New therapeutic strategies targeting transmembrane signal transduction in the immune system

Single-chain receptors and multichain immune recognition receptors (SRs and MIRRs, respectively) represent families of structurally related but functionally different surface receptors expressed on different cells. In contrast to SRs, a distinctive and common structural characteristic of MIRR family members is that the extracellular recognition domains and intracellular signaling domains are located on separate subunits. How extracellular ligand binding triggers MIRRs and initiates intracellular signal transduction processes is not clear. A novel model of immune signaling, the Signaling Chain HOmoOLigomerization (SCHOOL) model, suggests that the homooligomerization of receptor intracellular signaling domains represents a necessary and sufficient condition for receptor triggering. In this review, I demonstrate striking similarities between a consensus model of SR signaling and the SCHOOL model of MIRR signaling and show how these models, together with the lessons learned from viral pathogenesis, provide a molecular basis for novel pharmacological approaches targeting inter- and intrareceptor transmembrane interactions as universal therapeutic targets for a diverse variety of immune and other disorders.     

Lipid-binding activity of intrinsically unstructured cytoplasmic domains of multichain immune recognition receptor signaling subunits

Multichain immune recognition receptors (MIRRs) found on the surface of T cells, B cells, mast cells, natural killer cells, basophils, and other immune cells are formed by the association of several single-pass transmembrane proteins, with immunoglobulin-like ligand recognition domains and signal-transducing domains present on separate subunits. The MIRR signaling subunits all have cytoplasmic domains containing one or more copies of an immunoreceptor tyrosine-based activation motif (ITAM), tyrosine residues of which are phosphorylated upon receptor engagement in an early and obligatory event in the signaling cascade. Despite the proximity to the cell membrane and crucial role in transmembrane signal transduction, little is known about the structure and lipid-binding activity of the ITAM-containing cytoplasmic domains. Here we investigate the conformation and lipid-binding activity of several MIRR cytoplasmic domains, namely, T cell receptor zetacyt, CD3epsiloncyt, CD3deltacyt, and CD3gammacyt, B cell receptor Igalphacyt and Igbetacyt, and Fc receptor FcepsilonRIgammacyt, using purified recombinant proteins. Secondary structure prediction analysis and experimental circular dichroism spectra identify each of these cytoplasmic domains as natively unfolded proteins. We also report that zetacyt, CD3epsiloncyt, and FcepsilonRIgammacyt bind to acidic and mixed phospholipid vesicles and that the binding strength correlates with the protein net charge and the presence of clustered basic amino acid residues. Circular dichroism analysis reveals the lack of secondary structure for these domains in lipid-bound form. Phosphorylation of zetacyt and FcepsilonRIgammacyt does not alter their random-coil conformation but weakens binding to membranes. The implications of these results for transmembrane signal transduction by immune receptors are discussed.     

Isolated Toll-like Receptor Transmembrane Domains Are Capable of Oligomerization

Toll-like receptors (TLRs) act as the first line of defense against bacterial and viral pathogens by initiating critical defense signals upon dimer activation. The contribution of the transmembrane domain in the dimerization and signaling process has heretofore been overlooked in favor of the extracellular and intracellular domains. As mounting evidence suggests that the transmembrane domain is a critical region in several protein families, we hypothesized that this was also the case for Toll-like receptors. Using a combined biochemical and biophysical approach, we investigated the ability of isolated Toll-like receptor transmembrane domains to interact independently of extracellular domain dimerization. Our results showed that the transmembrane domains had a preference for the native dimer partners in bacterial membranes for the entire receptor family. All TLR transmembrane domains showed strong homotypic interaction potential. The TLR2 transmembrane domain demonstrated strong heterotypic interactions in bacterial membranes with its known interaction partners, TLR1 and TLR6, as well as with a proposed interaction partner, TLR10, but not with TLR4, TLR5, or unrelated transmembrane receptors providing evidence for the specificity of TLR2 transmembrane domain interactions. Peptides for the transmembrane domains of TLR1, TLR2, and TLR6 were synthesized to further study this subfamily of receptors. These peptides validated the heterotypic interactions seen in bacterial membranes and demonstrated that the TLR2 transmembrane domain had moderately strong interactions with both TLR1 and TLR6. Combined, these results suggest a role for the transmembrane domain in Toll-like receptor oligomerization and as such, may be a novel target for further investigation of new therapeutic treatments of Toll-like receptor mediated diseases.     

无脊椎动物清道夫受体的免疫调控

在长期进化的过程中,无脊椎动物逐渐形成了受体识别-信号传导-免疫应答为特征的天然免疫体系,以清除凋亡细胞或外界的病原微生物。清道夫受体（SRs）是一类位于细胞表面的跨膜受体,也是一类参与无脊椎动物天然免疫反应的重要模式识别受体。清道夫受体参与免疫反应的异己靶标识别,通过下游信号级联调控抗菌肽合成和吞噬作用。本文综述了无脊椎动物清道夫受体的种类、结构及其参与天然免疫的调控机制,探讨了无脊椎动物清道夫受体研究中尚待解决的问题。     

Fc Rgamma -independent signaling by the platelet collagen receptor glycoprotein VI

The platelet collagen receptor glycoprotein VI (GPVI) is structurally homologous to multisubunit immune receptors and signals through the immune receptor adaptor Fc Rgamma. Multisubunit receptors are composed of specialized subunits thought to be dedicated exclusively to ligand binding or signal transduction. However, recent studies of the intracellular region of GPVI, a ligand-binding subunit, have suggested the existence of protein-protein interactions that could regulate receptor signaling. In the present study we have investigated the signaling role of the GPVI intracellular domain by stably expressing GPVI mutants in RBL-2H3 cells, a model system that accurately reproduces the GPVI signaling events observed in platelets. Studies of mutant GPVI receptor protein-protein interaction and calcium signaling reveal the existence of discrete domains within the receptor's intracellular tail that mediate interaction with Fc Rgamma, calmodulin, and Src family tyrosine kinases. These receptor interactions are modular and mediated by non-overlapping regions of the receptor transmembrane and intracellular domains. GPVI signaling requires all three of these domains as receptor mutants able to couple to only two interacting proteins exhibited severe signaling defects despite normal surface expression. Our results demonstrate that the ligand-binding subunit of the GPVI-Fc Rgamma receptor participates directly in receptor signaling by interacting with downstream signaling molecules other than Fc Rgamma through an adaptor-like mechanism.     

The structural biology of Toll-like receptors

The membrane-bound Toll-like receptors (TLRs) trigger innate immune responses after recognition of a wide variety of pathogen-derived compounds. Despite the wide range of ligands recognized by TLRs, the receptors share a common structural framework in their extracellular, ligand-binding domains. These domains all adopt horseshoe-shaped structures built from leucine-rich repeat motifs. Typically, on ligand binding, two extracellular domains form an "m"-shaped dimer sandwiching the ligand molecule bringing the transmembrane and cytoplasmic domains in close proximity and triggering a downstream signaling cascade. Although the ligand-induced dimerization of these receptors has many common features, the nature of the interactions of the TLR extracellular domains with their ligands varies markedly between TLR paralogs.     

Observation of an unexpected third receptor molecule in the crystal structure of human interferon-gamma receptor complex

BACKGROUND: Molecular interactions among cytokines and cytokine receptors form the basis of many cell-signaling pathways relevant to immune function. Interferon-gamma (IFN-gamma) signals through a multimeric receptor complex consisting of two different but structurally related transmembrane chains: the high-affinity receptor-binding subunit (IFN-gammaRalpha) and a species-specific accessory factor (AF-1 or IFN-gammaRbeta). In the signaling complex, the two receptors probably interact with one another through their extracellular domains. Understanding the atomic interactions of signaling complexes enhances the ability to control and alter cell signaling and also provides a greater understanding of basic biochemical processes. RESULTS: The crystal structure of the complex of human IFN-gamma with the soluble, glycosylated extracellular part of IFN-gammaRalpha has been determined at 2.9 A resolution using multiwavelength anomalous diffraction methods. In addition to the expected 2:1 complex, the crystal structure reveals the presence of a third receptor molecule not directly associated with the IFN-gamma dimer. Two distinct intermolecular contacts, involving the edge strands of the C-terminal domains, are observed between this extra receptor and the 2:1 receptor-ligand complex thereby forming a 3:1 complex. CONCLUSIONS: The observed interactions in the 2:1 complex of the high-affinity cell-surface receptor with the IFN-gamma cytokine are similar to those seen in a previously reported structure where the receptor chains were not glycosylated. The formation of beta-sheet packing interactions between pairs of IFN-gammaRalpha receptors in these crystals suggests a possible model for receptor oligomerization of Ralpha and the structurally homologous Rbeta receptors in the fully active IFN-gamma signaling complex.     

A novel mechanism for Wnt activation of canonical signaling through the LRP6 receptor

LDL receptor-related protein 6 (LRP6) is a Wnt coreceptor in the canonical signaling pathway, which plays essential roles in embryonic development. We demonstrate here that wild-type LRP6 forms an inactive dimer through interactions mediated by epidermal growth factor repeat regions within the extracellular domain. A truncated LRP6 comprising its transmembrane and cytoplasmic domains is expressed as a constitutively active monomer whose signaling ability is inhibited by forced dimerization. Conversely, Wnts are shown to activate canonical signaling through LRP6 by inducing an intracellular conformational switch which relieves allosteric inhibition imposed on the intracellular domains. Thus, Wnt canonical signaling through LRP6 establishes a novel mechanism for receptor activation which is opposite to the general paradigm of ligand-induced receptor oligomerization.     

Single-spanning transmembrane domains in cell growth and cell-cell interactions: More than meets the eye?

As a whole, integral membrane proteins represent about one third of sequenced genomes, and more than 50% of currently available drugs target membrane proteins, often cell surface receptors. Some membrane protein classes, with a defined number of transmembrane (TM) helices, are receiving much attention because of their great functional and pharmacological importance, such as G protein-coupled receptors possessing 7 TM segments. Although they represent roughly half of all membrane proteins, bitopic proteins (with only 1 TM helix) have so far been less well characterized. Though they include many essential families of receptors, such as adhesion molecules and receptor tyrosine kinases, many of which are excellent targets for biopharmaceuticals (peptides, antibodies, et al.). A growing body of evidence suggests a major role for interactions between TM domains of these receptors in signaling, through homo and heteromeric associations, conformational changes, assembly of signaling platforms, etc. Significantly, mutations within single domains are frequent in human disease, such as cancer or developmental disorders. This review attempts to give an overview of current knowledge about these interactions, from structural data to therapeutic perspectives, focusing on bitopic proteins involved in cell signaling.Key words: bitopic membrane proteins, transmembrane domains, transmembrane signaling, helix-helix interactions, receptors     

Receptor tyrosine kinase activation: From the ligand perspective

Receptor tyrosine kinases (RTKs) are single-span transmembrane receptors in which relatively conserved intracellular kinase domains are coupled to divergent extracellular modules. The extracellular domains initiate receptor signaling upon binding to either soluble or membrane-embedded ligands. The diversity of extracellular domain structures allows for coupling of many unique signaling inputs to intracellular tyrosine phosphorylation. The combinatorial power of this receptor system is further increased by the fact that multiple ligands can typically interact with the same receptor. Such ligands often act as biased agonists and initiate distinct signaling responses via activation of the same receptor. Mechanisms behind such biased agonism are largely unknown for RTKs, especially at the level of receptor–ligand complex structure. Using recent progress in understanding the structures of active RTK signaling units, we discuss selected mechanisms by which ligands couple receptor activation to distinct signaling outputs.     

Syndecan transmembrane domain modulates intracellular signaling by regulating the oligomeric status of the cytoplasmic domain

Cell surface receptors must specifically recognize an extracellular ligand and then trigger an appropriate response within the cell. Their general structure enables this, as it comprises an extracellular domain that can bind an extracellular ligand, a cytoplasmic domain that can transduce a signal inside the cell to produce an appropriate response, and a transmembrane domain that links the two and is responsible for accurately delivering specific information on a binding event from the extracellular domain to the cytoplasmic domain, to trigger the proper response. A vast body of research has focused on elucidating the specific mechanisms responsible for regulating extracellular binding events and the subsequent interactions of the cytoplasmic domain with intracellular signaling. In contrast, far less work has focused on examining how the transmembrane domain links these domains and delivers the necessary information. In this review, we propose the importance of the transmembrane domain as a signal regulator. We highlight the cell adhesion receptor, syndecan, as a special case, and propose that the transmembrane domain-mediated oligomerization of the syndecan cytoplasmic domain is a unique regulatory mechanism in syndecan signaling.     

The assembly of diverse immune receptors is focused on a polar membrane-embedded interaction site

The majority of receptors responsible for activation of distinct cell types within the immune system assemble with dimeric signaling modules through interaction of a basic transmembrane residue with a pair of acidic residues of the signaling dimer. Because assembly of other membrane proteins requires specific interactions along extended stretches of transmembrane helices, we examined how transmembrane sequences flanking the polar interaction site contribute to assembly for three receptors that associate with different signaling modules—the natural killer cell receptors KIR and NKG2D and the Fc receptor for IgA, FcαRI. The KIR and NKG2D receptors assembled with the DAP12 and DAP10 dimers, respectively, even when the entire KIR or NKG2D transmembrane domains were replaced by polyleucine sequences with a properly positioned basic residue. In contrast, a high degree of specificity for the basic side chain could be observed because the KIR–DAP12 and FcαRI–Fcγ interactions favored lysine or arginine, respectively. Steric hindrance among incompatible extra-membranous domains and competition for signaling modules also contributed to specificity of assembly. These results demonstrate that these interactions are focused on the polar site created by three ionizable transmembrane residues, and explain how the DAP12 and Fcγ signaling modules can assemble with large, non-overlapping sets of receptors that have highly divergent transmembrane sequences.     

Activation of transmembrane cell‐surface receptors via a common mechanism? The “rotation model”

It has long been thought that transmembrane cell‐surface receptors, such as receptor tyrosine kinases and cytokine receptors, among others, are activated by ligand binding through ligand‐induced dimerization of the receptors. However, there is growing evidence that prior to ligand binding, various transmembrane receptors have a preformed, yet inactive, dimeric structure on the cell surface. Various studies also demonstrate that during transmembrane signaling, ligand binding to the extracellular domain of receptor dimers induces a rotation of transmembrane domains, followed by rearrangement and/or activation of intracellular domains. The paper here describes transmembrane cell‐surface receptors that are known or proposed to exist in dimeric form prior to ligand binding, and discusses how these preformed dimers are activated by ligand binding.     

Molecular modeling of nearly full-length ErbB2 receptor

Members of the epidermal growth factor receptor family play important roles in various cellular processes, both in physiological and in pathological conditions. Dimerization and autophosphorylation of these receptor tyrosine kinases are key events of signal transduction. Details of the molecular events of the signaling are not entirely known. To facilitate the understanding of receptor structure and function at the molecular level, a molecular model was built for the nearly full-length ErbB2 dimer. Modeling was based on the x-ray or nuclear-magnetic resonance structures of extracellular, transmembrane, and intracellular domains. The extracellular domain was positioned above the cell membrane based on the distance determined from experimentally measured fluorescence resonance energy transfer. Favorable dimerization interactions are predicted for the extracellular, transmembrane, and protein kinase domains in the model of a nearly full-length dimer of ErbB2, which may act in a coordinated fashion in ErbB2 homodimerization, and also in heterodimers of ErbB2 with other members of the ErbB family.     

Evidence of positive selection at codon sites localized in extracellular domains of mammalian CC motif chemokine receptor proteins

Background  

CC chemokine receptor proteins (CCR1 through CCR10) are seven-transmembrane G-protein coupled receptors whose signaling pathways are known for their important roles coordinating immune system responses through targeted trafficking of white blood cells. In addition, some of these receptors have been identified as fusion proteins for viral pathogens: for example, HIV-1 strains utilize CCR5, CCR2 and CCR3 proteins to obtain cellular entry in humans. The extracellular domains of these receptor proteins are involved in ligand-binding specificity as well as pathogen recognition interactions.     

The Transmembrane Domains of TNF-Related Apoptosis-Inducing Ligand (TRAIL) Receptors 1 and 2 Co-Regulate Apoptotic Signaling Capacity

TNF-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor (TNF) ligand family that exerts its apoptotic activity in human cells by binding to two transmembrane receptors, TRAILR1 and TRAILR2. In cells co-expressing both receptors the particular contribution of either protein to the overall cellular response is not well defined. Here we have investigated whether differences in the signaling capacities of TRAILR1 and TRAILR2 can be attributed to certain functional molecular subdomains. We generated and characterized various chimeric receptors comprising TRAIL receptor domains fused with parts from other members of the TNF death receptor family. This allowed us to compare the contribution of particular domains of the two TRAIL receptors to the overall apoptotic response and to identify elements that regulate apoptotic signaling. Our results show that the TRAIL receptor death domains are weak apoptosis inducers compared to those of CD95/Fas, because TRAILR-derived constructs containing the CD95/Fas death domain possessed strongly enhanced apoptotic capabilities. Importantly, major differences in the signaling strengths of the two TRAIL receptors were linked to their transmembrane domains in combination with the adjacent extracellular stalk regions. This was evident from receptor chimeras comprising the extracellular part of TNFR1 and the intracellular signaling part of CD95/Fas. Both receptor chimeras showed comparable ligand binding affinities and internalization kinetics. However, the respective TRAILR2-derived molecule more efficiently induced apoptosis. It also activated caspase-8 and caspase-3 more strongly and more quickly, albeit being expressed at lower levels. These results suggest that the transmembrane domains together with their adjacent stalk regions can play a major role in control of death receptor activation thereby contributing to cell type specific differences in TRAILR1 and TRAILR2 signaling.     

Cutting edge: human FcRL4 and FcRL5 are receptors for IgA and IgG

Fc receptor-like (FcRL) proteins are a family of cellular receptors homologous to FcγRI and are predominantly expressed by B cells. They function to costimulate or inhibit BCR signaling through consensus ITAMs and ITIMs; however, the extracellular ligands of these receptors remain unknown or controversial. In this study, we tested the ability of human FcRL proteins to bind Igs and found FcRL4 and FcRL5 to be bona fide Fc receptors. In cellular binding assays, FcRL4 bound efficiently to IgA and FcRL5 binds all IgG isotypes with varied efficiency. Additionally, we generated mAbs capable of specifically blocking these interactions. Given their expression on activated B cells and potential for inhibitory signaling, FcRL4 and FcRL5 are likely to be important for immune complex-dependent human B cell regulation, and they represent novel therapeutic targets for receptor blockade therapies.     

Dynamic equilibrium between multiple active and inactive conformations explains regulation and oncogenic mutations in ErbB receptors

The ErbB growth factor receptor family members are key players in vital physiological and pathological processes. Like other receptor tyrosine kinases, the ErbBs are bi-topic membrane proteins, whose extracellular and intracellular domains are connected by single transmembrane span. In recent years the crystal structures of the extracellular and intracellular domains of some ErbBs have been determined. We integrated the available structural information with phylogenetic, biochemical, biophysical, genetic, and computational data into a suggested model for the regulation and activation of these receptors. According to the model, regulation is maintained by a dynamic equilibrium between monomeric and dimeric states in various conformations. Along this dynamic equilibrium, variations in the points of interactions within the dimers alter the activation state and ligand-binding affinities. The active state was recently shown to be associated with an asymmetric dimer of the kinase domains. That finding enabled us to elucidate, in molecular terms, the directionality observed in the activation process of ErbB heterodimers; it can explain, for example, the preferential activation of ErbB2 by ErbB1 over activation of ErbB1 by ErbB2. Sequence alterations that reverse this directionality lead to aberrant signaling and cancer. Our model also offers molecular interpretations of the effects of various oncogenic alterations that interfere with the regulatory mechanism.     

Syndecans,signaling, and cell adhesion

Syndecans are transmembrane proteoglycans which can participate in diverse cell surface interactions, involving extracellular matrix macromolecules, growth factors, protease inhibitors, and even viral entry. Currently, all extracellular interactions are believed to be mediated by distinct structures within the heparan sulfate chains, leaving the roles of chondroitin sulfate chains and extracellular portion of the core proteins to be elucidated. Evidence that syndecans are a class of receptor involved in cell adhesion is mounting, and their small cytoplasmic domains may link with the microfilament cytoskeleton, thereby mediating signaling events. The molecular details are unknown, but the conservation of regions of syndecan cytoplasmic domains, and a strong tendency for homotypic association, support the idea that the ligand-induced clustering may be a discrete source of specific transmembrane signaling from matrix to cytoskeleton, as proposed for other classes of adhesion receptors. © 1996 Wiley-Liss, Inc.     

Conserved disulfide bond is not essential for the adenosine A2A receptor: Extracellular cysteines influence receptor distribution within the cell and ligand-binding recognition

G protein-coupled receptors (GPCRs) are integral membrane proteins involved in cellular signaling and constitute major drug targets. Despite their importance, the relationship between structure and function of these receptors is not well understood. In this study, the role of extracellular disulfide bonds on the trafficking and ligand-binding activity of the human A_2A adenosine receptor was examined. To this end, cysteine-to-alanine mutations were conducted to replace individual and both cysteines in three disulfide bonds present in the first two extracellular loops. Although none of the disulfide bonds were essential for the formation of plasma membrane-localized active GPCR, loss of the disulfide bonds led to changes in the distribution of the receptor within the cell and changes in the ligand-binding affinity. These results indicate that in contrast to many class A GPCRs, the extracellular disulfide bonds of the A_2A receptor are not essential, but can modulate the ligand-binding activity, by either changing the conformation of the extracellular loops or perturbing the interactions of the transmembrane domains.