Types Of Cell Surface Receptors Research Articles

Spatial Organization of Dectin-1 and TLR2 during Synergistic Crosstalk Revealed by Super-resolution Imaging.

Innate immune cells recognize and elicit responses against pathogens by integrating signals from different types of cell-surface receptors. How the receptors interact in the membrane to enable their signaling crosstalk is poorly understood. Here, we reveal the nanoscale organization of TLR2 and Dectin-1, a receptor pair known to cooperate in regulating antifungal immunity, through their synergistic signaling crosstalk at macrophage cell membranes. Using super-resolution single-molecule localization microscopy, we show that discrete noncolocalized nanoclusters of Dectin-1 and TLR2 are partially overlapped during their synergistic crosstalk. Compared to when one type of receptor is activated alone, the simultaneous activation of Dectin-1 and TLR2 leads to a higher percentage of both receptors being activated by their specific ligands and consequently an increased level of tyrosine phosphorylation. Our results depict, in nanoscale detail, how Dectin-1 and TLR2 achieve synergistic signaling through the spatial organization of their receptor nanoclusters.

Thoracic positron emission tomography: 18F-fluorodeoxyglucose and beyond.

Ongoing technologic and therapeutic advancements in medicine are now testing the limits of conventional anatomic imaging techniques. The ability to image physiology, rather than simply anatomy, is critical in the management of multiple disease processes, especially in oncology. Nuclear medicine has assumed a leading role in detecting, diagnosing, staging and assessing treatment response of various pathologic entities, and appears well positioned to do so into the future. When combined with computed tomography (CT) or magnetic resonance imaging (MRI), positron emission tomography (PET) has become the sine quo non technique of evaluating most solid tumors especially in the thorax. PET/CT serves as a key imaging modality in the initial evaluation of pulmonary nodules, often obviating the need for more invasive testing. PET/CT is essential to staging and restaging in bronchogenic carcinoma and offers key physiologic information with regard to treatment response. A more recent development, PET/MRI, shows promise in several specific lung cancer applications as well. Additional recent advancements in the field have allowed PET to expand beyond imaging with 18F-flurodeoxyglucose (FDG) alone, now with the ability to specifically image certain types of cell surface receptors. In the thorax this predominantly includes 68Ga-DOTATATE which targets the somatostatin receptors abundantly expressed in neuroendocrine tumors, including bronchial carcinoid. This receptor targeted imaging technique permits targeting these tumors with therapeutic analogues such as 177Lu labeled DOTATATE. Overall, the proper utilization of PET in the thorax has the ability to directly impact and improve patient care.

Two to Tango: Co-evolution of Hominid Natural Killer Cell Receptors and MHC.

Natural killer (NK) cells have diverse roles in hominid immunity and reproduction. Modulating these functions are the interactions between major histocompatibility complex (MHC) class I molecules that are ligands for two NK cell surface receptor types. Diverse killer cell immunoglobulin-like receptors (KIR) bind specific motifs encoded within the polymorphic MHC class I cell surface glycoproteins, while, in more conserved interactions, CD94:NKG2A receptors recognize MHC-E with bound peptides derived from MHC class I leader sequences. The hominid lineage presents a choreographed co-evolution of KIR with their MHC class I ligands. MHC-A, -B, and -C are present in all great apes with species-specific haplotypic variation in gene content. The Bw4 epitope recognized by lineage II KIR is restricted to MHC-B but also present on some gorilla and human MHC-A. Common to great apes, but rare in humans, are MHC-B possessing a C1 epitope recognized by lineage III KIR. MHC-C arose from duplication of MHC-B and is fixed in all great apes except orangutan, where it exists on approximately 50% of haplotypes and all allotypes are C1-bearing. Recent study showed that gorillas possess yet another intermediate MHC organization compared to humans. Like orangutans, but unlike the Pan-Homo species, duplication of MHC-B occurred. However, MHC-C is fixed, and the MHC-C C2 epitope (absent in orangutans) emerges. The evolution of MHC-C drove expansion of its cognate lineage III KIR. Recently, position −21 of the MHC-B leader sequence has been shown to be critical in determining NK cell educational outcome. In humans, methionine (−21M) results in CD94:NKG2A-focused education whereas threonine (−21T) produces KIR-focused education. This is another dynamic position among hominids. Orangutans have exclusively −21M, consistent with their intermediate stage in lineage III KIR-focused evolution. Gorillas have both −21M and −21T, like humans, but they are unequally encoded by their duplicated B genes. Chimpanzees have near-fixed −21T, indicative of KIR-focused NK education. Harmonious with this observation, chimpanzee KIR exhibit strong binding and, compared to humans, smaller differences between binding levels of activating and inhibitory KIR. Consistent between these MHC-NK cell receptor systems over the course of hominid evolution is the evolution of polymorphism favoring the more novel and dynamic KIR system.

Serotonin drives the acquisition of a profibrotic and anti-inflammatory gene profile through the 5-HT7R-PKA signaling axis

Peripheral serotonin (5-hydroxytryptamine, 5-HT) regulates cell growth and differentiation in numerous cell types through engagement of seven types of cell surface receptors (HTR1–7). Deregulated 5-HT/HTR levels contribute to pathology in chronic inflammatory diseases, with macrophages being relevant targets for the physio-pathological effects of 5-HT. In fact, 5-HT skews human macrophage polarization through engagement of 5-HT2BR and 5-HT7R receptors. We now report that 5-HT primes macrophages for reduced pro-inflammatory cytokine production and IFN type I-mediated signaling, and promotes an anti-inflammatory and pro-fibrotic gene signature in human macrophages. The acquisition of the 5-HT-dependent gene profile primarily depends on the 5-HT7R receptor and 5-HT7R-initiated PKA-dependent signaling. In line with the transcriptional results, 5-HT upregulates TGFβ1 production by human macrophages in an HTR7- and PKA-dependent manner, whereas the absence of Htr7 in vivo results in diminished macrophage infiltration and collagen deposition in a mouse model of skin fibrosis. Our results indicate that the anti-inflammatory and pro-fibrotic activity of 5-HT is primarily mediated through the 5-HT7R-PKA axis, and that 5-HT7R contributes to pathology in fibrotic diseases.

A central role for R7bp in the regulation of itch sensation.

Itch is a protective sensation producing a desire to scratch. Pathologic itch can be a chronic symptom of illnesses such as uremia, cholestatic liver disease, neuropathies and dermatitis, however current therapeutic options are limited. Many types of cell surface receptors, including those present on cells in the skin, on sensory neurons and on neurons in the spinal cord, have been implicated in itch signaling. The role of G protein signaling in the regulation of pruriception is poorly understood. We identify here 2 G protein signaling components whose mutation impairs itch sensation. R7bp (a.k.a. Rgs7bp) is a palmitoylated membrane anchoring protein expressed in neurons that facilitates Gαi/o -directed GTPase activating protein activity mediated by the Gβ5/R7-RGS complex. Knockout of R7bp diminishes scratching responses to multiple cutaneously applied and intrathecally-administered pruritogens in mice. Knock-in to mice of a GTPase activating protein-insensitive mutant of Gαo (Gnao1 G184S/+) produces a similar pruriceptive phenotype. The pruriceptive defect in R7bp knockout mice was rescued in double knockout mice also lacking Oprk1, encoding the G protein-coupled kappa-opioid receptor whose activation is known to inhibit itch sensation. In a model of atopic dermatitis (eczema), R7bp knockout mice showed diminished scratching behavior and enhanced sensitivity to kappa opioid agonists. Taken together, our results indicate that R7bp is a key regulator of itch sensation and suggest the potential targeting of R7bp-dependent GTPase activating protein activity as a novel therapeutic strategy for pathological itch.

Sprouty2 regulates proliferation and survival of multiple myeloma byinhibiting activation of the ERK1/2 pathway invitro and invivo.

Multiple myeloma (MM) is an incurable disease, and its pathogenesis remains unclear. MicroRNA (miR)-21 was detected at a high level in MM and plays a key role in the pathogenesis of MM. However, Sprouty2 (spry2), a downstream target of miR-21, has low expression, and its mechanism in MM is unknown. We investigated whether spry2 could exert an antimyeloma effect and further studied the potential pathogenesis and progression of MM. To address the functional consequences of spry2, we assessed the expression levels of spry2 in several myeloma cell lines and detected low expression levels in MMcells. Overexpression of spry2 suppressed growth and colony formation ability and decreased the phosphorylation of extracellular signal-regulated kinases 1 and 2. Spry2 also decreased secretion of vascular endothelial growth factor and partially enhanced the sensitivity of MMcells to an inhibitor of mitogen-activated protein kinases 1 and 2. Additionally, spry2 inhibited the tumorigenesis and angiogenesis of MMcells invivo. In summary, we report for the first time that spry2 can inhibit MMcell growth and survival with a concomitant reduction in phosphorylation of extracellular signal-regulated kinases 1 and 2 invitro and invivo.

ERK and β-Arrestin Interaction: A Converging Point of Signaling Pathways for Multiple Types of Cell Surface Receptors

β-Arrestin, a signal adaptor protein, mediates intracellular signal transductions through protein-protein interactions by bringing two or more proteins in proximity. Extracellular signal-regulated kinase (ERK), a protein kinase in the family of mitogen-activated protein kinases (MAPKs), is involved in various receptor signal pathways. Interaction of ERK with β-arrestin or formation of ERK/β-arrestin signal complex occurs in response to activation of a variety of cell surface receptors. The ERK/β-arrestin signal complex may be a common transducer to converge a variety of extracellular stimuli to similar downstream intracellular signaling pathways. By using a cell-based protein-protein interaction LinkLight assay technology, we demonstrate a direct interaction between ERK and β-arrestin in response to extracellular stimuli, which can be sensitively and quantitatively monitored. Activations of G protein–coupled receptors (GPCRs), receptor tyrosine kinases (RTKs), and cytokine receptors promote formation of the ERK/β-arrestin signal complex. Our data indicate that the ERK/β-arrestin signal complex is a common transducer that participates in a variety of receptor signaling pathways. Furthermore, we demonstrate that receptor antagonists or kinase inhibitors can block the agonist-induced ERK and β-arrestin interaction. Thus, the ERK/β-arrestin interaction assay is useful for screening of new receptor modulators.

Biochemical fractionation of membrane receptors in the nucleus.

Fractionation of cytoplasmic and nuclear proteins is a well-recognized biochemical technique to detect the intracellular distribution and expression level of proteins of interest. In the last decade, accumulating evidence shows that various types of cell surface receptors, such as receptor tyrosine kinases (RTKs), peptide hormone receptors, and cytokine receptors, are detected in the nuclei. Therefore, subcellular fractionation, including nonnuclear/nuclear extraction and the subsequent subnuclear fractionation without detectable cross-contamination during the process, is critical for studying membrane receptors that transit from the cell surface to the nucleus. Here, we utilize the epidermal growth factor receptor (EGFR) tyrosine kinase as an example of a comprehensive biochemical protocol for isolating membrane receptors in the nuclei of cancer cells.

Computational Modeling Predicts Phosphatase Oxidation as an Important Axis of Redox Regulation in IL-4 Signaling

Reactive oxygen species (ROS) are produced following activation of several types of cell surface receptors and can play an important role in modulating cell signaling. How simultaneous oxidative modifications of multiple proteins, sometimes with potentially opposite effects, regulate cell signaling is not well understood at the system level. We are using computational modeling based on quantitative experimental measurements to develop a systemic understanding of redox regulation of cell signaling in the context of the IL-4 signaling pathway. We observe that IL-4 signaling in Jurkat cells is accompanied by transient ROS production, and ROS augment signaling activity as measured by STAT6 phosphorylation. A number of candidate redox-regulated mechanisms exist in the IL-4 pathway that could contribute to the observed outcomes; however, it is technically challenging to directly measure redox modifications of the possibly redox-regulated proteins. To circumvent this issue, we have developed kinetic models of IL-4 signaling that incorporate competing hypotheses regarding redox regulatory mechanisms. With the guidance of measurable experimental data we are using innovative model selection strategies to determine the best candidate models. We have also acquired time course data for processes not directly related to redox regulation, such as transcriptional negative feedback regulation and proteasomal degradation as mechanisms for downregulating IL-4 signaling, that aid model selection and validation. Our results so far indicate that reversible oxidative inhibition of phosphatases and compartmentation of phosphatase activity between subcellular compartments may be the primary redox regulatory mechanisms in IL-4 signaling. These studies will help evolve an understanding of how oxidative modifications of different components of the signaling pathway operate in parallel along with better studied post-translational modification mechanisms of protein regulation to determine the overall dynamics of cell signaling.

Vitamin A Transport and the Transmembrane Pore in the Cell-Surface Receptor for Plasma Retinol Binding Protein

Vitamin A and its derivatives (retinoids) play diverse and crucial functions from embryogenesis to adulthood and are used as therapeutic agents in human medicine for eye and skin diseases, infections and cancer. Plasma retinol binding protein (RBP) is the principal and specific vitamin A carrier in the blood and binds vitamin A at 1∶1 ratio. STRA6 is the high-affinity membrane receptor for RBP and mediates cellular vitamin A uptake. STRA6 null mice have severely depleted vitamin A reserves for vision and consequently have vision loss, even under vitamin A sufficient conditions. STRA6 null humans have a wide range of severe pathological phenotypes in many organs including the eye, brain, heart and lung. Known membrane transport mechanisms involve transmembrane pores that regulate the transport of the substrate (e.g., the gating of ion channels). STRA6 represents a new type of membrane receptor. How this receptor interacts with its transport substrate vitamin A and the functions of its nine transmembrane domains are still completely unknown. These questions are critical to understanding the molecular basis of STRA6′s activities and its regulation. We employ acute chemical modification to introduce chemical side chains to STRA6 in a site-specific manner. We found that modifications with specific chemicals at specific positions in or near the transmembrane domains of this receptor can almost completely suppress its vitamin A transport activity. These experiments provide the first evidence for the existence of a transmembrane pore, analogous to the pore of ion channels, for this new type of cell-surface receptor.

Structure‐function Aspects of Extracellular Leucine‐rich Repeat‐containing Cell Surface Receptors in Plants

Plants exploit several types of cell surface receptors for perception of extracellular signals, of which the extracellular leucine-rich repeat (eLRR)-containing receptors form the major class. Although the function of most plant eLRR receptors remains unclear, an increasing number of these receptors are shown to play roles in innate immunity and a wide variety of developmental processes. Recent efforts using domain swaps, gene shuffling analyses, site-directed mutagenesis, interaction studies, and crystallographic analyses resulted in the current knowledge on ligand binding and the mechanism of activation of plant eLRR receptors. This review provides an overview of eLRR receptor research, specifically summarizing the recent understanding of interactions among plant eLRR receptors, their co-receptors and corresponding ligands. The functions of distinct eLRR receptor domains, and their role in structure, ligand perception and multimeric complex formation are discussed. [Figure: see text] Bart P.H.J. Thomma (Corresponding author).

A mechanism for different receptors coupled to the same G protein to generate different responses mediated by different second messengers

To respond to changes in the extracellular environment and maintain effective intercellular communication, a signal transduction system of astonishing complexity has evolved in eukaryotic cells. A cell can express a menagerie of literally thousands of different types of cell surface receptors, with

A mechanism for different receptors coupled to the same G protein to generate different responses mediated by different second messengers

To respond to changes in the extracellular environment and maintain effective intercellular communication, a signal transduction system of astonishing complexity has evolved in eukaryotic cells. A cell can express a menagerie of literally thousands of different types of cell surface receptors, with

Transcriptional Regulation of the Protocadherin β Cluster during Her-2 Protein-induced Mammary Tumorigenesis Results from Altered N-Glycan Branching

Changes in the levels of N-acetylglucosaminyltransferase V (GnT-V) can alter the function of several types of cell surface receptors and adhesion molecules by causing altered N-linked glycan branching. Using a her-2 mammary tumor mouse model, her-2 receptor signaling was down-regulated by GnT-V knock-out, resulting in a significant delay in the onset of her-2-induced mammary tumors. To identify the genes that contributed to this GnT-V regulation of early events in tumorigenesis, microarray analysis was performed using her-2 induced mammary tumors from wild-type and GnT-V-null mice. We found that 142 genes were aberrantly expressed (>2.0-fold) with 64 genes up-regulated and 78 genes down-regulated after deletion of GnT-V. Among differentially expressed genes, the expression of a subgroup of the cadherin superfamily, the protocadherin β (Pcdhβ) cluster, was up-regulated in GnT-V-null tumors. Altered expression of the Pcdhβ cluster in GnT-V-null tumors was not due to changes in promoter methylation; instead, impaired her-2-mediated signaling pathways were implicated at least in part resulting from reduced microRNA-21 expression. Overexpression of Pcdhβ genes inhibited tumor cell growth, decreased the proportion of tumor-initiating cells, and decreased tumor formation in vivo, demonstrating that expression of the Pcdhβ gene cluster can serve as an inhibitor of the transformed phenotype. Our results suggest the up-regulation of the Pcdhβ gene cluster as a mechanism for reduced her-2-mediated tumorigenesis resulting from GnT-V deletion.

Receptor-Mediated Cellular Uptake Mechanism That Couples to Intracellular Storage

Cells are known to take up molecules through membrane transport mechanisms such as active transport, channels, and facilitated transport. We report here a new membrane transport mechanism that employs neither cellular energy like active transport nor a preexisting electrochemical gradient of the free substrate like channels or facilitated transport. Through this mechanism, cells take up vitamin A bound with high affinity to retinol binding protein (RBP) in the blood. This mechanism is mediated by the RBP receptor STRA6, which defines a new type of cell-surface receptor. STRA6 is essential for the proper functioning of multiple human organs, but the mechanisms that enable and control its cellular vitamin A uptake activity are unknown. We found that STRA6-mediated vitamin A uptake is tightly coupled to specific intracellular retinoid storage proteins, but no single intracellular protein is absolutely required for its transport activity. By developing sensitive real-time monitoring techniques, we found that STRA6 is not only a membrane receptor but also catalyzes vitamin A release from RBP. However, vitamin A released from RBP by STRA6 inhibits further vitamin A release by STRA6 unless specific intracellular retinoid storage proteins relieve this inhibition. This mechanism is responsible for its coupling to intracellular storage proteins. The coupling of uptake to storage provides high specificity in cellular uptake of vitamin A and prevents the excessive accumulation of free vitamin A. We have also identified a robust small-molecule-based technique to specifically stimulate cellular vitamin A uptake. This technique has implications in treating human diseases.

Plasmin Is Essential in Preventing Periodontitis in Mice

Periodontitis involves bacterial infection, inflammation of the periodontium, degradation of gum tissue, and alveolar bone resorption, which eventually leads to loss of teeth. To study the role of the broad-spectrum protease plasmin in periodontitis, we examined the oral health of plasminogen (Plg)-deficient mice. In wild-type mice, the periodontium was unaffected at all time points studied; in Plg-deficient mice, periodontitis progressed rapidly, within 20 weeks. Morphological study results of Plg-deficient mice revealed detachment of gingival tissue, resorption of the cementum layer, formation of necrotic tissue, and severe alveolar bone degradation. IHC staining showed massive infiltration of neutrophils in the periodontal tissues. Interestingly, doubly deficient mice, lacking both tissue- and urokinase-type plasminogen activators, developed periodontal disease similar to that in Plg-deficient mice; however, mice lacking only tissue- or urokinase-type plasminogen activator remained healthy. Supplementation by injection of Plg-deficient mice with human plasminogen for 10 days led to necrotic tissue absorption, inflammation subsidence, and full regeneration of gum tissues. Notably, there was also partial regrowth of degraded alveolar bone. Taken together, our results show that plasminogen is essential for the maintenance of a healthy periodontium and plays an important role in combating the spontaneous development of chronic periodontitis. Moreover, reversal to healthy status after supplementation of Plg-deficient mice with plasminogen suggests the possibility of using plasminogen for therapy of periodontal diseases.

Synergistic Ca2+ Responses by Gαi- and Gαq-coupled G-protein-coupled Receptors Require a Single PLCβ Isoform That Is Sensitive to Both Gβγ and Gαq

Synergistic Ca2+ Responses by Gαi- and Gαq-coupled G-protein-coupled Receptors Require a Single PLCβ Isoform That Is Sensitive to Both Gβγ and Gαq

The nerve growth factor receptor TrkA exists as a pre‐formed homodimer before ligand binding

The nerve growth factor (NGF) family of neurotrophins plays a central role in the development, growth, and maintenance of the nervous system. These neurotrophins transduce their effects by interacting with two types of cell surface receptors, the Trk family of tyrosine kinase receptors, and p75NTR. Two classes of NGF‐binding sites, high and low affinities, are present on the surface of neurons. When TrkA and p75NTR receptors are co‐expressed, the association rate is dramatically increased, creating a new site consistent with the NGF high affinity binding site. These results suggest that the receptors interact each other prior to ligand binding.In the present study, we have analyzed the structures of the TrkA receptor for NGF by bimolecular fluorescence complementation and luciferase fragment complementation assays as well as by chemical crosslinking of the receptor expressed on the cell surface. These analyses demonstrated that before ligand binding, the majority of receptors exist as a homodimer in living cells. Using Brefeldin A, a lactone antibiotic that disassembles the Golgi apparatus and block anterograde transport of the receptors from ER to Golgi, furthermore, it has been found that TrkA homodimers are initially formed in ER before reaching to the cell surface. This work provides new insights into understanding of transmembrane signaling by TrkA receptors.

Sequence and expression analysis of extracellular signal-regulated kinase 1 from flounder ( Paralichthys olivaceous)

Extracellular signal-regulated kinases (ERKs) are a subgroup of mitogen-activated protein kinases (MAPK) that function as important intermediates in signal transduction pathways initiated by several types of cell surface receptors. We cloned a transcript of ERK1 from a cDNA library of flounder leukocytes stimulated with bacterial lipopolysaccharide and hemagglutinin lectin. Flounder ERK1 consists of 1502 nucleotides and encodes a polypeptide of 393 amino acids. Flounder ERK1 showed 90 and 89% amino acid sequence identity to ERK1 of carp and zebrafish, respectively, and over 85% to that of mammals. Multiple bands were detected by Southern blot analysis of flounder genomic DNA after digestion with various restriction enzymes, implying the presence of additional MAPK genes in flounder. Real-time PCR revealed the ubiquitous expression of flounder MAPK in all tissues with high levels of transcription in brain, gill, and fin, but not in muscle or skin. Flounder MAPK was successfully expressed in mammalian COS1 cells and phosphorylated myelin basic protein (MBP) substrate when the cells were stimulated with PMA or EGF, indicating that flounder MAPK is functional in animal cells.

Reconstruction of the P2X2 Receptor Reveals a Vase-Shaped Structure with Lateral Tunnels above the Membrane

In response to the intercellular messenger ATP, P2X receptors transfer various sensory information, including pain. Here we have reconstructed the structure of the P2X(2) receptor at 15 A resolution from more than 90,000 particle images, taken with a cryo-electron microscope equipped with a helium-cooled stage. This three-dimensional depiction, presumably in a closed state, revealed an elongated vase-shaped structure 202 A in height and 160 A in major diameter. The extracellular and transmembrane domains present a two-layered structure, in which a sparse outer layer surrounds a pore-forming inner density. The decreased diameter of a putative ion-conducting pathway at the middle of the membrane was considered to be the narrowest part of the pore, which has been predicted from electrophysiological studies. The sparse, extended structure of the P2X(2) receptor indicates a loose assembly of subunits, which could be a basis for the activation-dependent pore dilation of P2X receptors.