamma-aminobutyric acidA (GABAA) receptor subtypes have suggested that both the alpha and gamma subunits, but not the beta subunit, contribute to the benzodiazepine binding site. We took advantage of the different pharmacological properties conferred by the inclusion of different gamma subunits in the receptor macromolecule to identify amino acids gamma2Phe77 and gamma2Met130 as key determinants of the benzodiazepine binding site. gamma2Phe77 was required for high affinity binding of the benzodiazepine site ligands flumazenil, CL218,872, and methyl-beta-carboline-3-carboxylate but not flunitrazepam. This amino acid was, however, required for allosteric modulation by flunitrazepam, as well as other benzodiazepine site ligands. In contrast, gamma2Met130 was required for high affinity binding of flunitrazepam, clonazepam, and triazolam but not flumazenil, CL218, 872, or methyl-beta-carboline-3-carboxylate and did not affect benzodiazepine efficacy. Introduction of the phenylalanine and methionine into the appropriate positions of gamma1 was not sufficient to confer high affinity for the benzodiazepine site ligand zolpidem. These data show that gamma2Phe77 and gamma2Met130 are necessary for high affinity binding of a number of benzodiazepine site ligands. Although most previous studies have focused on the contribution of the alpha subunit, we demonstrated a critical role for the gamma subunit at the benzodiazepine binding site, indicating that this modulatory site is located at the interface of these two subunits. Furthermore, gamma2Phe77 is homologous to alpha1Phe64, which has been previously shown to be a key determinant of the GABA binding site, suggesting a conservation of motifs between different ligand binding sites on the GABAA receptor.     

gamma-Aminobutyric acid type A receptors in the rat brain can contain both gamma 2 and gamma 3 subunits, but gamma 1 does not exist in combination with another gamma subunit

Antibodies specific for the gamma 1, gamma 2, and gamma 3 subunits of the gamma-aminobutyric acid (GABA)A receptor have been used to probe the composition of naturally occurring GABAA receptors in the rat brain. Most GABAA receptors contain at least one of these three subunits. The percentage of each, determined by immunoprecipitation of [3H]muscimol binding, was 11 +/- 1%, 59 +/- 3%, and 14 +/- 2% for gamma 1, gamma 2, and gamma 3 subunits, respectively. Receptors containing gamma 2 or gamma 3 subunits were labeled by benzodiazepine site ligands with high affinity, whereas gamma 1-containing receptors could be labeled only by [3H]muscimol. Receptors immunoprecipitated by anti-gamma 2 or anti-gamma 3 antibodies were labeled with [3H]Ro 15-1788 with similar affinities (Kd for anti-gamma 2-immunoprecipitated receptors, 1.9 nM; Kd for anti-gamma 3-immunoprecipitated receptors, 1.7 nM). Immunoprecipitation or Western blot analysis of GABAA receptors solubilized from rat cerebellar or whole-brain preparations indicated that gamma 1 was not present coassembled with any other gamma subunit. Western blot analysis of receptors purified on alpha-specific immunoaffinity resins showed that gamma 1 was predominantly assembled with the alpha 2 subunit. Some GABAA receptors may contain more than one type of gamma subunit. Quantitative immunoprecipitation and Western blot analysis both indicated that gamma 2 and gamma 3 subunits can exist in the same receptor complex. A large proportion of GABAA receptors immunopurified on a gamma 3 affinity resin also appeared to contain a gamma 2 subunit. In contrast, when receptors were purified on a gamma 2 affinity resin a small proportion also appeared to contain a gamma 3 subunit. We conclude that most gamma 1-containing receptors have no other gamma subunit in the same receptor complex but some GABAA receptors contain both gamma 2 and gamma 3 subunits.     

Analysis of human IL-2/IL-2 receptor beta chain interactions: monoclonal antibody H2-8 and new IL-2 mutants define the critical role of alpha helix-A of IL-2

Interleukin 2 (IL-2) interacts with a receptor (IL-2R) composed of three subunits (IL-2R alpha, IL-2R beta and IL-2R gamma). IL-2R beta plays a critical role in signal transduction. An anti-human IL-2 mAb (H2-8) produced after immunization with peptide 1-30 of IL-2 was found to recognize the region occupied by Asp20, at the exposed interface between alpha-helices A and C. Muteins at position 17 and 20 are not recognized by mAb H2-8. mAb H2-8 specifically inhibits the IL-2 proliferation of TS1beta cells which are dependent on the expression of human IL-2R beta chain for IL-2 proliferation. Substitution at internal position Leu17 demonstrates that this position is essential for IL-2 binding and IL-2 bioactivity. New IL-2 mutants at position Asp20 have been analysed. Substitutions Asp --> Asn, Asp --> Lys, Asp --> Leu, show a correlation between diminished affinity for IL-2 receptor and reduced bioactivity measured on TS1beta cells. Mutein Asp Arg lose affinity for IL-2R and bioactivity simultaneously. Furthermore, during the course of the study we have found that mutein Asp20 --> Leu is an IL-2 antagonist. The biological effects of mAb H2-8 and the properties of new mutants at positions 17 and 20 demonstrate that this region of alpha helix-A is involved in IL-2-IL-2R beta interactions.     

The murine anti-human common gamma chain monoclonal antibody CP.B8 blocks the second step in the formation of the intermediate affinity IL-2 receptor

A murine monoclonal antibody, CP.B8, specific for the extracellular portion of the human common gamma (gammac) chain, and its Fab fragment are shown to block the binding of IL-2 to COS-7 cells transfected with the cDNA for the full-length IL-2 receptor beta (IL-2Rbeta) and gammac chains, components which together comprise the intermediate affinity IL-2 receptor (IL-2R) expressed on the surface of resting T cells, NK cells, and on certain intestinal epithelial cells. To investigate the mechanism of this inhibition, the extracellular portions of the IL-2Rbeta and gammac chains were expressed and purified, and their interactions with each other and with IL-2 were studied by gel filtration and by surface plasmon resonance (SPR). By gel filtration, a stable ternary complex was formed by association of the three proteins, while no stable binary complexes were detected between any two of the three proteins. By SPR analysis, IL-2 was shown to associate rapidly with IL-2Rbeta, forming a binary complex with an equilibrium dissociation constant (Kd) of 800 nM, which permitted subsequent association of the gammac chain. Dissociation of the IL-2/IL-2Rbeta/gammac chain complex was significantly slower than dissociation of the IL-2/IL-2Rbeta complex. Using these model systems, we tested the ability of mAb CP.B8 to inhibit the association of the gammac chain with IL-2 and IL-2Rbeta. By gel filtration, mAb CP.B8 formed a stable complex with the gammac chain, preventing its association with IL-2 and IL-2Rbeta. MAb CP.B8 was also capable of dissociating the gammac chain already complexed with IL-2 and IL-2Rbeta. SPR analysis confirmed these findings and showed, in addition, that the Fab fragment of CP.B8 was also capable of inhibiting the association of the gammac chain with the IL-2/IL-2Rbeta complex. We conclude that mAb CP.B8 blocks the second step in the formation of the intermediate affinity IL-2R on the surface of transfected COS-7 cells by binding at or close to a region on the gammac chain that is involved in contact with IL-2 and/or IL-2Rbeta.     

Reconstitution of a functional interleukin (IL)-7 receptor demonstrates that the IL-2 receptor gamma chain is required for IL-7 signal transduction

The interleukin (IL)-2 receptor gamma chain has recently been shown to be a component of the IL-7 and IL-4 receptors. Using a transient transfection assay and the trans-activation of reporter gene constructs which are under the control of cytokine-responsive promoter elements, we have studied signal transduction through the IL-7 receptor (IL-7R). The reporter gene expression was not stimulated by receptors that contained the cytoplasmic domain of the IL-7R, either as intact IL-7R or as part of a chimeric receptor. However, co-expression of the IL-7R with the IL-2 receptor gamma chain was able to stimulate gene activation. For maximal stimulation the intact cytoplasmic domains of each chain was required.     

Fc gamma RI blockade and modulation for immunotherapy

Splenectomy and corticosteroids are the treatment of choice for patients with immune thrombocytopenic purpura (ITP). However, for the 10%-15% of patients who do not respond to conventional therapy, high-dose i.v. IgG can induce life-saving transient responses. The benefits of i.v. IgG have been attributed to Fc receptor blockade; however, the involvement of the individual Fc receptors for IgG (Fc gamma R) in ITP remain to be more completely defined. Recently a mAb, designated mAb H22, which recognizes an epitope on Fc gamma RI (CD64) outside the ligand-binding domain, was humanized. Because mAb H22 is a human IgG1 and Fc gamma RI has a high affinity for human IgG1 antibodies, we predicted that mAb H22 would bind to the Fc gamma RI ligand-binding site through its Fc domain and to its external Fc gamma RI epitope through both Fab domains. These studies demonstrate that mAb H22 blocked Fc gamma RI-mediated phagocytosis of opsonized red blood cells more effectively than an irrelevant IgG. Moreover, cross-linking Fc gamma RI with mAb H22 down-modulated Fc gamma RI expression on monocytes, an effect seen within 2 h.     

Interaction affinity between cytokine receptor components on the cell surface

The anti-common gamma chain (gammac) mAb CP.B8 is shown to inhibit interleukin 4 (IL-4)-dependent proliferation of phytohemagglutinin (PHA) activated T cells noncompetitively with respect to cytokine by blocking the IL-4-induced heterodimerization of IL-4Ralpha and gammac receptor chains. Affinities for the binding of IL-4 to Cos-7 cells transfected with huIL-4Ralpha, and to PHA blasts expressing both IL-4Ralpha and gammac, were used to estimate the affinity of the key interaction between gammac and the binary IL-4Ralpha.IL-4 complex on the cell surface. This affinity was defined in terms of the dimensionless ratio [IL-4Ralpha.IL-4.gammac]/[IL-4Ralpha.IL-4], which we designate KR. The results show that on PHA blasts this interaction is relatively weak; KR approximately 9, implying that approximately 10% of the limiting IL-4Ralpha chain remains free of gammac even at saturating concentrations of IL-4. This quantitative treatment establishes KR as a key measure of the coupling between ligand binding and receptor activation, providing a basis for functional distinctions between different receptors that are activated by ligand-induced receptor dimerization.     

Human granulocyte-macrophage colony-stimulating factor receptor signal transduction requires the proximal cytoplasmic domains of the alpha and beta subunits

Human granulocyte-macrophage colony-stimulating factor (GM-CSF) controls the production, maturation, and function of cells in multiple hematopoietic lineages. These effects are mediated by a cell-surface receptor (GM-R) composed of alpha and beta subunits, each containing 378 and 881 amino acids, respectively. Whereas the alpha subunit exists as several isoforms that bind GM-CSF with low affinity, the beta common subunit (beta c) does not bind GM-CSF itself, but acts as a high-affinity converter for GM-CSF, interleukin-3 (IL-3), and IL-5 receptor alpha subunits. The cytoplasmic region of GM-R alpha consists of a membrane-proximal conserved region shared by the alpha 1 and alpha 2 isoforms and a C-terminal variable region that is divergent between alpha 1 and alpha 2. The cytoplasmic region of beta c contains membrane proximal serine and acidic domains. To investigate the amino acid sequences that influence signal transduction by this receptor complex, we constructed a series of cytoplasmic truncation mutants of the alpha 2 and beta subunits. To study these truncations, we stably transfected the IL-3-dependent murine cell line Ba/F3 with wild-type or mutant cDNAs. We found that the wild-type and mutant alpha subunits conferred similar low-affinity binding sites for human GM-CSF to Ba/F3, and the wild-type or mutant beta subunit converted some of these sites to high-affinity; the cytoplasmic domain of beta was unnecessary for this high-affinity conversion. Proliferation assays showed that the membrane-proximal conserved region of GM-R alpha and the serine-acidic domain of beta c are required for both cell proliferation and ligand-dependent phosphorylation of a 93-kD cytoplasmic protein. We suggest that these regions may represent an important signal transduction motif present in several cytokine receptors.     

Multiple regions of human Fc gamma RII (CD32) contribute to the binding of IgG

The low affinity receptor for IgG, Fc gamma RII (CD32), has a wide distribution on hematopoietic cells where it is responsible for a diverse range of cellular responses crucial for immune regulation and resistance to infection. Fc gamma RII is a member of the immunoglobulin superfamily, containing an extracellular region of two Ig-like domains. The IgG binding site of human Fc gamma RII has been localized to an 8-amino acid segment of the second extracellular domain, Asn154-Ser161. In this study, evidence is presented to suggest that domain 1 and two additional regions of domain 2 also contribute to the binding of IgG by Fc gamma RII. Chimeric receptors generated by exchanging the extracellular domains and segments of domain 2 between Fc gamma RII and the structurally related Fc epsilon RI alpha chain were used to demonstrate that substitution of domain 1 in its entirety or the domain 2 regions encompassing residues Ser109-Val116 and Ser130-Thr135 resulted in a loss of the ability of these receptors to bind hIgG1 in dimeric form. Site-directed mutagenesis performed on individual residues within and flanking the Ser109-Val116 and Ser130-Thr135 domain 2 segments indicated that substitution of Lys113, Pro114, Leu115, Val116, Phe129, and His131 profoundly decreased the binding of hIgG1, whereas substitution of Asp133 and Pro134 increased binding. These findings suggest that not only is domain 1 contributing to the affinity of IgG binding by Fc gamma RII but, importantly, that the domain 2 regions Ser109-Val116 and Phe129-Thr135 also play key roles in the binding of hIgG1. The location of these binding regions on a molecular model of the entire extracellular region of Fc gamma RII indicates that they comprise loops that are juxtaposed in domain 2 at the interface with domain 1, with the putative crucial binding residues forming a hydrophobic pocket surrounded by a wall of predominantly aromatic and basic residues.     

Out-of-hospital quantitative monitoring of end-tidal carbon dioxide pressure during CPR

BACKGROUND: Human growth hormone (hGH) binds to both the hGH and human prolactin (hPRL) receptors. Binding to the hPRL receptor, however, is approximately 50-fold tighter and requires a single Zn2+ cation, unlike binding of hGH to the hGH receptor. Previous mutational studies have identified putative ligands from hGH and the hPRL receptor responsible for coordinating the interfacial Zn2+. RESULTS: One of these ligands was introduced at a structurally analogous site in the extracellular domain of the hGH receptor by mutating Asn218 to His, and the resulting mutant protein showed a 20-fold increase in hGH binding in the presence of ZnCl2. Alanine-scanning mutagenesis showed that the binding site on hGH for the Asn218-->His hGH receptor in the presence of Zn2+ resembled that for the hPRL receptor. CONCLUSIONS: It is possible to introduce the metal-binding site from the hPRL receptor into the homologous hGH receptor. More generally, these studies indicate that affinity between two proteins may be enhanced by design of an interfacial metal-binding site.     

Interleukin-15 induces adhesion receptor redistribution in T lymphocytes

Chemotactic factors such as cytokines and chemokines direct the migration of leukocytes into inflammatory sites. Chemokines play a role regulating both the expression and adhesive properties of leukocyte integrins. We have recently described an additional function of chemokines in the induction of cell polarization and adhesion receptor redistribution during the initial step of leukocyte locomotion. We herein report that interleukin (IL)-15, a newly described cytokine with chemotactic properties, is able to induce uropod formation on T lymphoblasts to which intercellular adhesion molecule (ICAM)-3, a leukocyte-restricted counter-receptor for the lymphocyte function-associated antigen (LFA)-1 integrin, is redistributed. Other adhesion molecules, such as ICAM-1, ICAM-2, CD43 and CD44, also redistributed to the uropod, although in a lower proportion of the cells. The induction of uropod formation by IL-15 was observed on T lymphoblasts adhering to the integrin ligands fibronectin, vascular cell adhesion molecule (VCAM)-1 and ICAM-1, but not to bovine serum albumin or poly-L-lysine. The effect of IL-15 was dose dependent and specifically inhibited by a monoclonal antibody (mAb) against this cytokine. Blocking experiments with anti-IL-2 receptor beta chain mAb showed an inhibitory effect on IL-15-mediated redistribution of ICAM-3, whereas no effect was observed in the presence of anti-IL-2 receptor alpha chain mAb. The uropod induced by IL-15 is enriched in many different adhesion receptors and, being well exposed to the external milieu, is likely to modulate the adhesive properties of lymphocytes.     

The common cytokine receptor gamma chain controls survival of gamma/delta T cells

We have investigated the role of common gamma chain (gamma c)-signaling pathways for the development of T cell receptor for antigen (TCR)-gamma/delta T cells. TCR-gamma/delta-bearing cells were absent from the adult thymus, spleen, and skin of gamma c-deficient (gamma c-) mice, whereas small numbers of thymocytes expressing low levels of TCR-gamma/delta were detected during fetal life. Recent reports have suggested that signaling via interleukin (IL)-7 plays a major role in facilitating TCR-gamma/delta development through induction of V-J (variable-joining) rearrangements at the TCR-gamma locus. In contrast, we detected clearly TCR-gamma rearrangements in fetal thymi from gamma c- mice (which fail to signal in response to IL-7) and reduced TCR-gamma rearrangements in adult gamma c thymi. No gross defects in TCR-delta or TCR-beta rearrangements were observed in gamma c- mice of any age. Introduction of productively rearranged TCR V gamma 1 or TCR V gamma 1/V delta 6 transgenes onto mice bearing the gamma c mutation did not restore TCR-gamma/delta development to normal levels suggesting that gamma c-dependent pathways provide additional signals to developing gamma/delta T cells other than for the recombination process. Bcl-2 levels in transgenic thymocytes from gamma c- mice were dramatically reduced compared to gamma c+ transgenic littermates. We favor the concept that gamma c-dependent receptors are required for the maintenance of TCR-gamma/delta cells and contribute to the completion of TCR-gamma rearrangements primarily by promoting survival of cells committed to the TCR-gamma/delta lineage.     

Uptake of aluminum and gallium into tissues of the rat: influence of antibody against the transferrin receptor

A phosphatidylcholine-like phospholipid expressed in the outer leaflet of the cell membrane shortly after mitogenic activation of T-cells is described, based on the binding of monoclonal antibody 90. 60.3. Expression of the 90.60.3 phospholipid antigen in T-cells is activation-dependent. Once expressed, the 90.60.3 phospholipid is in direct physical association with the interleukin-2 (IL-2) binding domain of IL-2 receptor alpha subunits, but does not affect IL-2 binding. The association is specific, because the 90.60.3 phospholipid is not found in association with other domains of IL-2 receptor alpha subunits, or near IL-2 receptor beta or gamma subunits. Culturing cytokine-dependent cell lines in the presence of monoclonal antibody 90.60.3 potentiates IL-2-dependent cell survival and proliferation in a dose-dependent manner. In contrast, IL-4-dependent responses are not potentiated. Taken together, the data suggest that specific plasma membrane phospholipids expressed in the outer leaflet after T-cell activation associate with the IL-2 binding domain of IL-2 receptor alpha subunits (and perhaps other cytokine receptors), and may play a role in regulating receptor mobility or signal transduction.     

BAG-1 and Bcl-2 in IL-2 signaling

Some cytokines can prolong cell survival in hematolymphoid cells and thus may be crucial for regulation of hematolymphoid cell numbers. It has been shown that mitogenic cytokines can induce not only cellular proliferation but also cellular survival by inhibiting apoptosis in hematolymphoid cells. The signals transduced by these cytokines eventually go to the nucleus and induce expression of their specific target genes. In this context, the induction of anti-apoptotic molecules such as Bcl-2 oncoprotein and BAG-1 protein seems to be a key event for the anti-apoptotic function of cytokines. In T lymphocytes, the interaction of interleukin-2 (IL-2) with its receptor (IL-2R) induces both cellular proliferation and cellular survival. The IL-2R consists of three subunits, i.e., IL-2Ralpha, IL-2R(beta)c, and IL-2R(gamma)c chains. Structure-function analysis of the IL-2R(beta)c chain has revealed that there are at least two functional domains within the subunit. The serine-rich (S) region but not the acidic (A) region within the (beta)c chain is responsible for the mitogenic signaling of IL-2R. The S region is also crucial for the cellular survival signaling, which include the induction of anti-apoptotic gene expressions bcl-2 and bag-l. However, the cellular survival signaling is segregated from the mitogenic signaling in independence from the Jak-family protein kinase activation and rapamycin sensitivity. Segregation of the two signaling pathways of a cytokine receptor has also been shown in receptors of the other mitogenic cytokines. Current topics regarding signal transductions of cytokine receptors responsible for the suppression of apoptosis are discussed in this review.     

A potential role for interleukin-15 in the regulation of human natural killer cell survival

Resting lymphocyte survival is dependent upon the expression of Bcl-2, yet the factors responsible for maintaining lymphocyte Bcl-2 protein expression in vivo are largely unknown. Natural killer (NK) cells are bone marrow-derived lymphocytes that constitutively express the beta and common gamma(c) subunits of the IL-2 receptor (R) as a heterodimer with intermediate affinity for IL-2. IL-15 also binds to IL-2Rbeta gamma(c) and is much more abundant in normal tissues than IL-2. Mice that lack the IL-2 gene have NK cells, whereas mice and humans that lack IL-2R gamma(c) do not have NK cells. Further, treatment of mice with an antibody directed against IL-2Rbeta results in a loss of the NK cell compartment. These data suggest that a cytokine other than IL-2, which binds to IL-2Rbeta gamma(c), is important for NK cell development and survival in vivo. In the current report, we show that the recently described IL-15R(alpha) subunit cooperates with IL-2Rbeta gamma(c) to transduce an intracellular signal at picomolar concentrations of IL-15. We demonstrate that resting human NK cells express IL-15R(alpha) mRNA and further, that picomolar amounts of IL-15 can sustain NK cell survival for up to 8 d in the absence of serum. NK cell survival was not sustained by other monocyte-derived factors (i.e., TNF-alpha, IL-1beta, IL-10, IL-12) nor by cytokines known to use gamma(c) for signaling (i.e., IL-4, IL-7, IL-9, IL- 13). One mechanism by which IL-15 promotes NK cell survival may involve the maintenance of Bcl-2 protein expression. Considering these functional properties of IL-15 and the fact that it is produced by bone marrow stromal cells and activated monocytes, we propose that IL-15 may function as an NK cell survival factor in vivo.     

A novel ligand-binding site in the zeta-form 14-3-3 protein recognizing the platelet glycoprotein Ibalpha and distinct from the c-Raf-binding site

We reported previously that the zeta-form 14-3-3 protein (14-3-3zeta) binds to a platelet adhesion receptor, glycoprotein (GP) Ib-IX, and this binding is dependent on the SGHSL sequence at the C terminus of GPIbalpha. In this study, we have identified a binding site in the helix I region of 14-3-3zeta (residues 202-231) required for binding to GPIb-IX complex and to the cytoplasmic domain of GPIbalpha. We also show that phosphorylation-dependent binding of c-Raf to 14-3-3zeta requires helix G (residues 163-187) but not helix I. Thus, the GPIbalpha-binding site is distinct from the binding sites for RSXpSXP motif-dependent ligands. Furthermore, we show that wild type 14-3-3zeta has a higher affinity for GPIb-IX complex than recombinant GPIbalpha cytoplasmic domain. Deletion of helices A and B (residues 1-32) disrupts 14-3-3zeta dimerization and decreases its affinity for GPIb-IX. Disruption of 14-3-3zeta dimerization, however, does not reduce 14-3-3zeta binding to recombinant GPIbalpha cytoplasmic domain. This suggests a dual site recognition mechanism in which a 14-3-3zeta dimer interacts with both GPIbalpha and GPIbbeta (known to contain a phosphorylation-dependent binding site), resulting in high affinity binding.     

Determinants of the phagocytic signal mediated by the type IIIA Fc gamma receptor, Fc gamma RIIIA: sequence requirements and interaction with protein-tyrosine kinases

The Fc gamma receptor-associated gamma and zeta subunits contain a conserved cytoplasmic motif, termed the immunoglobulin gene tyrosine activation motif, which contains a pair of YXXL sequences. The tyrosine residues within these YXXL sequences have been shown to be required for transduction of a phagocytic signal. We have previously reported that the gamma subunit of the type IIIA Fc gamma receptor (Fc gamma RIIIA) is approximately 6 times more efficient in mediating phagocytosis than the zeta subunit of Fc gamma RIIIA. By exchanging regions of the cytoplasmic domains of the homologous gamma and zeta chains, we observed that the cytoplasmic area of the gamma chain bearing a pair of the conserved YXXL sequences is important in phagocytic signaling. Further specificity of phagocytic signaling is largely determined by the two internal XX amino acids in the YXXL sequences. In contrast, the flanking amino acids of the YXXL sequences including the seven intervening amino acids between the two YXXL sequences do not significantly affect the phagocytic signal. Furthermore, the protein-tyrosine kinase Syk, but not the related kinase ZAP-70, stimulated Fc gamma RIIIA-mediated phagocytosis. ZAP-70, however, increased phagocytosis when coexpressed with the Src family kinase Fyn. These data demonstrate the importance of the two specific amino acids within the gamma subunit YXXL cytoplasmic sequences in phagocytic signaling and explain the difference in phagocytic efficiency of the gamma and zeta chains. These results indicate the importance of Syk in Fc gamma RIIIA-mediated phagocytosis and demonstrate that ZAP-70 and syk differ in their requirement for a Src-related kinase in signal transduction.