Intra-membrane ligand diffusion and cell shape modulate juxtacrine patterning

A key problem in developmental biology is how pattern and planar polarity are transmitted in epithelial structures. Examples include Drosophila neuronal differentiation, ommatidia formation in the compound eye, and wing hair polarization. A key component for the generation of such patterns is direct cell-cell signalling by transmembrane ligands, called juxtacrine signalling. Previous models for this mode of communication have considered homogeneous distributions in the cell membrane, and the role of polarity has been largely ignored. In this paper we determine the role of inhomogeneous protein and receptor distributions in juxtacrine signalling. We explicitly include individual membrane segments, diffusive transport of proteins and receptors between these segments, and production terms with a combination of local and global responses to ligand binding. Our analysis shows that intra-membrane ligand transport is vital for the generation of long wavelength patterns. Moreover, with no ligand transport, there is no pattern formation for lateral induction, a process in which receptor activation up-regulates ligand production. Biased production of ligand also modulates patterning bifurcations and predicted wavelengths. In addition, biased ligand and receptor trafficking can lead to regular polarity across a lattice, in which each cell has the same orientation-directly analogous to patterns of hairs in the Drosophila wing. We confirm the trends in pattern wavelengths previously observed for patterns with cellular homogeneity-lateral inhibition tends to give short-range patterns, while lateral induction can give patterns with much longer wavelengths. Moreover, the original model can be recovered if intra-membrane bound receptor diffusion is included and rapid equilibriation between the sides is considered. Finally, we consider the role of irregular cell shapes and waves in such networks, including wave propagation past clones of non-signalling cells.     

Ryanodine受体间相互作用及其与钙释放功能的关系

在真核生物和原核生物的生物膜上都存在由同种受体蛋白相互连接在一起形成的紧密二维排列。最近的模型计算表明这种排列方式可能是一种新型信号转导机制的结构基础,相邻受体可通过功能上的耦联优化信号处理性能。Ryanodine受体（ryanodine receptor,RyR）／钙释放通道通常在肌肉的肌浆网膜上形成二维晶格排列,该蛋白成为研究受体二维排列及其生理功能的一个很好的模型。本文综述了近几年在RyR相互作用及其二维排列工作模式和生理功能研究方面的进展,着重介绍了我们实验室利用新方法对RyR相互作用及其调控进行的研究工作。我们研究中发现了RyR功能状态对其相互作用的调控,本文对据此提出的RyR二维排列的“动态耦联模型”及其可能的生理功能进行了详细讨论。     

Spatial organization of transmembrane receptor signalling

The spatial organization of transmembrane receptors is a critical step in signal transduction and receptor trafficking in cells. Transmembrane receptors engage in lateral homotypic and heterotypic cis‐interactions as well as intercellular trans‐interactions that result in the formation of signalling foci for the initiation of different signalling networks. Several aspects of ligand‐induced receptor clustering and association with signalling proteins are also influenced by the lipid composition of membranes. Thus, lipid microdomains have a function in tuning the activity of many transmembrane receptors by positively or negatively affecting receptor clustering and signal transduction. We review the current knowledge about the functions of clustering of transmembrane receptors and lipid–protein interactions important for the spatial organization of signalling at the membrane.     

SNX27 mediates retromer tubule entry and endosome-to-plasma membrane trafficking of signalling receptors

Endocytic sorting of signalling receptors between recycling and degradative pathways is a key cellular process controlling the surface complement of receptors and, accordingly, the cell's ability to respond to specific extracellular stimuli. The β2 adrenergic receptor (β2AR) is a prototypical seven-transmembrane signalling receptor that recycles rapidly and efficiently to the plasma membrane after ligand-induced endocytosis. β2AR recycling is dependent on the receptor's carboxy-terminal PDZ ligand and Rab4. This active sorting process is required for functional resensitization of β2AR-mediated signalling. Here we show that sequence-directed sorting occurs at the level of entry into retromer tubules and that retromer tubules are associated with Rab4. Furthermore, we show that sorting nexin 27 (SNX27) serves as an essential adaptor protein linking β2ARs to the retromer tubule. SNX27 does not seem to directly interact with the retromer core complex, but does interact with the retromer-associated Wiskott-Aldrich syndrome protein and SCAR homologue (WASH) complex. The present results identify a role for retromer in endocytic trafficking of signalling receptors, in regulating a receptor-linked signalling pathway, and in mediating direct endosome-to-plasma membrane traffic.     

Chemoreceptors in signalling complexes: shifted conformation and asymmetric coupling

Bacterial chemotaxis is mediated by signalling complexes of chemoreceptors, histidine kinase CheA and coupling protein CheW. Interactions in complexes profoundly affect the kinase. We investigated effects of these interactions on chemoreceptors by comparing receptors alone and in complexes. Assays of initial rates of methylation indicated that signalling complexes shifted receptor conformation towards the methylation-on, higher-ligand-affinity, kinase-off state, tuning receptors for greater sensitivity. In contrast, transmembrane and conformational signalling within chemoreceptors was essentially unaltered, consistent with other evidence identifying receptor dimers as the fundamental units of such signalling. In signalling complexes, coupling of ligand binding to kinase activity is cooperative and the dynamic range of kinase control expanded > 100-fold by receptor adaptational modification. We observed no cooperativity in influence of ligand on receptor conformation, only on kinase activity. However, receptor modification generated increased dynamic range in a stepwise fashion, partly in coupling ligand to receptor conformation and partly in coupling receptor conformation to kinase activity. Thus, receptors and kinase were not equivalently affected by interactions in signalling complexes or by ligand binding and adaptational modification, indicating asymmetrical coupling between them. This has implications for mechanisms of precise adaptation. Coupling might vary, providing a previously unappreciated locus for sensory control.     

Multimerization of polyomavirus middle-T antigen.

The oncogenic protein of polyomavirus, middle-T antigen, associated with cell membranes and interacts with a variety of cellular proteins involved in mitogenic signalling. Middle-T antigen may therefore mimic the function of cellular tyrosine kinase growth factor receptors, like the platelet-derived growth factor or epidermal growth factor receptor. Growth factor receptor signalling is initiated upon the binding of a ligand to the extracellular domain of the receptor. This results in activation of the intracellular tyrosine kinase domain of the receptor, followed by receptor phosphorylation, presumably as a consequence of dimerization of two receptor molecules. Similar to middle-T antigen, phosphorylation of growth factor receptors leads to recruitment of cellular signalling molecules downstream in the signalling cascade. In this study, we investigated whether middle-T antigen, similar to tyrosine kinase growth factor receptors, is able to form dimeric signalling complexes. We found that association with cellular membranes was a prerequisite for multimerization, most likely dimer formation. A chimeric middle-T antigen carrying the membrane-targeting sequence of the vesicular stomatitis virus G protein instead of the authentic polyomavirus sequence still dimerized. However, mutants of middle-T antigen unable to associate with 14-3-3 proteins, like d18 and S257A, did not form dimers but were still oncogenic. This indicates that both membrane association and binding of 14-3-3 are necessary for dimer formation of middle-T antigen but that only the former is essential for cell transformation.     

Mis-assembly of clathrin lattices on endosomes reveals a regulatory switch for coated pit formation

The clathrin-coated pit lattice is held onto the plasma membrane by an integral membrane protein that binds the clathrin AP-2 subunit with high affinity. In vitro studies have suggested that this protein controls the assembly of the pit because membrane bound AP-2 is required for lattice assembly. If so, the AP-2 binding site must be a resident protein of the coated pit and recycle with other receptors that enter cells through this pathway. Proper recycling, however, would require the switching off of AP-2 binding to allow the binding site to travel through the endocytic pathway unencumbered. Evidence for this hypothesis has been revealed by the cationic amphiphilic class of drugs (CAD), which have previously been found to inhibit receptor recycling. Incubation of human fibroblasts in the presence of these drugs caused clathrin lattices to assemble on endosomal membranes and at the same time prevented coated pit assembly at the cell surface. These effects suggest that CADs reverse an on/off switch that controls AP-2 binding to membranes. We conclude that cells have a mechanism for switching on and off AP-2 binding during the endocytic cycle.     

Molecular recognition in bone morphogenetic protein (BMP)/receptor interaction

Bone morphogenetic proteins (BMPs) and other members of the TGF-beta superfamily are secreted signalling proteins determining the development, maintenance and regeneration of tissues and organs. These dimeric proteins bind, via multiple epitopes, two types of signalling receptor chains and numerous extracellular modulator proteins that stringently control their activity. Crystal structures of free ligands and of complexes with type I and type II receptor extracellular domains and with the modulator protein Noggin reveal structural epitopes that determine the affinity and specificity of the interactions. Modelling of a ternary complex BMP/(BMPR-IA(EC))2 / (ActR-II(EC))2 suggests a mechanism of receptor activation that does not rely on direct contacts between extracellular domains of the receptors. Mutational and interaction analyses indicate that the large hydrophobic core of the interface of BMP-2 (wrist epitope) with the type I receptor does not provide a hydrophobic hot spot for binding. Instead, main chain amide and carbonyl groups that are completely buried in the contact region represent major binding determinants. The affinity between ligand and receptor chains is probably strongly increased by two-fold interactions of the dimeric ligand and receptor chains that exist as homodimers in the membrane (avidity effects). BMP muteins with disrupted epitopes for receptor chains or modulator proteins provide clues for drug design and development.     

Modulation of the oligomerization of isolated ryanodine receptors by their functional states

The calcium release channels/ryanodine receptors (RyRs) usually form two-dimensional regular lattices in the endoplasmic/sarcoplasmic reticulum membranes. However, the function and modulation of the interaction between neighboring RyRs are still unknown. Here, with an in vitro aqueous system, we demonstrate that the interaction between RyRs isolated from skeletal muscle (RyR1s) is modulated by their functional states by using photon correlation spectroscopy and [(3)H]ryanodine binding assay. High level of oligomerization is observed for resting closed RyR1s with nanomolar Ca(2+) in solution. Activation of RyR1s by micromolar Ca(2+) or/and millimolar AMP leads to the de-oligomerization of RyR1s. The oligomerization of RyR1s remains at high level when RyR1s are stabilized at closed state by Mg(2+). The modulation of RyR1-RyR1 interaction by the functional state is also observed under near-physiological conditions, suggesting that the interaction between arrayed RyR1s would be dynamically modulated during excitation-contraction coupling. These findings provide exciting new information to understand the function and operating mechanism of RyR arrays.     

Characterization of the Molecular Architecture of Human Caveolin-3 and Interaction with the Skeletal Muscle Ryanodine Receptor

Caveolin-3 (cav-3), an integral membrane protein, is a building block of caveolae as well as a regulator of a number of physiological processes by facilitating the formation of multiprotein signaling complexes. We report that the expression of cav-3 in insect (Sf9) cells induces caveola formation, comparable in size with those observed in native tissue. We have also purified the recombinant cav-3 determining that it forms an oligomer of ∼220 kDa. We present the first three-dimensional structure for cav-3 (using transmission electron microscopy and single particle analysis methods) and show that nine cav-3 monomers assemble to form a complex that is toroidal in shape, ∼16.5 nm in diameter and ∼ 5.5 nm in height. Labeling experiments and reconstitution of the purified cav-3 into liposomes have allowed a proposal for the orientation of the protein with respect to the membrane. We have identified multiple caveolin-binding motifs within the ryanodine receptor (RyR1) sequence employing a bioinformatic analysis. We have then shown experimentally that there is a direct interaction between recombinant cav-3 nonamers and purified RyR1 homotetramers that would imply that at least one of the predicted cav-3-binding sites is exposed within the fully assembled RyR1 structure. The cav-3 three-dimensional model provides new insights as to how a cav-3 oligomer can bind multiple partners in close proximity to form signaling complexes. Furthermore, a direct interaction with RyR1 suggests a possible role for cav-3 as a modifier of muscle excitation-contraction coupling and/or for localization of the receptor to regions of the sarcoplasmic reticulum.     

RanBPM associates with CD39 and modulates ecto-nucleotidase activity

CD39/ecto-NTPDase 1 (nucleoside triphosphate diphosphohydrolase 1) is an ecto-nucleotidase that influences P2 receptor activation to regulate vascular and immune cell adhesion and signalling events pivotal in inflammation. Whether CD39 interacts with other membrane or cytoplasmic proteins has not been established to date. Using the yeast two-hybrid system, we note that the N-terminus of CD39 binds to RanBPM (Ran binding protein M; also known as RanBP9), a multi-adaptor scaffolding membrane protein originally characterized as a binding protein for the small GTPase Ran. We confirm formation of complexes between CD39 and RanBPM in transfected mammalian cells by co-immunoprecipitation studies. Endogenous CD39 and RanBPM are also found to be co-expressed and abundant in cell membranes of B-lymphocytes. NTPDase activity of recombinant CD39, but not of N-terminus-deleted-CD39 mutant, is substantially diminished by RanBPM co-expression in COS-7 cells. The conserved SPRY [repeats in splA and RyR (ryanodine receptor)] moiety of RanBPM is insufficient alone for complete physical and functional interactions with CD39. We conclude that CD39 associations with RanBPM have the potential to regulate NTPDase catalytic activity. This intermolecular interaction may have important implications for the regulation of extracellular nucleotide-mediated signalling.     

Homer proteins and InsP(3) receptors co-localise in the longitudinal sarcoplasmic reticulum of skeletal muscle fibres

Striated muscle represents one of the best models for studies on Ca(2+) signalling. However, although much is known on the localisation and molecular interactions of the ryanodine receptors (RyRs), far less is known on the localisation and on the molecular interactions of the inositol trisphosphate receptors (InsP(3)Rs) in striated muscle cells. Recently, members of the Homer protein family have been shown to cluster type 1 metabotropic glutamate receptors (mGluR1) in the plasma membrane and to interact with InsP(3)R in the endoplasmic reticulum of neurons. Thus, these scaffolding proteins are good candidates for organising plasma membrane receptors and intracellular effector proteins in signalosomes involved in intracellular Ca(2+) signalling. Homer proteins are also expressed in skeletal muscle, and the type 1 ryanodine receptor (RyR1) contains a specific Homer-binding motif. We report here on the relative sub-cellular localisation of InsP(3)Rs and Homer proteins in skeletal muscle cells with respect to the localisation of RyRs. Immunofluorescence analysis showed that both Homer and InsP(3)R proteins present a staining pattern indicative of a localisation at the Z-line, clearly distinct from that of RyR1. Consistent herewith, in sub-cellular fractionation experiments, Homer proteins and InsP(3)R were both found in the fractions enriched in longitudinal sarcoplasmic reticulum (LSR) but not in fractions of terminal cisternae that are enriched in RyRs. Thus, in skeletal muscle, Homer proteins may play a role in the organisation of a second Ca(2+) signalling compartment containing the InsP(3)R, but are apparently not involved in the organisation of RyRs at triads.     

ESCRT proteins and cell signalling

Subunits of the endosomal sorting complex required for transport (ESCRT) were identified as components of a molecular machinery that sorts ubiquitinated membrane proteins into the intraluminal vesicles (ILVs) of multivesicular endosomes (MVEs) for subsequent delivery to the lumen of lysosomes or related organelles. As many of the membrane proteins that undergo ESCRT-mediated sorting are signalling receptors that are ubiquitinated in response to ligand binding, ESCRT subunits have been hypothesized to play a crucial role in attenuation of cell signalling by mediating ligand-induced receptor degradation. Here we discuss this concept based on the examples from loss-of-function studies in model organisms and cell lines. The emerging picture is that ESCRTs are indeed involved in downregulation of receptor signalling pathways associated with cell survival, proliferation and polarity. In addition, the recent discovery of a positive role for the ESCRT pathway in Wnt signalling through sequestration of an inhibitory cytosolic component into MVEs illustrates that ESCRTs may also control signalling in ways that are independent of degradative receptor sorting.     

Cytoplasmic domains determine signal specificity, cellular routing characteristics and influence ligand binding of epidermal growth factor and insulin receptors.

The cell surface receptors for insulin and epidermal growth factor (EGF) both employ a tyrosine-specific protein kinase activity to fulfil their distinct biological roles. To identify the structural domains responsible for various receptor activities, we have generated chimeric receptor polypeptides consisting of major EGF and insulin receptor structural domains and examined their biochemical properties and cellular signalling activities. The EGF-insulin receptor hybrids are properly synthesized and transported to the cell surface, where they form binding competent structures that are defined by the origin of their extracellular domains. While their ligand binding affinities are altered, we find that these chimeric receptors are fully functional in transmitting signals across the plasma membrane and into the cell. Thus, EGF receptor and insulin receptor cytoplasmic domain signalling capabilities are independent of their new heterotetrameric or monomeric environments respectively. Furthermore, the cytoplasmic domains carry the structural determinants that define kinase specificity, mitogenic and transforming potential, and receptor routing.     

Functional studies of membrane-bound and purified human Hedgehog receptor Patched expressed in yeast

The Sonic Hedgehog (Shh) signalling pathway plays an important role both in embryonic development and in adult stem cell function. Inappropriate regulation of this pathway is often due to dysfunction between two membrane receptors Patched (Ptc) and Smoothened (Smo), which lead to birth defects, cancer or neurodegenerative diseases. However, little is known about Ptc, the receptor of the Shh protein, and the way Ptc regulates Smo, the receptor responsible for the transduction of the signal. To develop structure-function studies of these receptors, we expressed human Ptc (hPtc) in the yeast Saccharomyces cerevisiae. We demonstrated that hPtc expressed in a yeast membrane fraction is able to interact with its purified ligand Shh, indicating that hPtc is produced in yeast in its native conformational state. Using Surface Plasmon Resonance technology, we showed that fluorinated surfactants preserve the ability of hPtc to interact with its ligand after purification. This is the first report on the heterologous expression and the purification of a native and stable conformation of the human receptor Ptc. This work will allow the scale-up of hPtc production enabling its biochemical characterization, allowing the development of new therapeutic approaches against diseases induced by Shh signalling dysfunction.     

Two-dimensional crystallization of the ryanodine receptor Ca2+ release channel on lipid membranes

The ryanodine receptor (RyR) is the largest known membrane protein with a total molecular mass of 2.3 x 10(3) kDa. Well ordered, two-dimensional (2D) crystals are an essential prerequisite to enable RyR structure determination by electron crystallography. Conventionally, the 2D crystallization of membrane proteins is based on a 'trial-and-error' strategy, which is both time-consuming and chance-directed. By adopting a new strategy that utilizes protein sequence information and predicted transmembrane topology, we successfully crystallized the RyR on positively charged lipid membranes. Image processing of negatively stained crystals reveals that they are well ordered, with diffraction spots of IQ < or = 4 extending to approximately 20 angstroms, the resolution attainable in negative stain. The RyR crystals obtained on the charged lipid membrane have characteristics consistent with 2D arrays that have been observed in native sarcoplasmic reticulum of muscle tissues. These crystals provide ideal materials to enable structural analysis of RyR by high-resolution electron crystallography. Moreover, the reconstituted native-like 2D array provides an ideal model system to gain structural insights into the mechanism of RyR-mediated Ca2+ signaling processes, in which the intrinsic ability of RyR oligomers to organize into a 2D array plays a crucial role.     

Molecular mechanisms of cytokine receptor activation

Cytokine receptors are transmembrane proteins that transmit a signal into the cell upon ligand binding. Commonly, these molecules have one hydrophobic segment of about 20-26 amino acids that is believed to span the membrane as a helix and this divides these receptors into extra- and intracellular components. By utilizing two different epitopes, the cytokines bridge two receptor chains, resulting in a close proximity of the intracellular component and thereby initiating the intracellular signalling cascade. The dimerization event is believed to be the mechanism by which the signal is transmitted across a membrane. In the light of new results obtained for the erythropoietin receptor, James A. Wells questioned whether any dimer would be sufficient. This review will expand upon the above question by discussing the more complex signal-transducing receptor subunits of the Interleukin-6 type family of cytokines. Based on the recently solved quaternary structure of the Insulin receptor, possible analogies will be confronted.     

Latent membrane protein 1 of Epstein-Barr virus mimics a constitutively active receptor molecule.

Latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) is an integral membrane protein which has transforming potential and is necessary but not sufficient for B-cell immortalization by EBV. LMP1 molecules aggregate in the plasma membrane and recruit tumour necrosis factor receptor (TNF-R) -associated factors (TRAFs) which are presumably involved in the signalling cascade leading to NF-kappaB activation by LMP1. Comparable activities are mediated by CD40 and other members of the TNF-R family, which implies that LMP1 could function as a receptor. LMP1 lacks extended extracellular domains similar to beta-adrenergic receptors but, in contrast, it also lacks any motifs involved in ligand binding. By using LMP1 mutants which can be oligomerized at will, we show that the function of LMP1 in 293 cells and B cells is solely dependent on oligomerization of its carboxy-terminus. Biochemically, oligomerization is an intrinsic property of the transmembrane domain of wild-type LMP1 and causes a constitutive phenotype which can be conferred to the signalling domains of CD40 or the TNF-2 receptor. In EBV, immortalized B cells cross-linking in conjunction with membrane targeting of the carboxy-terminal signalling domain of LMP1 is sufficient for its biological activities. Thus, LMP1 acts like a constitutively activated receptor whose biological activities are ligand-independent.     

The insulin receptor and type I IGF receptor: Comparison of structure and function

The insulin receptor and type I IGF receptor are closely related in structure and function. The receptors are heterotetrameric glycoproteins, of structure αββα, which are widely distributed in mammalian tissues. A third member of this receptor family has been described, the insulin receptor-related receptor, for which a ligand has still to be identified. It has also been demonstrated that the insulin receptor and IGF receptor form αββ′α′ hybrids in cells expressing both receptors.The key elements in the function of any receptor are recognition of ligand and transmission of an intracellular signal. In the insulin and IGF receptors, determinants of binding specificity are contained within amino-terminal and cysteine-rich domains of the extracellular α-subunit. Intracellular signalling is dependent on ligand activated tyrosine kinase activity in the transmembrane β-subunit, which phosphorylates both the receptor itself and the specific substrate insulin receptor substrate-1 (IRS-1). Phosphorylated IRS-1 binds the enzyme phosphatidylinositol 3-kinase and may act as a multivalent docking site for SH2 domains of other proteins involved in signalling. The possibility that some signalling molecules interact directly with the receptors has not been ruled out.The specificity of action of insulin and IGFs in vivo depends on differences between the respective receptors in tissue distribution, ligand binding specificity and intrinsic signalling capacity. However, the detailed aspects of gene and receptor structure which underly these functional differences are still poorly understood. Moreover, the issue of specificity is complicated by the existence of hybrid and atypical receptors, which in principle could bind and respond to both insulin and IGF-I, although the physiological significance of these receptor subtypes is at present unclear.     

Adaptational modification and ligand occupancy have opposite effects on positioning of the transmembrane signalling helix of a chemoreceptor

Sensory systems adapt to persistent stimulation. In the transmembrane receptors of bacterial chemotaxis, adaptation is mediated by methylation at specific glutamyl residues in the cytoplasmic domain. Methylation counteracts effects of ligand binding on functional activities of that domain. Both ligand binding and adaptational modification are thought to act through conformational changes. As characterized for Escherichia coli chemoreceptors, a mechanistically crucial feature of the ligand-induced conformational change is piston sliding towards the cytoplasm of a signalling helix in the periplasmic/transmembrane domain. Adaptational modification could counteract this signalling movement by blocking its influence on the cytoplasmic domain or by reversing it. To investigate, we characterized effects of adaptational modification on the position of the signalling helix in chemoreceptor Trg using rates of disulphide formation between introduced cysteines. We utilized an intact cell procedure in which receptors were in their native, functional state. In vivo rates of disulphide formation between diagnostic cysteine pairs spanning a signalling helix interface changed as a function of adaptational modification. Strikingly, those changes were opposite those caused by ligand occupancy for each diagnostic pair tested. This suggests that adaptational modification resets the receptor complex to its null state by reversal of the conformational change generated by ligand binding.