Functional regions of the mouse interleukin-10 receptor cytoplasmic domain.

The functions of wild-type and mutant mouse interleukin-10 receptors (mIL-10R) expressed in murine Ba/F3 cells were studied. As observed previously, IL-10 stimulates proliferation of IL-10R-expressing Ba/F3 cells. Accumulation of viable cells in the proliferation assay is to a significant extent balanced by concomitant cell death. Moreover, growth in IL-10 also induces a previously unrecognized response, differentiation of the cells, as evidenced both by formation of large clusters of cells in cultures with IL-10 and by induction or enhancement of expression of several cell surface antigens, including CD32/16, CD2, LECAM-1 (v-selectin), and heat-stable antigen. Two distinct functional regions near the C terminus of the mIL-10R cytoplasmic domain which mediate proliferation were identified; one of these regions also mediates the differentiation response. A third region proximal to the transmembrane domain was identified; removal of this region renders the cell 10- to 100-fold more sensitive to IL-10 in the proliferation assay. In cells expressing both wild-type and mutant IL-10R, stimulation with IL-10 leads to tyrosine phosphorylation of the kinases JAK1 and TYK2 but not JAK2 or JAK3 under the conditions tested.     

Characterization of the interaction between interleukin-13 and interleukin-13 receptors

Interleukin-13 (IL-13) possesses two types of receptor: the heterodimer, composed of the IL-13Ralpha1 chain (IL-13Ralpha1) and the IL-4Ralpha chain (IL-4Ralpha), transducing the IL-13 signals; and the IL-13Ralpha2 chain (IL-13Ralpha2), acting as a nonsignaling "decoy" receptor. Extracellular portions of both IL-13Ralpha1 and IL-13Ralpha2 are composed of three fibronectin type III domains, D1, D2, and D3, of which the last two comprise the cytokine receptor homology modules (CRHs), a common structure of the class I cytokine receptor superfamily. Thus far, there has been no information about the critical amino acids of the CRHs or the role of the D1 domains of IL-13Ralpha1 and IL-13Ralpha2 in binding to IL-13. In this study, we first built the homology modeling of the IL-13.hIL-13 receptor complexes and then predicted the amino acids involved in binding to IL-13. By incorporating mutations into these amino acids, we identified Tyr-207, Asp-271, Tyr-315, and Asp-318 in the CRH of human IL-13Ralpha2, and Leu-319 and Tyr-321 in the CRH of human IL-13Ralpha1, as critical residues for binding to IL-13. Tyr-315 in IL-13Ralpha2 and Leu-319 in IL-13Ralpha1 are positionally conserved hydrophobic amino acid residues. Furthermore, by using D1 domain-deleted mutants, we found that the D1 domain is needed for the expression of IL-13Ralpha2, but not IL-13Ralpha1, and that the D1 domain of IL-13Ralpha1 is important for binding to IL-13, but not to IL-4. These results provide the basis for a precise understanding of the interaction between IL-13 and its receptors.     

Characterization of mpl cytoplasmic domain sequences required for myeloproliferative leukemia virus pathogenicity.

v-mpl is a truncated form of a receptor-like chain which belongs to the cytokine receptor superfamily. This sequence has been transduced in the myeloproliferative leukemia virus as an env-mpl fusion gene responsible for an acute myeloproliferative disorder in mice. We constructed a series of viral mutants in the mpl sequence. Analysis of their oncogenic potential in vivo indicated that a critical 69-amino-acid-long cytoplasmic domain of v-Mpl is required for myoproliferative leukemia virus pathogenicity. We also developed an in vitro assay and showed that expression of the env-mpl gene confers growth factor independence to murine as well as to human hematopoietic growth factor-dependent cell lines. These findings strongly suggest that v-Mpl delivers a constitutive proliferative signal through a limited region of its cytoplasmic domain.     

Identification of an essential region for growth signal transduction in the cytoplasmic domain of the human interleukin-4 receptor.

Interleukin-4 (IL-4) is a pleiotropic lymphokine which plays an important role in the immune system by regulating proliferation and differentiation of a wide variety of lymphoid and myeloid cells. These biological effects are manifested via binding of IL-4 to specific membrane-associated high affinity receptors. While the IL-4 receptor (IL-4R) cDNA expresses high affinity binding sites when transfected in COS7 cells, its intracellular domain lacks consensus motifs for known signal transducing molecules such as a tyrosine kinase. In this study, we use a DNA deletion approach to explore the mechanism of signal transduction utilized by the human IL-4R cDNA expressed in a murine pro-B cell line, Ba/F3 cells. Using this system, we have identified the critical region of the cytoplasmic domain of human IL-4R for human IL-4-induced transduction of a growth signal in these cells. Our data indicate that the critical region for signal transduction is located between amino acid residues 433-473 numbering from the carboxyl terminus. This region is highly conserved between mouse and human IL-4R but lacks homology with other cytokine receptors. Our studies additionally demonstrate that the cytoplasmic domain is not essential for forming high affinity IL-4-binding sites nor for ligand internalization.     

Functionally essential cytoplasmic domain of the erythropoietin receptor.

The cytoplasmic domains of the erythropoietin receptor essential for signal transduction were identified by assessing a series of truncated and deletional mutant receptors. A 91-amino acid region proximal to the transmembrane domain was required for growth signaling. In this region, residues between 353Pro and 362His and between 278Gln and 308Leu appeared to constitute the essential cytoplasmic domains. These two domains contain the conserved amino acids common in the cytokine receptor superfamily, which indicates that these domains in the cytoplasmic regions of the erythropoietin receptor may be important for interaction with common signal transducers or protein tyrosine kinases.     

Characterization of the reversible conformational equilibrium of the cytoplasmic domain of erythrocyte membrane band 3

The cytoplasmic domain of the erythrocyte membrane protein, band 3, contains binding sites for hemoglobin, several glycolytic enzymes, and ankyrin, the linkage to the cytoskeleton. In an earlier study, we found evidence which suggested that band 3 might undergo a native conformational change. We demonstrate here that the cytoplasmic domain of band 3 does exist in a reversible, pH-dependent conformational equilibrium among 3 native states. At physiological salt concentrations this equilibrium is characterized by apparent pKa values of 7.2 and 9.2; however, these apparent pKa values change if the domain's sulfhydryl groups are modified. A major component of the structural change appears to involve the pivoting of two subdomains of the cytoplasmic domain at a central hinge, as evidenced by both hydrodynamic and fluorescence energy transfer measurements. The probable site of this hinge is between residues 176 and 191, a region highly accessible to proteases and also rich in proline. These structural rearrangements also apparently extend to the cluster of tryptophan residues near the N terminus, since the domain's intrinsic fluorescence more than doubles between pH 6.5 and 9.5. No measurable change in band 3 secondary or quaternary structure could be detected during the conformational transitions. A structural model of the cytoplasmic domain of band 3 is presented to show the possible spatial relationships between the regions of conformational change and the sites of peripheral protein binding.     

Characterization of four mammalian numb protein isoforms. Identification of cytoplasmic and membrane-associated variants of the phosphotyrosine binding domain.

Numb is a membrane-associated, phosphotyrosine binding (PTB) domain-containing protein that functions as an intrinsic determinant of cell fate during Drosophila development. We have identified four isoforms of mammalian Numb with predicted molecular masses of 65, 66, 71, and 72 kDa that are generated by alternative splicing of the Numb mRNA. The different isoforms result from the presence of two sequence inserts within the PTB domain and the central region of the protein. The endogenous expression pattern of these isoforms, examined using specific antisera, varied in different tissues and cell lines. In addition, differentiation of P19 cells with retinoic acid leads to the specific loss of expression of the 71- and 72-kDa Numb proteins, suggesting that the expression of certain forms of Numb protein is regulated in a cell type-specific manner. Expression of Numb proteins fused to green fluorescent protein revealed that the form of the PTB domain with the alternatively spliced insert constitutively associated with the plasma membrane in polarized Madin-Darby canine kidney cells. In contrast, the isoform without the insert was cytoplasmic, suggesting that different PTB domain isoforms may regulate the subcellular localization of Numb proteins. The membrane localization may be due, in part, to differential affinity for acidic phospholipids. The distinct expression and localization patterns of the different mammalian Numb isoforms suggest that they have distinct functional properties.     

Sequences determining the cytoplasmic localization of a chemoreceptor domain.

The Escherichia coli serine chemoreceptor (Tsr) is a protein with a simple topology consisting of two membrane-spanning sequences (TM1 and TM2) separating a large periplasmic domain from N-terminal and C-terminal cytoplasmic regions. We analyzed the contributions of several sequence elements to the cytoplasmic localization of the C-terminal domain by using chemoreceptor-alkaline phosphatase gene fusions. The principal findings were as follows. (i) The cytoplasmic localization of the C-terminal domain depended on TM2 but was quite tolerant of mutations partially deleting or introducing charged residues into the sequence. (ii) The basal level of C-terminal domain export was significantly higher in proteins with the wild-type periplasmic domain than in derivatives with a shortened periplasmic domain, suggesting that the large size of the wild-type domain promotes partial membrane misinsertion. (iii) The membrane insertion of deletion derivatives with a single spanning segment (TM1 or TM2) could be controlled by either an adjacent positively charged sequence or an adjacent amphipathic sequence. The results provide evidence that the generation of the Tsr membrane topology is an overdetermined process directed by an interplay of sequences promoting and opposing establishment of the normal structure.     

The role of interleukin-6 and interleukin-6/interleukin-6 receptor-alpha complex in the pathogenesis of multiple myeloma

Multiple myeloma (MM) is a plasma-cell disorder in which malignant plasma cells accumulate in the bone marrow and usually produce a monoclonal immunoglobulin. Usual presenting features of overt MM include recurrent osteolytic lesions, bacterial infections, anemia and renal insufficiency. MM is responsible for about 1 percent of all cancer-related deaths in Western countries. Its epidemiologic pattern remains obscure, and its cause unknown [1]. The presence of somatic mutations within the immunoglobulin genes of myeloma cells indicate that the putative myeloma-cell precursors have been stimulated by antigens within germinal centers and are either memory B cells or migrating plasmablasts. Myeloma cells proliferate slowly in the bone marrow and display a weak apoptotic index in vivo [2]. This suggest that some defects in the apoptotic process could be involved in this neoplasia. Interleukin-6 (IL-6) is known to be an essential survival factor of myeloma cells and to protect them from apoptosis induced by different stimuli (e.g. dexamethasone, CD95, serum starvation, gamma-irradiation). More recently, important works have been devoted to the biology of the soluble form of the IL-6R alpha i.e., sIL-6R alpha. These works give IL-6/sIL-6R alpha complex an important role in the biology of IL-6. The purpose of the current review is to emphasize the role of this complex in the pathogenesis of MM.     

Characterization of the reversible conformational equilibrium in the cytoplasmic domain of human erythrocyte membrane band 3

The cytoplasmic domain of erythrocyte membrane band 3 (cdb3) serves as a center of membrane organization, interacting with such proteins as ankyrin, protein 4.1, protein 4.2, hemoglobin, several glycolytic enzymes, and a tyrosine kinase, p72syk. cdb3 exists in a reversible, pH-dependent conformational equilibrium characterized by large changes in Stokes radius (11 A) and intrinsic fluorescence (2-fold). Based on the crystallographic structure of the cdb3 dimer, we hypothesized that the above conformational equilibrium might involve the movement of flanking peripheral protein binding domains away from a shared dimerization domain. To test this hypothesis, we have mutated both donor (W105L) and acceptor (D316A) residues of a prominent H bond that bridges the above two domains and have examined the effect on the resulting conformational equilibrium. Analysis of the intrinsic fluorescence, Stokes radius, thermal stability, urea stability, and segmental mobility of these mutants reveals that the above H bond is indeed present in the low pH conformation of cdb3 and broken in a higher pH conformation. The data further reveal that cdb3 exists in three native pH-dependent conformations and that rupture of the aforementioned H bond occurs only during conversion of the low pH conformation to the mid-pH conformation. Conversion of the mid-pH conformation to the high pH conformation would now appear to involve structural changes primarily in the peripheral protein binding domain. Because ankyrin associates avidly with the low pH conformation of cdb3, ankyrin occupancy should strongly influence this structural equilibrium and thereby affect band 3 and perhaps global membrane properties.     

Solution structure of the dimeric cytoplasmic domain of syndecan-4.

The syndecans, transmembrane proteoglycans which are involved in the organization of cytoskeleton and/or actin microfilaments, have important roles as cell surface receptors during cell-cell and/or cell-matrix interactions. Since previous studies indicate that the function of the syndecan-4 cytoplasmic domain is dependent on its oligomeric status, the conformation of the syndecan-4 cytoplasmic domain itself is important in the understanding of its biological roles. Gel filtration results show that the syndecan-4 cytoplasmic domain (4L) itself forms a dimer stabilized by ionic interactions between peptides at physiological pH. Commensurately, the NMR structures demonstrate that syndecan-4L is a compact intertwined dimer with a symmetric clamp shape in the central variable V region with a root-mean-square deviation between backbone atom coordinates of 0.95 A for residues Leu(186)-Ala(195). The molecular surface of the 4L dimer is highly positively charged. In addition, no intersubunit NOEs in membrane proximal amino acid resides (C1 region) have been observed, demonstrating that the C1 region is mostly unstructured in the syndecan-4L dimer. Interestingly, two parallel strands of 4L form a cavity in the center of the dimeric twist similar to our previously reported 4V structure. The overall topology of the central variable region within the 4L structure is very similar to that of 4V complexed with the phosphatidylinositol 4,5-bisphosphate; however, the intersubunit interaction mode is affected by the presence of C1 and C2 regions. Therefore, we propose that although the 4V region in the full cytoplasmic domain has a tendency for strong peptide--peptide interaction, it may not be enough to overcome the repulsion of the C1 regions of syndecan-4L.     

Rac-dependent anti-apoptotic signaling by the insulin receptor cytoplasmic domain.

Mutations in the cytoplasmic domain of the insulin receptor that block the ability of the receptor to stimulate glucose uptake do not block the receptor's ability to inhibit apoptosis (Boehm, J. E., Chaika, O. V., and Lewis, R. E. (1998) J. Biol. Chem. 273, 7169-7176). To characterize this survival pathway we used a chimeric receptor (CSF1R/IR) consisting of the ligand-binding domain of the colony-stimulating factor-1 receptor spliced to the cytoplasmic domain of the insulin receptor and a mutated version of the chimeric receptor containing a 12-amino acid deletion of the juxtamembrane domain (CSF1R/IRDelta960). In addition to the inhibition of apoptosis, activation of either the CSF1R/IR or the CSF1R/IRDelta960 rapidly induced membrane ruffling in Rat1 fibroblasts. The small GTPase Rac mediates membrane ruffling. Activated and dominant-inhibitory mutants of Rac and other small GTPases were expressed in Rat1 fibroblasts to examine a potential link between the intracellular pathways that induce membrane ruffling and promote cell survival. The anti-apoptotic action of the CSF1R/IRDelta960 was reversed by dominant-inhibitory Rac(N17), but not by Ras(N17) or Cdc42(N17). Activated Rac(V12), but not Ras(D12) or Cdc42(V12), promoted cell survival in the absence of insulin. These data implicate Rac as a mediator of an unique anti-apoptotic signaling pathway activated by the insulin receptor cytoplasmic domain.     

Regulation of embryonic cell adhesion by the cadherin cytoplasmic domain.

Differential adhesion between embryonic cells has been proposed to be mediated by a family of closely related glycoproteins called the cadherins. The cadherins mediate adhesion in part through an interaction between the cadherin cytoplasmic domain and intracellular proteins, called the catenins. To determine whether these interactions could regulate cadherin function in embryos, a form of N-cadherin was generated that lacks an extracellular domain. Expression of this mutant in Xenopus embryos causes a dramatic inhibition of cell adhesion. Analysis of the mutant phenotype shows that at least two regions of the N-cadherin cytoplasmic domain can inhibit adhesion and that the mutant cadherin can inhibit catenin binding to E-cadherin. These results suggest that cadherin-mediated adhesion can be regulated by cytoplasmic interactions and that this regulation may contribute to morphogenesis when emerging tissues coexpress several cadherin types.     

Specialized functional properties of the integrin alpha 4 cytoplasmic domain.

For functional studies of the integrin alpha 4 cytoplasmic domain, we have expressed the following in K562 and Chinese hamster ovary (CHO) cells: 1) wild-type alpha 4 (called X4C4), 2) two chimeric forms of alpha 4 (called X4C2 and X4C5) that contain the cytoplasmic domains of alpha 2 and alpha 5, respectively, and 3) alpha 4 with no cytoplasmic domain (X4C0). Cytoplasmic domain exchange had no effect on VLA-4-dependent static cell adhesion or tethering to VCAM-1 in conditions of shear flow. However, the presence of the alpha 2 or alpha 5 tails markedly enhanced VLA-4-dependent K562 cells spreading (X4C2 > X4C5 > X4C4 > X4C0), increased localization of VLA-4 into focal adhesion-like complexes in CHO cells (X4C2 > X4C5 > X4C4), and strengthened CHO and K562 cell resistance to detachment from VCAM-1 in conditions of shear flow (X4C2 > X4C5 > X4C4 > X4C0). Conversely, the alpha 4 tail supported greater VLA-4-dependent haptotactic and chemotactic cell migration. In the absence of any alpha tail (i.e., X4C0), robust focal adhesions were observed, even though cell spreading and adhesion strengthening were minimal. Thus, such focal adhesions may have relatively little functional importance, and should not be compared with focal adhesions formed when alpha tails are present. Together, these results indicate that all three alpha-chain tails exert defined positive effects (compared with no tail at all), but suggest that the alpha 4 cytoplasmic domain may be specialized to engage in weaker cytoskeletal interactions, leading to diminished focal adhesion formation, cell spreading, and adhesion strengthening, while augmenting cell migration and facilitating rolling under shear flow. These properties of the alpha 4 tail are consistent with the role of alpha 4 integrins on highly motile lymphocytes, monocytes, and eosinophils.     

Phosphorylation of the human interleukin-2 receptor and a synthetic peptide identical to its C-terminal, cytoplasmic domain

The recombinant human interleukin-2 (IL-2) receptor was expressed in mouse mammary epithelial cells following the transfection of these cells with an expression vector containing the human IL-2 receptor cDNA. The recombinant IL-2 receptor in these cells was rapidly phosphorylated in response to phorbol myristate acetate (PMA), but its phosphorylation could not be detected in the absence of PMA or upon addition of human IL-2. The C-terminal, cytoplasmic peptide domain of the IL-2 receptor, Gln-Arg-Arg-Gln-Arg-Lys-Ser-Arg-Arg-Thr-Ile, was synthesized and used as a substrate for protein kinase C. The Km for phosphorylation of the peptide by protein kinase C was 23 microM. The stoichiometry of phosphorylation was 1 mol of phosphate/mol of peptide and serine was the predominant amino acid phosphorylated. Because this peptide was a good substrate for protein kinase C in vitro, it was possible that the same serine (serine 247) was also phosphorylated in the receptor in the cell. The IL-2 receptor gene in the expression vector was therefore altered by site-directed mutagenesis to code for an IL-2 receptor containing an alanine in the place of serine 247. The IL-2 receptor expressed by these cells was not phosphorylated in the presence of PMA. These data suggest that protein kinase C, in response to PMA, phosphorylates the C-terminal serine residue (serine 247) in the human IL-2 receptor.     

Characterization of cytoplasmic DNA of mouse-myeloma cells.

Cytoplasmic DNA from mouse myeloma cells comprised between 1% and 2% of the total cellular DNA. Detergent-prepared cytoplasmic lysate consisted mainly of 8-S and 22-S species. While these DNA species were present in the 13000 times g pellet of the detergent-prepared cytoplasmic lysate, only the light DNA species was present in the 13000 times g supernatant fraction. In neutral CsCl gradients the DNA of both cytoplasmic fractions had a buoyant density of 1700 g/cm3, which is identical to that of nuclear DNA. The similarity between the cytoplasmic and nuclear DNA was also demonstrated by analysis on alkaline CsCl gradients. A small proportion of closed-circular DNA, presumably of mitochondrial origin, was demonstrated only in cytoplasmic fraction obtained from mechanically disrupted cells and not in detergent-prepared cytoplasmic lysate. It was found that poly (A)-containing mRNA and 28-S ribosomal RNA hybridized to about the same extent to the cytoplasmic DNA as compared to nuclear DNA. The results indicate that most of the cytoplasmic DNA in myeloma cells is similar to nuclear DNA and does not consist of mitochondrial DNA.     

Characterization of residues in the cytoplasmic domain of the LDL receptor required for exit from the endoplasmic reticulum

Newly synthesized low density lipoprotein receptors (LDLRs) exit the endoplasmic reticulum (ER) as the first step in the secretory pathway. In this study we have generated truncating deletions and substitutions within the 50 amino acid cytoplasmic domain of the LDLR in order to identify residues required for the exit from the ER. Western blot analysis was used to determine the relative amounts of the 120 kDa precursor form of the LDLR located in the ER and the 160 kDa mature form that has exited the ER. These studies have shown that the exit of an LDLR lacking the cytoplasmic domain, is markedly reduced. Moreover, the longer the cytoplasmic domain, the more efficient is the exit from the ER. At least 30 residues were required for the LDLR to efficiently exit the ER. Mutations in the two di-acidic motifs ExE₈₁₄ and/or ExD₈₃₇ had only a small effect on the exit from the ER. The requirement for a certain length of the cytoplasmic domain for efficient exit from the ER, could reflect the distance needed to interact with the COPII complex of the ER membrane or the requirement for the LDLR to undergo dimerization.     

Characterization of interleukin-13 receptor in carcinoma cell lines and human blood cells and comparison with the interleukin-4 receptor

Abstract

The interleukin-13 receptor is characterized by ligand-binding and crosslinking studies and compared with the interleukin-4 receptor. Crosslinking of radio-labeled hIL-4 and hIL-13 to the receptors on human carcinoma and mast cell lines demonstrated a predominant subunit at 130 kDa with two other minor bands of lower molecular mass (75 kDa and 65 kDa) in autoradiography. All binding of ¹²⁵I-IL-13 was specifically blocked when the carcinoma cell suspensions were incubated with an excess of unlabeled hIL-4. However, unlabeled hIL-13 was unable to completely displace ¹²⁵I-hIL-4 from the 130 kDa protein. In addition, ¹²⁵I-hIL-13 showed no binding to mouse fibroblast cells transfected with human 130 kDa hIL-4 receptor c-DNA. Using weighted nonlinear computer modeling of the data from several equilibrium binding studies with human mast cells, a model of two binding sites for IL-4 (K_d = 50 and 190 pmol/L) and one site for IL-13 (K_d=100 pmol/L) fitted better than a one site model with a very high level of significance (F = 10.66, P < 0.0001). It can be concluded that human IL-4R and hIL-13R are similar but distinct. This conclusion is supported here for the first time by a strong statistical criterion.