Receptor Assembly Research Articles

The Shaping of a B Cell Pool Maximally Responsive to Infections.

B cell subsets differ in development, tissue distribution, and mechanisms of activation. In response to infections, however, all can differentiate into extrafollicular plasmablasts that rapidly provide highly protective antibodies, indicating that these plasmablasts are the main humoral immune response effectors. Yet, the effectiveness of this response type depends on the presence of antigen-specific precursors in the circulating mature B cell pool, a pool that is generated initially through the stochastic processes of B cell receptor assembly. Importantly, germinal centers then mold the repertoire of this B cell pool to be increasingly responsive to pathogens by generating a broad array of antimicrobial memory B cells that act as highly effective precursors of extrafollicular plasmablasts. Such B cell repertoire molding occurs in two ways: continuously via the chronic germinal centers of mucosal lymphoid tissues, driven by the presence of the microbiome, and via de novo generated germinal centers following acute infections. For effectively evaluating humoral immunity as a correlate of immune protection, it might be critical to measure memory B cell pools in addition to antibody titers.

A multiscale study on the mechanisms of spatial organization in ligand-receptor interactions on cell surfaces

The binding of cell surface receptors with extracellular ligands triggers distinctive signaling pathways, leading into the corresponding phenotypic variation of cells. It has been found that in many systems, these ligand-receptor complexes can further oligomerize into higher-order structures. This ligand-induced oligomerization of receptors on cell surfaces plays an important role in regulating the functions of cell signaling. The underlying mechanism, however, is not well understood. One typical example is proteins that belong to the tumor necrosis factor receptor (TNFR) superfamily. Using a generic multiscale simulation platform that spans from atomic to subcellular levels, we compared the detailed physical process of ligand-receptor oligomerization for two specific members in the TNFR superfamily: the complex formed between ligand TNFα and receptor TNFR1 versus the complex formed between ligand TNFβ and receptor TNFR2. Interestingly, although these two systems share high similarity on the tertiary and quaternary structural levels, our results indicate that their oligomers are formed with very different dynamic properties and spatial patterns. We demonstrated that the changes of receptor’s conformational fluctuations due to the membrane confinements are closely related to such difference. Consistent to previous experiments, our simulations also showed that TNFR can preassemble into dimers prior to ligand binding, while the introduction of TNF ligands induced higher-order oligomerization due to a multivalent effect. This study, therefore, provides the molecular basis to TNFR oligomerization and reveals new insights to TNFR-mediated signal transduction. Moreover, our multiscale simulation framework serves as a prototype that paves the way to study higher-order assembly of cell surface receptors in many other bio-systems.

The cryo-EM structure of the endocytic receptor DEC-205

DEC-205 (CD205), a member of the macrophage mannose receptor protein family, is the prototypic endocytic receptor of dendritic cells, whose ligands include phosphorothioated cytosine–guanosine oligonucleotides, a motif often seen in bacterial or viral DNA. However, despite growing biological and clinical significance, little is known about the structural arrangement of this receptor or any of its family members. Here, we describe the 3.2 Å cryo-EM structure of human DEC-205, thereby illuminating the structure of the mannose receptor protein family. The DEC-205 monomer forms a compact structure comprising two intercalated rings of C-type lectin-like domains, where the N-terminal cysteine-rich and fibronectin domains reside at the central intersection. We establish a pH-dependent oligomerization pathway forming tetrameric DEC-205 using solution-based techniques and ultimately solved the 4.9 Å cryo-EM structure of the DEC-205 tetramer to identify the unfurling of the second lectin ring which enables tetramer formation. Furthermore, we suggest the relevance of this oligomerization pathway within a cellular setting, whereby cytosine–guanosine binding appeared to disrupt this cell-surface oligomer. Accordingly, we provide insight into the structure and oligomeric assembly of the DEC-205 receptor.

How the biomimetic assembly of membrane receptors into multivalent domains is regulated by a small ligand.

In living cells, communication requires the action of membrane receptors that are activated following very small environmental changes. A binary all-or-nothing behavior follows, making the organism extremely efficient at responding to specific stimuli. Using a minimal system composed of lipid vesicles, chemical models of a membrane receptor and their ligands, we show that bio-mimetic ON/OFF assembly of high avidity, multivalent domains is triggered by small temperature changes. Moreover, the intensity of the ON signal at the onset of the switch is modulated by the presence of small, weakly binding divalent ligands, reminiscent of the action of primary messengers in biological systems. Based on the analysis of spectroscopic data, we develop a mathematical model that rigorously describes the temperature-dependent switching of the membrane receptor assembly and ligand binding. From this we derive an equation that predicts the intensity of the modulation of the ON signal by the ligand-messenger as a function of the pairwise binding parameters, the number of binding sites that it features and the concentration. The behavior of our system, and the model derived, highlight the usefulness of weakly binding ligands in the regulation of membrane receptors and the pitfalls inherent to their binding promiscuity, such as non-specific binding to the membrane. Our model, and the equations derived from it, offer a valuable tool for the study of membrane receptors in both biological and biomimetic settings. The latter can be exploited to program membrane receptor avidity on sensing vesicles, create hierarchical protocell tissues or develop highly specific drug delivery vehicles.

M4, the Outermost Helix, is Extensively Involved in Opening of the α4β2 nACh Receptor.

Nicotinic acetylcholine receptors (nAChR) are the archetypal members of the pentameric ligand-gated ion channel (pLGIC) family, an important class of cell signaling proteins. In all members of this family, each of the five subunits has four transmembrane α-helices (M1-M4), with M2 lining the pore, then M1 and M3, and with M4 outermost and adjacent to the membrane lipids. Despite its remote location, M4 contributes both to receptor assembly and gating in pLGICs where it has been examined. This study probes the role of M4 residues in the α4β2 nAChR using site-directed mutagenesis to individually mutate each residue to alanine, followed by expression in HEK293 cells and then characterization using membrane potential sensitive dye and radioligand binding. Two of the resulting mutant receptors showed altered EC50s, while 13 were nonfunctional, although coexpression with the chaperones RIC3 and nAChO resulted in 4 of these responding to agonist. Of the remaining 9, radioligand binding with epibatidine showed that 8 were expressed, suggesting these residues may play a role in channel opening. These data differ from similar studies in other pLGIC, where few or no Ala mutants in M4 ablate function, and they suggest that the α4β2 nAChR M4 may play a more significant role than in related receptors.

Distinct Assemblies of Heterodimeric Cytokine Receptors Govern Stemness Programs in Leukemia

Leukemia stem cells (LSC) possess distinct self-renewal and arrested differentiation properties that are responsible for disease emergence, therapy failure and recurrence in acute myeloid leukemia (AML). Pleiotropic cytokines like interleukin (IL)-3 dimerize their receptors to signal multiple functional activities of AML cells, however, the mechanistic link between cytokine receptor dimerization and LSC fate regulation is unknown. Here, we show that the Class I heterodimeric IL3Rα/βc receptors (IL-3R) assemble as hexamers and dodecamers that segregate proliferation versus differentiation signals, respectively. Receptor stoichiometry varies across the individual cells that make up the AML hierarchy, with LSCs presenting with the highest IL3Rα/βc ratio thereby driving resultant hexamer-mediated stemness programs and poor patient survival. Our study establishes a new signaling mechanism of cytokine pleiotropy and the biased instruction of cell fate that directs the AML hierarchy; a signaling mechanism that may be generalizable to other normal and transformed cellular hierarchies.

Functional α6β4 acetylcholine receptor expression enables pharmacological testing of nicotinic agonists with analgesic properties.

The α6β4 nicotinic acetylcholine receptor (nAChR) is enriched in dorsal root ganglia neurons and is an attractive non-opioid therapeutic target for pain. However, difficulty expressing human α6β4 receptors in recombinant systems has precluded drug discovery. Here, genome-wide screening identified accessory proteins that enable reconstitution of human α6β4 nAChRs. BARP, an auxiliary subunit of voltage-dependent calcium channels, promoted α6β4 surface expression while IRE1α, an unfolded protein response sensor, enhanced α6β4 receptor assembly. Effects on α6β4 involve BARP's N-terminal region and IRE1α's splicing of XBP1 mRNA. Furthermore, clinical efficacy of nicotinic agents in relieving neuropathic pain best correlated with their activity on α6β4. Finally, BARP-knockout, but not NACHO-knockout mice lacked nicotine-induced antiallodynia, highlighting the functional importance of α6β4 in pain. These results identify roles for IRE1α and BARP in neurotransmitter receptor assembly and unlock drug discovery for the previously elusive α6β4 receptor.

Transcription Factor RBPJ as a Molecular Switch in Regulating the Notch Response.

The Notch signal transduction cascade requires cell-to-cell contact and results in the proteolytic processing of the Notch receptor and subsequent assembly of a transcriptional coactivator complex containing the Notch intracellular domain (NICD) and transcription factor RBPJ. In the absence of a Notch signal, RBPJ remains at Notch target genes and dampens transcriptional output. Like in other signaling pathways, RBPJ is able to switch from activation to repression by associating with corepressor complexes containing several chromatin-modifying enzymes. Here, we focus on the recent advances concerning RBPJ-corepressor functions, especially in regard to chromatin regulation. We put this into the context of one of the best-studied model systems for Notch, blood cell development. Alterations in the RBPJ-corepressor functions can contribute to the development of leukemia, especially in the case of acute myeloid leukemia (AML). The versatile role of transcription factor RBPJ in regulating pivotal target genes like c-MYC and HES1 may contribute to the better understanding of the development of leukemia.

IL-12 and IL-23-Close Relatives with Structural Homologies but Distinct Immunological Functions.

Cytokines of the IL-12 family show structural similarities but have distinct functions in the immune system. Prominent members of this cytokine family are the pro-inflammatory cytokines IL-12 and IL-23. These two cytokines share cytokine subunits and receptor chains but have different functions in autoimmune diseases, cancer and infections. Accordingly, structural knowledge about receptor complex formation is essential for the development of new therapeutic strategies preventing and/or inhibiting cytokine:receptor interaction. In addition, intracellular signaling cascades can be targeted to inhibit cytokine-mediated effects. Single nucleotide polymorphisms can lead to alteration in the amino acid sequence and thereby influencing protein functions or protein–protein interactions. To understand the biology of IL-12 and IL-23 and to establish efficient targeting strategies structural knowledge about cytokines and respective receptors is crucial. A highly efficient therapy might be a combination of different drugs targeting extracellular cytokine:receptor assembly and intracellular signaling pathways.

Inhibition of the Activity of Cyclophilin A Impedes Prolactin Receptor-Mediated Signaling, Mammary Tumorigenesis, and Metastases

SummaryProlactin (PRL) and its receptor (PRLr) play important roles in the pathogenesis of breast cancer. Cyclophilin A (CypA) is a cis-trans peptidyl-prolyl isomerase (PPI) that is constitutively associated with the PRLr and facilitates the activation of the tyrosine kinase Jak2. Treatment with the non-immunosuppressive prolyl isomerase inhibitor NIM811 or CypA short hairpin RNA inhibited PRL-stimulated signaling, breast cancer cell growth, and migration. Transcriptomic analysis revealed that NIM811 inhibited two-thirds of the top 50 PRL-induced genes and a reduction in gene pathways associated with cancer cell signaling. In vivo treatment of NIM811 in a TNBC xenograft lessened primary tumor growth and induced central tumor necrosis. Deletion of CypA in the MMTV-PyMT mouse model demonstrated inhibition of tumorigenesis with significant reduction in lung and lymph node metastasis. The regulation of PRLr/Jak2-mediated biology by NIM811 demonstrates that a non-immunosuppressive prolyl isomerase inhibitor can function as a potential breast cancer therapeutic.

Heterologous expression of concatenated nicotinic ACh receptors: Pros and cons of subunit concatenation and recommendations for construct designs.

Concatenation of Cys-loop receptor subunits is a commonly used technique to ensure experimental control of receptor assembly. However, we recently demonstrated that widely used constructs did not lead to the expression of uniform pools of ternary and more complex receptors. The aim was therefore to identify viable strategies for designing concatenated constructs that would allow strict control of resultant receptor pools. Concatenated dimeric, tetrameric, and pentameric α4β2-containing nicotinic ACh (nACh) receptor constructs were designed with successively shorter linker lengths and expressed in Xenopus laevis oocytes. Resulting receptor stoichiometries were investigated by functional analysis in two-electrode voltage-clamp experiments. Molecular dynamics simulations were performed to investigate potential effects of linkers on the 3D structure of concatemers. Dimeric constructs were found to be unreliable and should be avoided for expression of ternary receptors. By introducing two short linkers, we obtained efficient expression of uniform receptor pools with tetrameric and pentameric constructs. However, linkers should not be excessively short as that introduces strain on the 3D structure of concatemers. The data demonstrate that design of concatenated Cys-loop receptors requires a compromise between the desire for control of assembly and avoiding introduction of strain on the resulting protein. The overall best strategy was found to be pentameric constructs with carefully optimised linker lengths. Our findings will advance studies of ternary or more complex Cys-loop receptors as well as enabling detailed analysis of how pharmacological agents interact with stoichiometry-specific binding sites.

NACHO Engages N-Glycosylation ER Chaperone Pathways for α7 Nicotinic Receptor Assembly

The α7 nicotinic acetylcholine receptor participates in diverse aspects of brain physiology and disease. Neurons tightly control α7 assembly, which relies upon NACHO, an endoplasmic reticulum (ER)-localized integral membrane protein. By constructing α7 chimeras and mutants, we find that NACHO requires the α7 ectodomain to promote receptor assembly and surface trafficking. Also critical are two amino acids in the α7 second transmembrane domain. NACHO-mediated assembly is independent and separable from that induced by cholinergic ligands or RIC-3 protein, the latter of which acts on the large α7 intracellular loop. Proteomics indicates that NACHO associates with the ER oligosaccharyltransferase machinery and with calnexin. Accordingly, NACHO-mediated effects on α7 assembly and channel function require N-glycosylation and calnexin chaperone activity. These studies identify ER pathways that mediate α7 assembly by NACHO and provide insights into novel pharmacological strategies for these crucial nicotinic receptors.

Synergy among Exocyst and SNARE Interactions Identifies a Functional Hierarchy in Secretion during Vegetative Growth.

Vesicle exocytosis underpins signaling and development in plants and is vital for cell expansion. Vesicle tethering and fusion are thought to occur sequentially, with tethering mediated by the exocyst and fusion driven by assembly of soluble NSF attachment protein receptor (SNARE) proteins from the vesicle membrane (R-SNAREs or vesicle-associated membrane proteins [VAMPs]) and the target membrane (Q-SNAREs). Interactions between exocyst and SNARE protein complexes are known, but their functional consequences remain largely unexplored. We now identify a hierarchy of interactions leading to secretion in Arabidopsis (Arabidopsis thaliana). Mating-based split-ubiquitin screens and in vivo Förster resonance energy transfer analyses showed that exocyst EXO70 subunits bind preferentially to cognate plasma membrane SNAREs, notably SYP121 and VAMP721. The exo70A1 mutant affected SNARE distribution and suppressed vesicle traffic similarly to the dominant-negative truncated protein SYP121ΔC, which blocks secretion at the plasma membrane. These phenotypes are consistent with the epistasis of exo70A1 in the exo70A1 syp121 double mutant, which shows decreased growth similar to exo70A1 single mutants. However, the exo70A1 vamp721 mutant showed a strong, synergy, suppressing growth and cell expansion beyond the phenotypic sum of the two single mutants. These data are best explained by a hierarchy of SNARE recruitment to the exocyst at the plasma membrane, dominated by the R-SNARE and plausibly with the VAMP721 longin domain as a nexus for binding.

Modulation of Signaling Mediated by TSLP and IL-7 in Inflammation, Autoimmune Diseases, and Cancer.

Thymic Stromal Lymphopoietin (TSLP) and Interleukin-7 (IL-7) are widely studied cytokines within distinct branches of immunology. On one hand, TSLP is crucially important for mediating type 2 immunity at barrier surfaces and has been linked to widespread allergic and inflammatory diseases of the airways, skin, and gut. On the other hand, IL-7 operates at the foundations of T-cell and innate lymphoid cell (ILC) development and homeostasis and has been associated with cancer. Yet, TSLP and IL-7 are united by key commonalities in their structure and the structural basis of the receptor assemblies they mediate to initiate cellular signaling, in particular their cross-utilization of IL-7Rα. As therapeutic targeting of TSLP and IL-7 via diverse approaches is reaching advanced stages and in light of the plethora of mechanistic and structural data on receptor signaling mediated by the two cytokines, the time is ripe to provide integrated views of such knowledge. Here, we first discuss the major pathophysiological roles of TSLP and IL-7 in autoimmune diseases, inflammation and cancer. Subsequently, we curate structural and mechanistic knowledge about receptor assemblies mediated by the two cytokines. Finally, we review therapeutic avenues targeting TSLP and IL-7 signaling. We envision that such integrated view of the mechanism, structure, and modulation of signaling assemblies mediated by TSLP and IL-7 will enhance and fine-tune the development of more effective and selective approaches to further interrogate the role of TSLP and IL-7 in physiology and disease.

Participation of Ca2+-Permeable AMPA Receptors in Synaptic Plasticity

AMPA receptors are the key molecules of excitatory and inhibitory synapses and are involved in synaptic plasticity. Cognitive functions of the brain such as signal perception, processing and analysis of information, memory, storage and exchange of information are reduced when the processes controlling the assembly of AMPA receptors, membrane trafficking and synapse-specific expression are impaired. The content of the receptors in synapses is regulated by exocytosis, endocytosis, and receptor recycling. Auxiliary subunits and partners modulate the function of AMPA receptors. Ca2+-permeable AMPA receptors (CP-AMPAR) not containing the GluA2 subunit are involved in multiple forms of the synaptic plasticity, including long-term potentiation and depression, and play an important role in maintaining the correct balance between excitation and inhibition in the brain. The activation of CP-AMPAR in neurons provides a fast postsynaptic Ca2+ entry, which triggers the processes modifying synaptic functions through the interaction with other Ca2+-transporting systems. The purpose of this review is to draw the attention of researchers to recent advances in the participation of CP-AMPA receptors in synaptic plasticity.

From Orai to E-Cadherin: Subversion of Calcium Trafficking in Cancer to Drive Proliferation, Anoikis-Resistance, and Metastasis.

The common currency of epithelial differentiation and homeostasis is calcium, stored primarily in the endoplasmic reticulum, rationed according to need, and replenished from the extracellular milieu via store-operated calcium entry (SOCE). This currency is disbursed by the IP3 receptor in response to diverse extracellular signals. The rate of release is governed by regulators of proliferation, autophagy, survival, and programmed cell death, the strength of the signal leading to different outcomes. Intracellular calcium acts chiefly through intermediates such as calmodulin that regulates growth factor receptors such as epidermal growth factor receptor (EGFR), actin polymerization, and adherens junction assembly and maintenance. Here we review this machinery and its role in differentiation, then consider how cancer cells subvert it to license proliferation, resist anoikis, and enable metastasis, either by modulating the level of intracellular calcium or its downstream targets or effectors such as EGFR, E-cadherin, IQGAP1, TMEM16A, CLCA2, and TRPA1. Implications are considered for the roles of E-cadherin and growth factor receptors in circulating tumor cells and metastasis. The discovery of novel, cell type-specific modulators and effectors of calcium signaling offers new possibilities for cancer chemotherapy.

Flexibility of intrinsically disordered degrons in AUX/IAA proteins reinforces auxin co-receptor assemblies

Cullin RING-type E3 ubiquitin ligases SCFTIR1/AFB1-5 and their AUX/IAA targets perceive the phytohormone auxin. The F-box protein TIR1 binds a surface-exposed degron in AUX/IAAs promoting their ubiquitylation and rapid auxin-regulated proteasomal degradation. Here, by adopting biochemical, structural proteomics and in vivo approaches we unveil how flexibility in AUX/IAAs and regions in TIR1 affect their conformational ensemble allowing surface accessibility of degrons. We resolve TIR1·auxin·IAA7 and TIR1·auxin·IAA12 complex topology, and show that flexible intrinsically disordered regions (IDRs) in the degron’s vicinity, cooperatively position AUX/IAAs on TIR1. We identify essential residues at the TIR1 N- and C-termini, which provide non-native interaction interfaces with IDRs and the folded PB1 domain of AUX/IAAs. We thereby establish a role for IDRs in modulating auxin receptor assemblies. By securing AUX/IAAs on two opposite surfaces of TIR1, IDR diversity supports locally tailored positioning for targeted ubiquitylation, and might provide conformational flexibility for a multiplicity of functional states.

The M4 Helix Is Involved in α7 nACh Receptor Function.

Nicotinic acetylcholine receptors (nAChR) are the archetypal members of the pentameric ligand-gated ion channel (pLGIC) family, an important class of cell signaling proteins. In all members of this family, each of the five subunits has four transmembrane α-helices (M1-M4) with M2 lining the pore and then M1 and M3, with M4 outermost and adjacent to the membrane lipids. M4 has a variety of roles: its interaction with neighboring M1 and M3 helices is important for receptor assembly, it can a transmit information on the lipid content of the membrane to the gating mechanism, and it may form a vital link to the extracellular domain via the Cys-loop. This study examines the role of M4 receptor residues in the α7 nAChR using site-directed mutagenesis and subsequent expression in Xenopus oocytes. The data indicate that many of the residues in M4 play a role in receptor function, as substitution with Ala can modify functional parameters; 11 of 24 mutants showed a small gain of function (<10-fold decrease in EC50), and 1 (D446A) did not respond to the agonist; it was also not expressed at the cell surface. Removal or addition of aromatic residues had small or no effects. These results demonstrate the α7 nAChR M4 has a role in receptor function, and a structural model suggests possible interactions of some of these residues with their neighbors.

Immunohistochemical distribution of 10 GABAA receptor subunits in the forebrain of the rhesus monkey Macaca mulatta.

GABAA receptors are composed of five subunits arranged around a central chloride channel. Their subunits originate from different genes or gene families. The majority of GABAA receptors in the mammalian brain consist of two α‐, two β‐ and one γ‐ or δ‐subunit. This subunit organization crucially determines the physiological and pharmacological properties of the GABAA receptors. Using immunohistochemistry, we investigated the distribution of 10 GABAA receptor subunits (α1, α2, α3, α4, α5, β1, β2, β3, γ2, and δ) in the fore brain of three female rhesus monkeys (Macaca mulatta). Within the cerebral cortex, subunits α1, α5, β2, β3, and γ2 were found in all layers, α2, α3, and β1 were more concentrated in the inner and outer layers. The caudate/putamen was rich in α1, α2, α5, all three β‐subunits, γ2, and δ. Subunits α3 and α5 were more concentrated in the caudate than in the putamen. In contrast, α1, α2, β1, β2, γ2, and δ were highest in the pallidum. Most dorsal thalamic nuclei contained subunits α1, α2, α4, β2, β3, and γ2, whereas α1, α3, β1, and γ2 were most abundant in the reticular nucleus. Within the amygdala, subunits α1, α2, α5, β1, β3, γ2, and δ were concentrated in the cortical nucleus, whereas in the lateral and basolateral amygdala α1, α2, α5, β1, β3, and δ, and in the central amygdala α1, α2, β3, and γ2 were most abundant. Interestingly, subunit α3‐IR outlined the intercalated nuclei of the amygdala. In the hippocampus, subunits α1, α2, α5, β2, β3, γ2, and δ were highly expressed in the dentate molecular layer, whereas α1, α2, α3, α5, β1, β2, β3, and γ2 were concentrated in sector CA1 and the subiculum. The distribution of GABAA receptor subunits in the rhesus monkey was highly heterogeneous indicating a high number of differently assembled receptors. In most areas investigated, notably in the striatum/pallidum, amygdaloid nuclei and in the hippocampus it was more diverse than in the rat and mouse indicating a more heterogeneous and less defined receptor assembly in the monkey than in rodent brain.

Molecular T-Cell Repertoire Analysis as Source of Prognostic and Predictive Biomarkers for Checkpoint Blockade Immunotherapy.

The T cells are key players of the response to checkpoint blockade immunotherapy (CBI) and monitoring the strength and specificity of antitumor T-cell reactivity remains a crucial but elusive component of precision immunotherapy. The entire assembly of T-cell receptor (TCR) sequences accounts for antigen specificity and strength of the T-cell immune response. The TCR repertoire hence represents a “footprint” of the conditions faced by T cells that dynamically evolves according to the challenges that arise for the immune system, such as tumor neo-antigenic load. Hence, TCR repertoire analysis is becoming increasingly important to comprehensively understand the nature of a successful antitumor T-cell response, and to improve the success and safety of current CBI.