Activation of erythropoietin signaling by receptor dimerization

The hormone erythropoietin (Epo) is essential for red blood cell development. Epo binds a high affinity receptor on the surface of erythroid progenitor cells, stimulating receptor dimerization and activation of the intracellular signal transduction pathways that support erythroid cell survival, proliferation and differentiation. Biochemical and structural analysis of the erythropoietin receptor (EpoR) is revealing the molecular mechanisms of EpoR function, leading the way to the development of small molecule Epo mimetics. This review focuses on the role EpoR dimerization plays in receptor function.     

Probing fibroblast growth factor dimerization and role of heparin-like glycosaminoglycans in modulating dimerization and signaling

For a number of growth factors and cytokines, ligand dimerization is believed to be central to the formation of an active signaling complex. In the case of fibroblast growth factor-2 (FGF2) signaling, heparin/heparan sulfate-like glycosaminoglycans (HLGAGs) are involved through interaction with both FGF2 and its receptors (FGFRs) in assembling a tertiary complex and modulating FGF2 activity. Biochemical data have suggested different modes of HLGAG-induced FGF2 dimerization involving specific protein-protein contacts. In addition, several recent x-ray crystallography studies of FGF.FGFR and FGF.FGFR.HLGAG complexes have revealed other modes of molecular assemblage, with no FGF-FGF contacts. All these different biochemical and structural findings have clarified less and in fact raised more questions as to which mode of FGF2 dimerization, if any, is essential for signaling. In this study, we address the issue of FGF2 dimerization in signaling using a combination of biochemical, biophysical, and site-directed mutagenesis approaches. Our findings presented here provide direct evidence of FGF2 dimerization in mediating FGF2 signaling.     

Platelet-derived growth factor (PDGF)-induced disulfide-linked dimerization of PDGF receptor in living cells.

It is well established that epidermal growth factor and platelet-derived growth factor (PDGF) are able to induce noncovalent dimerization of their surface receptors. It is thought that receptor dimerization plays an important role in activation of the tyrosine kinase function and in the process of receptor autophosphorylation. Here we show that the addition of either PDGF-BB or PDGF-AA to intact 3T3 cells induces formation of 400- and 430-kDa species, respectively, recognized by either anti-PDGF receptor antibodies or anti-phosphotyrosine antibodies. Interestingly, the 400- and the 430-kDa species are detected in nonreducing gels but not in reducing gels. Moreover, an alkylating agent, N-ethylmaleimide, inhibits PDGF-induced formation of high-molecular-mass species. Comparisons of V8 protease peptide maps of [35S]methionine-labeled PDGF receptors and high-molecular-mass proteins indicate that they represent dimers of PDGF receptors. It appears therefore that in addition to noncovalent dimerization, PDGF receptors undergo ligand-dependent disulfide-linked dimerization.     

Efficient leukocyte Ig-like receptor signaling and crystal structure of disulfide-linked HLA-G dimer

HLA-G is a nonclassical major histocompatibility complex class I (MHCI) molecule, which is expressed in trophoblasts and confers immunological tolerance in the maternal-fetal interface by binding to leukocyte Ig-like receptors (LILRs, also called as LIR/ILT/CD85) and CD8. HLA-G is expressed in disulfide-linked dimer form both in solution and at the cell surface. Interestingly, MHCI dimer formations have been involved in pathogenesis and T cell activation. The structure and receptor binding characteristics of MHCI dimers have never been evaluated. Here we performed binding studies showing that the HLA-G dimer exhibited higher overall affinity to LILRB1/2 than the monomer by significant avidity effects. Furthermore, the cell reporter assay demonstrated that the dimer formation remarkably enhanced the LILRB1-mediated signaling at the cellular level. We further determined the crystal structure of the wild-type dimer of HLA-G with the intermolecular Cys(42)-Cys(42) disulfide bond. This dimer structure showed the oblique configuration to expose two LILR/CD8-binding sites upward from the membrane easily accessible for receptors, providing plausible 1:2 (HLA-G dimer:receptors) complex models. These results indicated that the HLA-G dimer conferred increased avidity in a proper structural orientation to induce efficient LILR signaling, resulting in the dominant immunosuppressive effects. Moreover, structural and functional implications for other MHCI dimers observed in activated T cells and the pathogenic allele, HLA-B27, are discussed.     

Analysis of G-protein-coupled receptor dimerization following chemokine signaling

An abundance of information has been generated in recent decades on the signaling events triggered through G-protein-coupled receptors (GPCRs). Nonetheless, the structural changes at the cell surface that provoke receptor activation are only now beginning to be understood. It is becoming clear that receptors are not isolated entities that are activated following ligand binding, but that they interact with other molecules already present or recruited to the vicinity, which results in a wide variety of new signaling possibilities. Understanding receptor interactions with relatives and/or friends on the cell surface is thus critical. The most important point is to determine which of these interactions are "casual" and which give rise to functional consequences.     

Differential regulation and role of interleukin-1 receptor associated kinase-M in innate immunity signaling

Toll-like-receptor mediated signaling is finely regulated by a complex intracellular protein network including the interleukin-1 receptor associate kinases (IRAKs). IRAK-4, 1, and 2 may positively regulate innate immunity signaling through the activation of various downstream kinases such as MAPKs. In contrast, IRAK-M plays an inhibitory role through unknown mechanism. In this report, we show that IRAK-M is ubiquitously present in the cell, and becomes exclusively cytoplasmic upon bacterial lipoprotein Pam(3)CSK(4) challenge. Furthermore, using bone marrow derived macrophages (BMDM) from wild type, IRAK1(-/-), and IRAK-M(-/-) mice, we have herein demonstrated that IRAK-M selectively attenuates bacterial lipopeptide Pam(3)CSK(4)-induced p38 activation, but not ERK or JNK. IRAK1(-/-) and IRAK-M(-/-)BMDM display distinct activation profile of various MAP kinases upon Pam(3)CSK(4) challenge, indicating that IRAK-M exerts its inhibitory effect through an IRAK1 independent pathway. Pam(3)CSK(4) challenge leads to rapid decrease of MKP-1 protein level in IRAK-M(-/-)BMDM as well as THP-1 cells with decreased IRAK-M expression through siRNA interference. Our findings indicate that IRAK-M selectively attenuates p38 activation and inhibits innate immunity through stabilizing MKP-1.     

Ryanodine receptor signaling is required for anti-CD3-induced T cell proliferation, interleukin-2 synthesis, and interleukin-2 receptor signaling

Ryanodine receptors (RyR) are involved in regulating intracellular Ca(++) mobilization in T lymphocytes. However, the importance of RyR signaling during T cell activation has not yet been determined. In this study, we have used the RyR-selective antagonists, ruthenium red and dantrolene, to determine the effect of RyR blockade on T cell receptor-mediated activation events and cytokine-dependent T cell proliferation. Both ruthenium red and dantrolene inhibited DNA synthesis and cell division, as well as the synthesis of interleukin (IL)-2 by T lymphocytes responding to mitogenic anti-CD3 antibody. Blockade of RyR at initiation of culture or as late as 24 h after T cell receptor stimulation inhibited T cell proliferation, suggesting a requirement for sustained RyR signaling during cell cycle progression. Although flow cytometry revealed that RyR blockade had little effect on activation-induced expression of the alpha chain (CD25) of the high affinity IL-2 receptor, the inhibitory effect of RyR antagonists could not be reversed by the addition of exogenous IL-2 at initiation of culture. In addition, both ruthenium red and dantrolene had a strong inhibitory effect on IL-2-dependent proliferation of CTLL-2 T cells. These data indicate that RyR are involved in regulating IL-2 receptor signaling that drives T cell progression through the cell cycle. We conclude that RyR-associated Ca(++) signaling regulates T cell proliferation by promoting both IL-2 synthesis and IL-2-dependent cell cycle progression.     

The role of TRADD in death receptor signaling

TRADD (TNFR1-associated death domain protein) was initially identified as an adaptor molecule that transduces the signal downstream of the TNFR1 (tumor necrosis factor receptor 1). TNFR1 belongs to the so-called death receptor (DR) family of receptors that depending on the context can induce either apoptosis or proliferation, as well as NF-κB and MAP kinase activation. The receptors of this group contain death domain (DD) that is necessary for the induction of apoptosis. This review summarizes the recent advances in the field of DR signaling and in particular the role of TRADD.     

Identification of the disulfide-linked homodimer of apolipoprotein E3 in plasma. Impact on receptor binding activity

The nature of disulfide-linked structures of apolipoprotein (apo) E3 in the plasma of E3/3 subjects was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis performed under nonreducing conditions followed by immunoblotting with apoE-specific antibodies. In addition to the expected presence of the heterodimer apoE3-A-II and monomeric apoE3, a band with an apparent Mr approximately 100,000 was also observed in plasma that had been treated with sulfhydryl-trapping reagents. This band and apoE3-A-II were both eliminated by disulfide reduction, which produced a corresponding increase in monomeric apoE3. Both bands were absent in plasma from a subject with the E4/4 phenotype. In spite of its apparent molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the high molecular weight band was demonstrated to represent the disulfide-linked homodimer of apoE3. It was isolated from purified apoE3 preparations that had undergone oxygen-mediated dimerization and shown to elute from a Sephacryl S-300 column in a position with the expected molecular weight of a homodimer. The apoE3 dimer displayed a preference for high density lipoproteins, as determined by agarose chromatography of E3/3 plasma but was stripped from high density lipoproteins by ultracentrifugation. Quantitation of the relative ratios of homodimer, apoE3-A-II, and monomer in the plasma of 22 normolipidemic E3/3 subjects by immunoblotting revealed that the disulfide-linked structures accounted for the majority (approximately 55%) of plasma apoE. Both the homodimer and apoE3-A-II displayed a reduced ability to compete with low density lipoproteins for fibroblast low density lipoprotein receptors (20 and 30% of monomeric apoE3 binding activity, respectively). These results raise the possibility that the amount or availability of receptor-active apoE3 in E3/3 subjects may be rate limiting for metabolic events involving the low density lipoprotein receptor.     

The role of TRADD in death receptor signaling

TRADD (TNFR1-associated death domain protein) was initially identified as an adaptor molecule that transduces the signal downstream of the TNFR1 (tumor necrosis factor receptor 1). TNFR1 belongs to the so-called death receptor (DR) family of receptors that depending on the context can induce either apoptosis or proliferation, as well as NF-κB and MAP kinase activation. The receptors of this group contain death domain (DD) that is necessary for the induction of apoptosis. This review summarizes the recent advances in the field of DR signaling and in particular the role of TRADD.     

Structure-activity relationship study of human interleukin-3: role of the C-terminal region for biological activity.

A structure-activity relationship study of human interleukin-3 (huIL-3) was performed by functional analysis of huIL-3 deletion and substitution variants combined with epitope mapping of huIL-3 specific neutralizing monoclonal antibodies (mAb). Analysis of the huIL-3 variants was accomplished by defining their capacity to compete with wild-type huIL-3 for binding to the huIL-3 receptor and to induce the proliferation of the huIL-3 dependent cell line M-O7. HuIL-3 variants with either 14 amino acids (aa) deleted from the N-terminus or eight aa from the C-terminus retained full biological activity in vitro. An huIL-3 variant, with 18 N-terminal aa deleted, exhibited a greater than 7-fold reduced receptor binding capacity and proliferative activity. No biological activity could be detected with a variant where 22 C-terminal aa have been deleted. Neutralizing mAb recognizing presumed discontinuous epitopes failed to interact with the latter deletion variant indicating a possible location of their epitopes within the C-terminal region. Computer-aided structure prediction and sequence homology analysis of this region indicated the presence of an amphiphilic alpha-helix with highly conserved residues like Lys110 and Leu111. Substitution of Lys110 with either Glu or Ala resulted in variants with a 10-fold reduced activity in the receptor binding assay and the proliferation assay. Further variants, where Leu111 was substituted by Pro or Met, were totally inactive in these assays. Analysis of the binding of the two neutralizing mAb to these substitution variants showed that they did not bind to either of the Leu111 variants suggesting that Leu111 is part of an active site. Based on our results, a possible model for the structure of the huIL-3 molecule can be constructed with two active sites in close proximity.     

The role of lymphotoxin receptor signaling in diseases

LT, LIGHT, and TNF are core family members of the TNFR superfamily of cytokines. LT and LIGHT, produced primarily by lymphocytes, interact with LTbetaR expressed by stromal and epithelial cells. Extensive studies over the last decade have revealed a critical role of LT-LTbetaR interactions for organogenesis and maintenance of the secondary lymphoid organs and in the generation of an efficient humoral immune response to various pathogens. LTbetaR's function beyond the lymphoid organs shows valuable potential yet remains largely undefined. Recent studies indicate that LTbetaR signaling is required for liver regeneration, hepatitis, and hepatic lipid metabolism. The balance of beneficial and detrimental effects of LTbetaR is critical for understanding the mechanisms of autoimmune disease and liver function and may open a new avenue for therapeutic intervention. This review will discuss recent advances in understanding LTbetaR's role in various human and murine disease models while focusing on its regulation of and implications in various liver related diseases.     

A point mutation uncouples human interleukin-1 beta biological activity and receptor binding

Interleukin-1 proteins elicit a number of biological activities, but the molecular events following formation of a cell surface receptor-ligand complex have not been well defined. Conversion of Arg127 to Gly127 in the mature human interleukin-1 beta protein reduces bioactivity by 100-fold while the receptor binding affinity decreases by only 25%. The results suggest that the mutant IL-1 beta protein is defective in activating signal transduction events and indicate that binding of interleukin-1 beta protein to receptor is necessary but insufficient for biological activity. The finding that the features of the IL-1 beta protein responsible for receptor binding and biological activity are at least in part distinct may be clinically relevant to the design of interleukin-1 antagonists.     

Human interleukin-3 (IL-3) induces disulfide-linked IL-3 receptor alpha- and beta-chain heterodimerization, which is required for receptor activation but not high-affinity binding.

The human interleukin-3 receptor (IL-3R) is a heterodimer that comprises an IL-3 specific alpha chain (IL-3R alpha) and a common beta chain (beta C) that is shared with the receptors for granulocyte-macrophage colony-stimulating factor (GM-CSF) and IL-5. These receptors belong to the cytokine receptor superfamily, but they are structurally and functionally more related to each other and thus make up a distinct subfamily. Although activation of the normal receptor occurs only in the presence of ligand, the underlying mechanisms are not known. We show here that human IL-3 induces heterodimerization of IL-3R alpha and beta c and that disulfide linkage of these chains is involved in receptor activation but not high-affinity binding. Monoclonal antibodies (MAb) to IL-3R alpha and beta c were developed which immunoprecipitated, in the absence of IL-3, the respective chains from cells labelled with 125I on the cell surface. However, in the presence of IL-3, each MAb immunoprecipitated both IL-3R alpha and beta c. IL-3-induced receptor dimers were disulfide and nondisulfide linked and were dependent on IL-3 interacting with both IL-3R alpha and beta c. In the presence of IL-3 and under nonreducing conditions, MAb to either IL-3R alpha or beta c immunoprecipitated complexes with apparent molecular weights of 215,000 and 245,000 and IL-3R alpha and beta c monomers. Preincubation with iodoacetamide prevented the formation of the two high-molecular-weight complexes without affecting noncovalent dimer formation or high-affinity IL-3 binding. Two-dimensional gel electrophoresis and Western blotting (immunoblotting) demonstrated the presence of both IL-3R alpha and beta c in the disulfide-linked complexes. IL-3 could also be coimmunoprecipitated with anti-IL-3R alpha or anti-beta c MAB, but it was not covalently attached to the receptor. Following IL-3 stimulation, only the disulfide-linked heterodimers exhibited reactivity with antiphosphotyrosine antibodies, with beta c but not IL-3R alpha being the phosphorylated species. A model of IL-3R activation is proposed which may be also applicable to the related GM-CSF and IL-5 receptors.     

Regulated dimerization of the thyrotropin-releasing hormone receptor affects receptor trafficking but not signaling

To investigate the function of dimerization of the TRH receptor, a controlled dimerization system was developed. A variant FK506 binding protein (FKBP) domain was fused to the receptor C terminus and dimerization induced by incubating cells with dimeric FKBP ligand, AP20187. The TRH receptor-fusion bound hormone and signaled normally. Addition of dimerizer to cells expressing the receptor-FKBP fusion dramatically increased the fraction of receptor running as dimer on SDS-PAGE. AP20187 caused dimerization in a time- and concentration-dependent manner, acting within 1 min. Dimerizer had no effect on TRH receptors lacking the FKBP domain, and its effects were blocked by excess monomeric FKBP ligand. AP20187-induced dimerization did not cause receptor phosphorylation, inositol phosphate production, or ERK1/2 activation, and dimerizer did not alter signaling by TRH. Induced dimerization did, however, alter TRH receptor trafficking. TRH promoted greater receptor internalization in cells treated with AP20187 but not monomeric ligand, based on loss of surface binding sites and immunostaining. Dimerization increased the rate of internalization of TRH receptors and decreased the apparent rate of receptor recycling. AP20187 enhanced the small amount of TRH-induced receptor internalization when the receptor-FKBP fusion protein was expressed in cells lacking beta-arrestins. The results show that controlled dimerization of the TRH receptor potentiates hormone-induced receptor trafficking.     

The role of receptor internalization in CD95 signaling

Activation of the cell surface CD95 receptor triggers a cascade of signaling events, including assembly of the death-inducing signaling complex (DISC), that culminate in cellular apoptosis. In this study, we demonstrate a general requirement of receptor internalization for CD95 ligand-mediated DISC amplification, caspase activation and apoptosis in type I cells. Recruitment of DISC components to the activated receptor predominantly occurs after the receptor has moved into an endosomal compartment and blockade of CD95 internalization impairs DISC formation and apoptosis. In contrast, CD95 ligand stimulation of cells unable to internalize CD95 results in activation of proliferative Erk and NF-kappaB signaling pathways. Hence, the subcellular localization and internalization pathways of CD95 play important roles in controlling activation of distinct signaling cascades to determine divergent cellular fates.     

Polarization vision and its role in biological signaling

Visual pigments, the molecules in photoreceptors that initiatethe process of vision, are inherently dichroic, differentiallyabsorbing light according to its axis of polarization. Manyanimals have taken advantage of this property to build receptorsystems capable of analyzing the polarization of incoming light,as polarized light is abundant in natural scenes (commonly beingproduced by scattering or reflection). Such polarization sensitivityhas long been associated with behavioral tasks like orientationor navigation. However, only recently have we become aware thatit can be incorporated into a high-level visual perception akinto color vision, permitting segmentation of a viewed scene intoregions that differ in their polarization. By analogy to colorvision, we call this capacity polarization vision. It is apparentlyused for tasks like those that color vision specializes in:contrast enhancement, camouflage breaking, object recognition,and signal detection and discrimination. While color is veryuseful in terrestrial or shallow-water environments, it is anunreliable cue deeper in water due to the spectral modificationof light as it travels through water of various depths or ofvarying optical quality. Here, polarization vision has specialutility and consequently has evolved in numerous marine species,as well as at least one terrestrial animal. In this review,we consider recent findings concerning polarization vision andits significance in biological signaling.