Membrane-localized Receptors Research Articles

Hybrid Live Cell-Supported Membrane Interfaces for Signaling Studies.

A wide range of cell-microenvironmental interactions are mediated by membrane-localized receptors that bind ligands present on another cell or the extracellular matrix. This situation introduces a number of physical effects including spatial organization of receptor-ligand complexes and development of mechanical forces in cells. Unlike traditional experimental approaches, hybrid live cell-supported lipid bilayer (SLB) systems, wherein a live cell interacts with a synthetic substrate supported membrane, allow interrogation of these aspects of receptor signaling. The SLB system directly offers facile control over the identity, density, and mobility of ligands used for engaging cellular receptors. Further, application of various nano- and micropatterning techniques allows for spatial patterning of ligands. In this review, we describe the hybrid live cell-SLB system and its application in uncovering a range of spatial and mechanical aspects of receptor signaling. We highlight the T cell immunological synapse, junctions formed between EphA2- and ephrinA1-expressing cells, and adhesions formed by cadherin and integrin receptors.

Screening and identification of a non-peptide antagonist for the peptide hormone receptor in Arabidopsis

Intercellular signaling mediated by peptide hormones and membrane-localized receptor kinases plays crucial roles in plant developmental processes. Because of their diverse functions, agonistic or antagonistic modulation of peptide signaling holds enormous promise for agricultural applications. Here we established a high-throughput screening system using a bead-immobilized receptor kinase and fluorescent-labeled peptide ligand to identify small molecules that bind peptide hormone receptors in competition with natural ligands. We used the Arabidopsis CLE9-BAM1 ligand-receptor pair to screen a library of ≈30,000 chemicals and identified NPD12704 as an antagonist for BAM1. NPD12704 also inhibited CLV3 binding to BAM1 but only minimally interfered with CLV3 binding to CLV1, the closest homolog of BAM1, demonstrating preferential receptor specificity. Treatment of clv1-101 mutant seedlings with NPD12704 enhanced the enlarged shoot apical meristem phenotype. Our results provide a technological framework enabling high-throughput identification of small non-peptide chemicals that specifically control receptor kinase–mediated peptide hormone signaling in plants.

Pollen tube integrity regulation in flowering plants: insights from molecular assemblies on the pollen tube surface.

Contents Summary 687 I. Introduction 687 II. Pollen tube membrane-localized receptors coordinate cell integrity and sperm release 689 III. RALF peptides mediate autocrine and paracrine signaling 689 IV. ROS and ion channel signaling mediate intracellular response 690 V. Involvements from pollen tube cell wall components 690 VI. Concluding remarks 691 Acknowledgements 692 Author contributions 692 References 692 SUMMARY: Unlike in animals, sperm in flowering plants are immotile and they are embraced as passive cargoes by a pollen tube which embarks on a long journey in the pistil to deliver them to the female gametophyte for fertilization. How the pollen tube switches from a rapid polarized growth towards its target to an abrupt disintegration for sperm cell release inside the female gametophyte is puzzling. Recent studies have shown that members of the Catharanthus roseus RLK1-like (CrRLK1L) receptor kinase family and their ligands, 5-kDa cysteine-rich peptide rapid alkalinization factors (RALFs), engage in an intricate balancing act involving autocrine and paracrine signaling to maintain pollen tube growth and induce timely tube rupture at the spatially confined pollen tube-female gametophyte interface. Here, we review recent progress related to pollen tube integrity control, mainly focusing on the molecular understanding of signaling as well as intracellular signaling nodes in Arabidopsis. Some missing links and future perspectives are also discussed.

Patched1–ArhGAP36–PKA–Inversin axis determines the ciliary translocation of Smoothened for Sonic Hedgehog pathway activation

The Sonic Hedgehog (Shh) pathway conducts primarily in the primary cilium and plays important roles in cell proliferation, individual development, and tumorigenesis. Shh ligand binding with its ciliary membrane-localized transmembrane receptor Patched1 results in the removal of Patched1 from and the translocation of the transmembrane oncoprotein Smoothened into the cilium, leading to Shh signaling activation. However, how these processes are coupled remains unknown. Here, we show that the Patched1-ArhGAP36-PKA-Inversin axis determines the ciliary translocation of Smoothened. We find that Patched1 interacts with and stabilizes the PKA negative regulator ArhGAP36 to the centrosome. Activating the Shh pathway results in the removal of ArhGAP36 from the mother centriole and the centrosomal PKA accumulation. This PKA then phosphorylates Inversin and promotes its interaction with and the ciliary translocation of Smoothened. Knockdown of Inversin disrupts the ciliary translocation of Smoothened and Shh pathway activation. These findings reveal a regulatory molecular mechanism for the initial step of Shh pathway activation.

Four tyrosine residues of the rice immune receptor XA21 are not required for interaction with the co-receptor OsSERK2 or resistance to Xanthomonas oryzae pv. oryzae.

Tyrosine phosphorylation has emerged as an important regulator of plasma membrane-localized immune receptors activity. Here, we investigate the role of tyrosine phosphorylation in the regulation of rice XANTHOMONAS RESISTANCE 21 (XA21)-mediated immunity. We demonstrate that the juxtamembrane and kinase domain of Escherichia coli–expressed XA21 (XA21JK) autophosphorylates on tyrosine residues. Directed mutagenesis of four out of the nine tyrosine residues in XA21JK reduced autophosphorylation. These sites include Tyr698 in the juxtamembrane domain, and Tyr786, Tyr907, and Tyr909 in the kinase domain. Rice plants expressing XA21-GFP fusion proteins or proteins with these tyrosine residues individually mutated to phenylalanine (XA21YF-GFP), which prevents phosphorylation at these sites, maintain resistance to Xanthomonas oryzae pv. oryzae. In contrast, plants expressing phosphomimetic XA21 variants with tyrosine mutated to aspartate (XA21YD-GFP) were susceptible. In vitro purified XA21JKY698F, XA21JKY907F, and XA21JKY909F variants are catalytically active, whereas activity was not detected in XA21JKY768F and the four XA21JKYD variants. We previously demonstrated that interaction of XA21 with the co-receptor OsSERK2 is critical for biological function. Four of the XA21JKYF variants maintain interaction with OsSERK2 as well as the XA21 binding (XB) proteins XB3 and XB15 in yeast, suggesting that these four tyrosine residues are not required for their interaction. Taken together, these results suggest that XA21 is capable of tyrosine autophosphorylation, but the identified tyrosine residues are not required for activation of XA21-mediated immunity or interaction with predicted XA21 signaling proteins.

EBP1: A crucial growth regulator downstream of receptor kinases across kingdoms.

Controlling organ growth and development is crucial for all multicellular organisms and is controlled by plasma membrane localized receptor kinases (RKs) across kingdoms, including animals and plants. A central RK in plants is FERONIA (FER), which perceives endogenous rapid alkalinization factor (RALF) peptides to regulate a plethora of biological responses, including growth and development. However, it remained largely unknown how RALF sensing by FER at the plasma membrane is translated into a nuclear response. A key step forward is presented by Li and colleagues, who show that FER increases ERBB3 binding protein 1 (EBP1) mRNA translation and directly phosphorylates EBP1 to shift its subcellular localization from the cytoplasm to the nucleus where it controls growth and development through its regulation of transcription. Importantly, EBP1 is described as a transcriptional and translational regulator in mammals by acting downstream of epidermal growth factor receptor (EGFR) signaling, suggesting that animals and plants use similar conserved pathways to fine-tune growth and development. Furthermore, this work highlights the importance of protein translation as a direct output of RK signaling, a mechanism that is largely unknown in plants.

Palmitoylation of caveolin-1 is regulated by the same DHHC acyltransferases that modify steroid hormone receptors

Palmitoylation is a reversible post-translational addition of a 16-carbon lipid chain involved in trafficking and compartmentalizing target proteins. It is important for many cellular functions, including signaling via membrane-localized estrogen receptors (ERs). Within the nervous system, palmitoylation of ERα is necessary for membrane surface localization and mediation of downstream signaling through the activation of metabotropic glutamate receptors (mGluRs). Substitution of the single palmitoylation site on ERα prevents its physical association with the integral membrane protein caveolin-1 (CAV1), required for the formation of the ER/mGluR signaling complex. Interestingly, siRNA knockdown of either of two palmitoyl acyltransferases, zinc finger DHHC type-containing 7 (DHHC7) or DHHC21, also eliminates this signaling mechanism. Because ERα has only one palmitoylation site, we hypothesized that one of these DHHCs palmitoylates CAV1. We investigated this possibility by using an acyl-biotin exchange assay in HEK293 cells in conjunction with DHHC overexpression and found that DHHC7 increases CAV1 palmitoylation. Substitution of the palmitoylation sites on CAV1 eliminated this effect but did not disrupt the ability of the DHHC enzyme to associate with CAV1. In contrast, siRNA-mediated knockdown of DHHC7 alone was not sufficient to decrease CAV1 palmitoylation but rather required simultaneous knockdown of DHHC21. These findings provide additional information about the overall influence of palmitoylation on the membrane-initiated estrogen signaling pathway and highlight the importance of considering the influence of palmitoylation on other CAV1-dependent processes.

Differentiation of the granulosa layer from hen prehierarchal follicles associated with follicle-stimulating hormone receptor signaling.

Recruitment of a single follicle into the preovulatory hierarchy of the domestic hen ovary occurs from a small cohort of prehierarchal follicles measuring 6-8 mm in diameter. We have previously reported that granulosa cells (GCs) collected from prehierarchal follicles express highest levels of membrane-localized follicle-stimulating hormone receptor (FSHR) during follicle development, yet fail to initiate signaling via cAMP following short-term incubation with FSH. Consequently, GC from prehierarchal follicles remain in an undifferentiated state and lack the capacity for steroidogenesis due to a deficiency of cAMP-dependent STAR protein and CYP11A1 gene expression. The present studies investigate FSH responsiveness in GC before and after the transition from undifferentiated to a differentiated state at follicle recruitment. Before recruitment focus is directed toward the inhibition of FSHR-signaling by β-ARRESTIN (βARR). Specifically, knockdown of βARR messenger RNA in cultured, undifferentiated GC using small interfering RNA facilitated FSH-induced cAMP formation, STAR expression, and progesterone production. Furthermore, overexpression of bovine βARR1 and G-protein-coupled receptor kinase2 in actively differentiating GC significantly decreased cAMP accumulation and progesterone production following a challenge with FSH. We propose that a βARR-mediated mechanism maintains FSHR unresponsiveness in undifferentiated GC from prehierarchal follicles, and as a result prevents GC differentiation until the time of follicle recruitment.

The Making and Breaking of Inositol 1,4,5-Trisphosphate Receptor Tetramers.

Mammalian cells express three highly conserved inositol 1,4,5-trisphosphate (IP3) receptor types (IP3R1, IP3R2 and IP3R3), which have broadly similar characteristics, but markedly different distributions, and form homo- or heterotetrameric Ca2+ channels in endoplasmic reticulum (ER) membranes. A vast array of published work details how mature, ER membrane-located IP3 receptor tetramers are regulated, but much less attention has been paid to the intriguing questions of how the tetramers are assembled and destroyed as part of their natural life cycle. Are they assembled at the ER membrane from nascent, or completely translated polypeptides? How are they disassembled and degraded? These questions and other recently defined modes of IP3 receptor processing will be briefly reviewed.

Probing Plant Receptor Kinase Functions with Labeled Ligands.

Plant cells rely on numerous membrane-localized receptor kinases for communication with each other as well as with the environment to ensure coordinated growth, development and survival. Additionally, several families of small secreted peptides have been identified in plants that possibly regulate various developmental processes through activation of specific membrane receptor kinases. In research efforts to elucidate the underlying signaling processes in plants, it has become obvious that knowledge of both the extracellular ligand and its corresponding receptor(s) is a prerequisite to understand cell communication. Of more than 1,000 orphan peptides in plants, only a few have been functionally characterized and paired with their respective receptors. Here, we discuss recent advances made in the field of receptor-ligand pairing and visualization in plant cells by means of chemically labeled ligands. We summarize and compare various chemical strategies from the mammalian field that can be employed to extend our understanding of the plant receptor kinase functions in plants.

Classification and phylogenetic analyses of the Arabidopsis and tomato G-type lectin receptor kinases

BackgroundPathogen perception by plants is mediated by plasma membrane-localized immune receptors that have varied extracellular domains. Lectin receptor kinases (LecRKs) are among these receptors and are subdivided into 3 classes, C-type LecRKs (C-LecRKs), L-type LecRKs (L-LecRKs) and G-type LecRKs (G-LecRKs). While C-LecRKs are represented by one or two members in all plant species investigated and have unknown functions, L-LecRKs have been characterized in a few plant species and have been shown to play roles in plant defense against pathogens. Whereas Arabidopsis G-LecRKs have been characterized, this family of LecRKs has not been studied in tomato.ResultsThis investigation updates the current characterization of Arabidopsis G-LecRKs and characterizes the tomato G-LecRKs, using LecRKs from the monocot rice and the basal eudicot columbine to establish a basis for comparisons between the two core eudicots. Additionally, revisiting parameters established for Arabidopsis nomenclature for LecRKs is suggested for both Arabidopsis and tomato. Moreover, using phylogenetic analysis, we show the relationship among and between members of G-LecRKs from all three eudicot plant species. Furthermore, investigating presence of motifs in G-LecRKs we identified conserved motifs among members of G-LecRKs in tomato and Arabidopsis, with five present in at least 30 of the 38 Arabidopsis members and in at least 45 of the 73 tomato members.ConclusionsThis work characterized tomato G-LecRKs and added members to the currently characterized Arabidopsis G-LecRKs. Additionally, protein sequence analysis showed an expansion of this family in tomato as compared to Arabidopsis, and the existence of conserved common motifs in the two plant species as well as conserved species-specific motifs.

Cytokinin signaling in plant development.

The phytohormone cytokinin plays diverse roles in plant development, influencing many agriculturally important processes, including growth, nutrient responses and the response to biotic and abiotic stresses. Cytokinin levels in plants are regulated by biosynthesis and inactivation pathways. Cytokinins are perceived by membrane-localized histidine-kinase receptors and are transduced through a His-Asp phosphorelay to activate a family of transcription factors in the nucleus. Here, and in the accompanying poster, we summarize the current understanding of cytokinin metabolism, transport and signaling, and discuss how this phytohormone regulates changes in gene expression to mediate its pleiotropic effects.

Leucine-rich repeat receptor-like gene screen reveals that Nicotiana RXEG1 regulates glycoside hydrolase 12 MAMP detection

Activation of innate immunity by membrane-localized receptors is conserved across eukaryotes. Plant genomes contain hundreds of such receptor-like genes and those encoding proteins with an extracellular leucine-rich repeat (LRR) domain represent the largest family. Here, we develop a high-throughput approach to study LRR receptor-like genes on a genome-wide scale. In total, 257 tobacco rattle virus-based constructs are generated to target 386 of the 403 identified LRR receptor-like genes in Nicotiana benthamiana for silencing. Using this toolkit, we identify the LRR receptor-like protein Response to XEG1 (RXEG1) that specifically recognizes the glycoside hydrolase 12 protein XEG1. RXEG1 associates with XEG1 via the LRR domain in the apoplast and forms a complex with the LRR receptor-like kinases BAK1 and SOBIR1 to transduce the XEG1-induced defense signal. Thus, this genome-wide silencing assay is demonstrated to be an efficient toolkit to pinpoint new immune receptors, which will contribute to developing durable disease resistance.

Convergent and Divergent Signaling in PAMP-Triggered Immunity and Effector-Triggered Immunity.

Plants use diverse immune receptors to sense pathogen attacks. Recognition of pathogen-associated molecular patterns (PAMPs) by pattern recognition receptors localized on the plasma membrane leads to PAMP-triggered immunity (PTI). Detection of pathogen effectors by intracellular or plasma membrane-localized immune receptors results in effector-triggered immunity (ETI). Despite the large variations in the magnitude and duration of immune responses triggered by different PAMPs or pathogen effectors during PTI and ETI, plasma membrane-localized immune receptors activate similar downstream molecular events such as mitogen-activated protein kinase activation, oxidative burst, ion influx, and increased biosynthesis of plant defense hormones, indicating that defense signals initiated at the plasma membrane converge at later points. On the other hand, activation of ETI by immune receptors localized to the nucleus appears to be more directly associated with transcriptional regulation of defense gene expression. Here, we review recent progress in signal transductions downstream of different groups of plant immune receptors, highlighting the converging and diverging molecular events.

New roles for nuclear EGFR in regulating the stability and translation of mRNAs associated with VEGF signaling.

Cell membrane-associated epidermal growth factor receptor (EGFR) translocates into a perinuclear/nuclear location upon stimulation, where it complexes with mRNAs. Treatment with radiation and cisplatin decreases the amounts of mRNAs present within this complex. Gene array analyses of mRNAs in complex with immunoprecipitated nEGFR revealed significant enrichment of different mRNA species compared to the control immunoprecipitation. Functional annotation with help of DAVID Gene Ontology Analysis identified under other terms the HIF-1A/VEGF signaling pathway as one of the top scoring KEGG pathways. RT-PCR and western blots revealed the radiation-induced expression of mRNAs and proteins involved in HIF-1A/VEGF signaling. Simultaneously, the levels of the corresponding validated miRNAs within the complex containing nEGFR and mRNAs were decreased. This finding argues that an mRNA/miRNA/nEGFR complex regulates protein expression. Indeed, we detected the GW182, AGO2, PABPC1 and cNOT1 proteins, which belong to the deadenylase complex, in a complex with nuclear EGFR. Erlotinib-mediated inhibition of EGFR kinase reduced the radiation-induced increase in mRNA expression. In this context, erlotinib reduced AGO2 phosphorylation by the EGFR kinase at residue Y393, which was associated with increased cNOT1 deadenylase activity and reduced mRNA stability. To prove the roles of miRNAs in this context, we transfected cells with an inhibitor of Hsa-mir-1180p5, which targets the NFATC4 mRNA, an mRNA associated with VEGF signaling, or pretreated cells with erlotinib. Indeed, Hsa-mir-1180p5 knockdown increased and the erlotinib treatment decreased the expression of the NFATC4 protein. The expression of the NFATC4 protein controlled the cloning efficiency and radiosensitivity of A549 and FaDu tumor cells. Thus, this study is the first to show that a membrane-located tyrosine kinase receptor, such as EGFR, is internalized to a nuclear/perinuclear location upon exposure to stress and modulates the stability and translation of miRNA-selected mRNAs. This mechanism enables cells to directly express proteins in response to EGFR activation and may contribute to treatment resistance in EGFR-overexpressing tumors.

Glucocorticoids: The mode of action in bullous pemphigoid.

Bullous pemphigoid (BP) is the most common of pemphigoid diseases caused by autoantibodies against the structures of dermoepidermal junction followed by complement activation, innate immune cell infiltration, neutrophil proteinase secretion and subepidermal blister formation. The first-line treatment of BP is topical and systemic glucocorticoids (GC). Regulation of the immune system and inflammatory cells is the main target of GC actions. GCs act through genomic and non-genomic mechanisms. The human glucocorticoid receptor (GR) mediates most of the biologic effects of GC: cytosolic GR binds GCs and is capable to bind to glucocorticoid response elements in DNA and either transactivate or transrepress genes depending on the tissue and cell type. In addition, GR exerts rapid, non-genomic effects possibly mediated by membrane-localized receptors or by translocation to mitochondria. GCs can also interact directly with several enzymes and cytokines. As a target treatment for BP, the production of autoantibodies should be discontinued. GCs, in spite of their wide immunosuppressive actions, are weak to stop immunoglobulin G (IgG) autoantibody formation. However, both systemic and topical GCs are able to reduce the clinical symptoms of BP. GCs are used to inhibit the secondary inflammation and symptoms, such as blistering and pruritus, and it is shown that GC treatment will gradually decrease also the autoantibody formation. Our review article analyses the mode of action of GC treatment in BP, as far it is possible due to paucity of modern immunological studies.

Antibody-induced dimerization of FGFR1 promotes receptor endocytosis independently of its kinase activity

Fibroblast growth factors (FGFs) and their plasma membrane-localized receptors (FGFRs) play a key role in the regulation of developmental processes and metabolism. Aberrant FGFR signaling is associated with the progression of serious metabolic diseases and human cancer. Binding of FGFs to FGFRs induces receptor dimerization and transphosphorylation of FGFR kinase domains that triggers activation of intracellular signaling pathways. Following activation, FGFRs undergo internalization and subsequent lysosomal degradation, which terminates transmission of signals. Although factors that regulate FGFR endocytosis are continuously discovered, little is known about the molecular mechanism that initiates the internalization of FGFRs. Here, we analyzed the internalization of antibody fragments in various formats that target FGFR1. We show that FGFR1-specific antibody fragments in the monovalent scFv format bind to FGFR1, but are not internalized into cells that overproduce FGFR1. In contrast, the same scFv proteins in the bivalent scFv-Fc format are efficiently internalized via FGFR1-mediated, clathrin and dynamin dependent endocytosis. Interestingly, the receptor tyrosine kinase activity is dispensable for endocytosis of scFv-Fc-FGFR1 complexes, suggesting that only dimerization of receptor is required to trigger endocytosis of FGFR1 complexes.

Cross-talk of Brassinosteroid signaling in controlling growth and stress responses.

Plants are faced with a barrage of stresses in their environment and must constantly balance their growth and survival. As such, plants have evolved complex control systems that perceive and respond to external and internal stimuli in order to optimize these responses, many of which are mediated by signaling molecules such as phytohormones. One such class of molecules called Brassinosteroids (BRs) are an important group of plant steroid hormones involved in numerous aspects of plant life including growth, development and response to various stresses. The molecular determinants of the BR signaling pathway have been extensively defined, starting with the membrane-localized receptor BRI1 and co-receptor BAK1 and ultimately culminating in the activation of BES1/BZR1 family transcription factors, which direct a transcriptional network controlling the expression of thousands of genes enabling BRs to influence growth and stress programs. Here, we highlight recent progress in understanding the relationship between the BR pathway and plant stress responses and provide an integrated view of the mechanisms mediating cross-talk between BR and stress signaling.

The autoinhibitory function of D1 domain of FGFR1 goes beyond the inhibition of ligand binding

Fibroblast growth factors (FGFs) and their plasma membrane-localized receptors (FGFRs) transduce signals that regulate developmental processes and metabolism. In numerous cancer types genetic aberrations of FGFR1 lead to its uncontrolled activation. To circumvent the unrestrained signal transduction, several intramolecular inhibitory mechanisms within FGFR1 have evolved. In vitro experiments with receptor truncation have demonstrated that the N-terminal D1 domain of FGFR1 negatively regulates ligand binding to the receptor. Here, we show that D1-specific monovalent antibody fragments can activate FGFR1 and its downstream signaling cascades in the absence of ligand. These data suggest that the D1 domain of FGFR1 may play autoinhibitory role not only by controlling ligand binding, but also by regulating the overall conformation of FGFR1, keeping it in a state that disfavors autoactivation in the absence of its cognate growth factor.

Genome Wide Identification and Expression Profiling of Ethylene Receptor Genes during Soybean Nodulation.

It has long been known that the gaseous plant hormone ethylene plays a key role in nodulation in legumes. The perception of ethylene by a family of five membrane-localized receptors is necessary to trigger the ethylene signaling pathway, which regulates various biological responses in Arabidopsis. However, a systematic analysis of the ethylene receptors in leguminous plants and their roles in nodule development is lacking. In this study, we performed a characterization of ethylene receptor genes based on the latest Glycine max genome sequence and a public microarray database. Eleven ethylene receptor family genes were identified in soybean through homology searches, and they were divided into two subgroups. Exon–intron analysis showed that the gene structures are highly conserved within each group. Further analysis of their expression patterns showed that these ethylene receptor genes are differentially expressed in various soybean tissues and organs, including functional nodules. Notably, the ethylene receptor genes showed different responses to rhizobial infection and Nod factors, suggesting a possible role for ethylene receptors and ethylene signaling in rhizobia–host cell interactions and nodulation in soybean. Together, these data indicate the functional divergence of ethylene receptor genes in soybean, and that some of these receptors mediate nodulation, including rhizobial infection, nodule development, and nodule functionality. These findings provide a foundation for further elucidation of the molecular mechanism by which the ethylene signaling pathway regulates nodulation in soybean, as well as other legumes.