Human atrial natriuretic peptide receptor defines a new paradigm for second messenger signal transduction.

We isolated cDNAs encoding a 115 kd human atrial natriuretic peptide (alpha ANP) receptor (ANP-A receptor) that possesses guanylate cyclase activity, by low-stringency hybridization with sea urchin Arbacia punctulata membrane guanylate cyclase probes. The human ANP-A receptor has a 32 residue signal sequence followed by a 441 residue extracellular domain homologous to the 60 kd ANP-C receptor. A 21 residue transmembrane domain precedes a 568 residue cytoplasmic domain with homology to the protein kinase family and to a subunit of the soluble guanylate cyclase. COS-7 cells transfected with an ANP-A receptor expression vector displayed specific [125I]alpha ANP binding, and exhibited alpha ANP stimulated cGMP production. These data demonstrate a new paradigm of cellular signal transduction where extracellular ligand binding allosterically regulates cyclic nucleotide second-messenger production by a receptor cytoplasmic catalytic domain.     

A new model for chemotactic signal transduction in Dictyostelium discoideum

Dictyostelium discoideum amoebae were employed to study the refractoriness and adaptation of the rapid (5sec) accumulation of actin in their Triton-insoluble cytoskeletons following stimulation with specific chemoattractants. Amoebae became refractory within 10sec for this response but no adaptation occurred during this period. Amoebae desensitized for one attractant were not desensitized for another and responses to stimulation with a mixture of attractants were approximately additive. The characteristics of these processes are compared to published studies of adaptation in other chemoattractant-induced responses and a new model for the chemotactic signal transduction pathway is formulated. We conclude that intracellular cGMP accumulation may be on a separate branch of the pathway from the actin response.     

Kinetic model for FGF, FGFR, and proteoglycan signal transduction complex assembly

The current working model for fibroblast growth factor receptor (FGFR) dimerization and activation requires the assembly of a ternary complex of fibroblast growth factor (FGF), FGFR, and heparin or heparan sulfate proteoglycan (HSPG) on the plasma membrane. The recent FGF2-FGFR1-heparin crystal structure provides a detailed but static view of the FGF-FGFR-heparin complex. However, the kinetics of ternary complex assembly has yet to be investigated. Here, we characterize FGF2, FGFR1, and heparin interactions using surface plasmon resonance (SPR). Binding constants for binary FGF2/FGFR1 (KD = 62 nM), FGF2/heparin (KD = 39 nM), and FGFR1/heparin (KD = 3.2 microM) interactions correlate to the magnitude of binding interface observed in the FGF2-FGFR1-heparin crystal structure. Interestingly, comparison of sensorgrams of sequential injections of FGF2 and FGFR1 and equimolar FGF2-FGFR1 injections onto a heparin neoproteoglycan surface demonstrates that FGF2 dramatically enhances the association of FGFR1 with heparin and leads us to propose a model for the stepwise assembly of a ternary FGF-FGFR-HSPG complex. The weak binding affinity of the FGFR1-heparin interaction suggests that in this model, FGFR and HSPG are unbound in the absence of FGF ligand. The availability of FGF results in formation of initial FGF-HSPG complexes, which promotes the rapid binding of FGFR and creates a ternary complex capable of undergoing dimerization and subsequent FGFR activation. In contrast, alternative models for the kinetic assembly of a ternary complex in which binary FGF-FGFR or FGFR-HSPG complexes are intermediates do not conform well with the experimental data.     

Bacteriophytochromes: new tools for understanding phytochrome signal transduction

The recent discovery of phytochrome-like photoreceptors, collectively called bacteriophytochromes, in a number of bacteria has greatly expanded our understanding of the origins and modes of action of phytochromes in higher plants. These primitive receptors contain an N-terminal domain homologous to the chromophore-binding pocket of phytochromes, and like phytochromes, they bind a variety of bilins to generate photochromic holoproteins. Following the chromophore pocket is a domain similar to two-component histidine kinases, suggesting that these bacterial photoreceptors function in phosphorelay cascades that respond to the light environment. Their organization and distribution support the views that higher-plant phytochromes evolved from a cyanobacterial precursor and that they act as light-regulated kinases. With the ability to exploit bacterial genetics, these bacteriophytochromes now offer simple models to help unravel the biochemical and biophysical events that initiate phytochrome signal transmission.     

Peptide microarrays for the detection of molecular interactions in cellular signal transduction

The formation of protein complexes is a hallmark of cellular signal transduction. Here, we show that peptide microarrays provide a robust and quantitative means to detect signalling-dependent changes of molecular interactions. Recruitment of a protein into a complex upon stimulation of a cell leads to the masking of an otherwise exposed binding site. In cell lysates this masking can be detected by reduced binding to a microarray carrying a peptide that corresponds to the binding motif of the respective interaction domain. The method is exemplified for the lymphocyte-specific tyrosine kinase 70 kDa zeta-associated protein binding to a bis-phosphotyrosine-motif of the activated T-cell receptor via its tandem SH2 domain. Compared to established techniques, the method provides a significant shortcut to the detection of molecular interactions.     

A pathway of sequential arginine-serine-rich domain-splicing signal interactions during mammalian spliceosome assembly

Serine-arginine (SR) proteins are general splicing factors and can function through binding to exonic splicing enhancers (ESEs). SR proteins and several other mammalian splicing factors contain an arginine-serine-rich (RS) domain required to promote splicing. We have recently found that the ESE bound RS domain functions by contacting the branchpoint. Here, we perform RNA-protein crosslinking experiments to show that the branchpoint is sequentially contacted first in complex E by the RS domain of the essential splicing factor U2AF(65) and then in the prespliceosome by the ESE bound RS domain. Although the ESE bound RS domain can promote formation of the prespliceosome, at least one additional SR protein is required for complete spliceosome assembly. We show that the RS domain of this additional SR protein contacts the 5' splice site specifically in the mature spliceosome. We propose that direct contact with splicing signals is a general mechanism by which RS domains promote splicing.     

Cytokine receptors and signal transduction.

Most of the recently cloned cytokine receptors that operate in the immune and hematopoietic systems contain no tyrosine kinase domains in their cytoplasmic regions, unlike the family of growth factor receptors defined earlier. However, they can be assigned to several new types of receptor families based on structural similarities among them. It is characteristic of these receptors that many of them require a receptor-associated molecule in order to achieve high-affinity ligand binding and/or transmission of cytoplasmic signals. Receptor-associated molecules have been found that transduce cytoplasmic signals and are shared by different cytokine receptors. Phosphorylation of the receptors and of various cytoplasmic proteins after ligand stimulation seems to be a common event in cytokine systems. Insight into the pleiotropic and redundant nature of cytokine action is provided by the discovery of several new cytokine receptor families and of shared signal transduction molecules and by the idea that several cytoplasmic kinases may be able to functionally substitute for one another in transmitting cytokine signals.     

A unified model for signal transduction reactions in cellular membranes.

An analytical solution is obtained for the steady-state reaction rate of an intracellular enzyme, recruited to the plasma membrane by active receptors, acting upon a membrane-associated substrate. Influenced by physical and chemical effects, such interactions are encountered in numerous signal-transduction pathways. The generalized modeling framework is the first to combine reaction and diffusion limitations in enzyme action, the finite mean lifetime of receptor-enzyme complexes, reactions in the bulk membrane, and constitutive and receptor-mediated substrate insertion. The theory is compared with other analytical and numerical approaches, and it is used to model two different signaling pathway types. For two-state mechanisms, such as activation of the Ras GTPase, the diffusion-limited activation rate constant increases with enhanced substrate inactivation, dissociation of receptor-enzyme complexes, or crowding of neighboring complexes. The latter effect is only significant when nearly all of the substrate is in the activated state. For regulated supply and turnover pathways, such as phospholipase C-mediated lipid hydrolysis, an additional influence is receptor-mediated substrate delivery. When substrate consumption is rapid, this process significantly enhances the effective enzymatic rate constant, regardless of whether enzyme action is diffusion limited. Under these conditions, however, enhanced substrate delivery can result in a decrease in the average substrate concentration.     

Integrin-mediated signal transduction pathways.

Integrins serve as adhesion receptors for extracellular matrix proteins and also transduce biochemical signals into the cell. They regulate a variety of cellular functions, including spreading, migration, proliferation and apoptosis. Many signaling pathways downstream of integrins have been identified and characterized and are discussed here. In particular, integrins regulate many protein tyrosine kinases and phosphatases, such as FAK and Src, to coordinate many of the cell processes mentioned above. The regulation of MAP kinases by integrins is important for cell growth or other functions, and the putative roles of Ras and FAK in these pathways are discussed. Phosphatidylinositol lipids and their modifying enzymes, particularly PI 3-kinase, are strongly implicated as mediators of integrin-regulated cytoskeletal changes and cell migration. Similarly, actin cytoskeleton regulation by the Rho family of GTPases is coordinated with integrin signaling to regulate cell spreading and migration, although the exact relationship between these pathways is not clear. Finally, intracellular pH and calcium fluxes by integrins are suggested to affect a variety of cellular proteins and functions.     

The role of receptor interactions in regulating ethylene signal transduction

The phytohormone ethylene is perceived in Arabidopsis by a five-member receptor family. Earlier work has demonstrated that the basic functional unit for an ethylene receptor is a disulfide-linked homodimer. We recently reported in The Journal of Biological Chemistry that the ethylene-receptor ETR1 physically associates with other ethylene receptors through higher order interactions, suggesting the existence of receptor clusters. Here we consider the implications of such clusters upon the mechanism of ethylene signal transduction. In particular, we consider how such clustering provides a cooperative mechanism, akin to what has been found for the prokaryotic chemoreceptors, by which plant sensitivity to ethylene may be increased. In addition, we consider how the dominant ethylene insensitivity conferred by some receptor mutations, such as etr1-1, may also be propagated by interactions among members of the ethylene receptor family.Key words: ethylene, receptor, ETR1, cooperativity, ArabidopsisThe plant hormone ethylene regulates growth and development, and is perceived by a five-member family of receptors (ETR1, ERS1, ETR2, ERS2 and EIN4) in Arabidopsis.¹ Genetic analysis indicates that ethylene receptors are functionally redundant and negatively regulate ethylene responses through interactions with the Raf-like kinase CTR1.^2–5 The functional unit of an ethylene receptor in a disulfide-linked homodimer, with each homodimer capable of binding one ethylene molecule.^6,7 However, several observations suggest that propagation of the ethylene signal through the receptors is likely to involve more than just ethylene-induced changes within individual receptor homodimers. First, Arabidopsis is amazingly sensitive to ethylene and can respond to ethylene concentrations as low as 0.2 nl/L,⁸ 300-fold lower than the K_d of the receptors for ethylene, which suggests that some mechanism exists for amplifying the input signal.^7,9 Second, ethylene-insensitive mutations in the binding sites of the receptors exhibit greater dominance than would be predicted solely from a lesion within one member of the receptor family.¹⁰In our paper published in The Journal of Biological Chemistry,¹¹ we demonstrate that the Arabidopsis ethylene receptor ETR1 physically associates with other ethylene receptors through higher order interactions. Such physical interactions suggest that the receptors exist in plants as clusters, and that models for cooperative signaling previously applied to the histidine-kinaselinked chemoreceptors of bacteria may also be applicable to the evolutionarily related ethylene receptors of plants. In bacteria, the highly packed chemoreceptors are found in clusters at one or both poles of the cell.^12,13 Structural studies indicate that chemoreceptors can associate to form a ‘trimer of dimers’^14,15 and also support the possibility that domain swapping may occur to produce a large interconnected array of receptors. 16 Our studies indicate that ethylene receptors can interact through their cytosolic GAF domains, identifying one possible interface through which conformational changes could be propagated in an ethylene receptor cluster.A higher-order cooperative mechanism among the ethylene receptors may explain the high sensitivity of plants to ethylene. In this model, the ethylene receptors amplify ethylene signaling by lateral signal output. Binding of ethylene to one receptor induces the conformation change of the receptor from a tense state (T) to a relaxed state (R). This conformational change is then propagated to other empty receptors in the cluster due to their physical associations with the receptor in the R state. As a result empty receptors also adopt the relaxed state (R′), resulting in amplification of the initial signal. It should be noted here that mutational evidence supports the unbound state of the receptors (T state) as being the lower energy conformation of the receptors.¹⁷ Thus, according to this model, part of the energy from ligand binding would be used to transmit conformational changes to the neighboring receptors.An alternative model that may also explain the high sensitivity of ethylene responsiveness in plants, and one that is not necessarily incompatible with the previous model, is a conjugation model.¹⁸ Here it is hypothesized that, due to the physical proximity of the ethylene receptors, that ethylene released from one receptor then binds to another receptor rather than diffusing away. Through this conjugation mechanism, one ethylene molecule could amplify its signal by converting the conformations of multiple ethylene receptors from the ethylene-unbound state (T) to the ethylene-bound state (R). This model is based on several assumptions. One assumption is that a single ethylene molecule can bind ethylene receptors in the same cluster multiple times due to the dynamic binding of ethylene and ethylene receptor. A second assumption is that, after ethylene is released from one ethylene receptor, the recovery time for that receptor to resume the T state is longer than the time required for the released ethylene to bind to and convert another receptor from the T to the R state.Models for cooperativity need to also explain the dominant ethylene insensitivity of various mutant receptors such as etr1-1, in which a missense mutation results in a receptor incapable of binding ethylene. Several studies indicate that the etr1-1 mutant receptor acts cooperatively to affect the signal output from other wild-type receptors (i.e., the presence of the etr1-1 receptor in its T state increases the likelihood of other receptors adopting the T state).^10,11 This observation can be most readily explained if the dominant ethylene-insensitive mutations result in a receptor that requires more energy to undergo the T to R transition than do the wild-type receptors. For example, the etr1-1 mutation may increase the stability of the T form (a T′ state). There is evidence to support this possibility. The etr1-1 missense mutation results in a receptor unable to chelate a copper cofactor necessary for ethylene binding,¹⁹ but the effects of this mutation on signaling are different from wild-type receptors that lack their copper cofactor. The etr1-1 mutant receptor appears locked in its T state, whereas wild-type receptors lacking the copper cofactor appear to be in the R state.²⁰ Thus etr1-1 is truly a gain-of-function mutation that alters the conformation of the receptor in ways not necessarily predicted from just the loss of the copper cofactor.In conclusion, we have attempted here to provide models that can resolve an apparent contradiction in the cooperative signaling behavior exhibited by ethylene receptors. The high sensitivity of plants to ethylene suggest cooperative changes in which an R state can be propagated within a receptor cluster, but the dominance of the ethylene ethylene-insensitive mutant etr1-1 suggests that the T state can also be propagated within a receptor cluster. It should be born in mind, however, that ethylene signaling is mediated by multiple signaling components. The ethylene receptors regulate ethylene responses through interaction with and modulation of CTR1 kinase activity. Thus, the total kinase activity of CTR1 represents the signal output from the receptors. This situation is very similar to that of the bacterial chemoreceptors, which regulate the activity of an associated histidine kinase, and, as with the chemoreceptors, the stoichiometry of CTR1 interactions with the ethylene receptors and the means by which its kinase activity is regulated are important for the elucidation of the mechanism of ethylene signal transduction.     

Analysis of proteinase-activated receptor 2 and TLR4 signal transduction: a novel paradigm for receptor cooperativity

Proteinase-activated receptor 2 (PAR(2)), a seven-transmembrane G protein-coupled receptor, is activated at inflammatory sites by proteolytic cleavage of its extracellular N terminus by trypsin-like enzymes, exposing a tethered, receptor-activating ligand. Synthetic agonist peptides (AP) that share the tethered ligand sequence also activate PAR(2), often measured by Ca(2+) release. PAR(2) contributes to inflammation through activation of NF-kappaB-regulated genes; however, the mechanism by which this occurs is unknown. Overexpression of human PAR(2) in HEK293T cells resulted in concentration-dependent, PAR(2) AP-inducible NF-kappaB reporter activation that was protein synthesis-independent, yet blocked by inhibitors that uncouple G(i) proteins or sequester intracellular Ca(2+). Because previous studies described synergistic PAR(2)- and TLR4-mediated cytokine production, we hypothesized that PAR(2) and TLR4 might interact at the level of signaling. In the absence of TLR4, PAR(2)-induced NF-kappaB activity was inhibited by dominant negative (DN)-TRIF or DN-TRAM constructs, but not by DN-MyD88, findings confirmed using cell-permeable, adapter-specific BB loop blocking peptides. Co-expression of TLR4/MD-2/CD14 with PAR(2) in HEK293T cells led to a synergistic increase in AP-induced NF-kappaB signaling that was MyD88-dependent and required a functional TLR4, despite the fact that AP exhibited no TLR4 agonist activity. Co-immunoprecipitation of PAR(2) and TLR4 revealed a physical association that was AP-dependent. The response to AP or lipopolysaccharide was significantly diminished in TLR4(-/-) and PAR (-/-)(2) macrophages, respectively, and SW620 colonic epithelial cells exhibited synergistic responses to co-stimulation with AP and lipopolysaccharide. Our data suggest a unique interaction between two distinct innate immune response receptors and support a novel paradigm of receptor cooperativity in inflammatory responses.     

Molecular modelling of protein-carbohydrate interactions. Docking of monosaccharides in the binding site of concanavalin A.

A general procedure is described for addressing the computer simulation of protein-carbohydrate interactions. First, a molecular mechanical force field capable of performing conformational analysis of oligosaccharides has been derived by the addition of new parameters to the Tripos force field; it is also compatible with the simulation of protein. Second, a docking procedure which allows for a systematic exploration of the orientations and positions of a ligand into a protein cavity has been designed. This so-called 'crankshaft' method uses rotations and variations about/of virtual bonds connecting, via dummy atoms, the ligand to the protein binding site. Third, calculation of the relative stability of protein ligand complexes is performed. This strategy has been applied to search for all favourable interactions occurring between a lectin [concanavalin A (ConA)] and methyl alpha-D-mannopyranoside or methyl alpha-D-glucopyranoside. For each monosaccharide, different stable orientations and positions within the binding site can be distinguished. Among them, one corresponds to very favourable interactions, not only in terms of hydrogen bonding, but also in terms of van der Waals interactions. It corresponds precisely to the binding mode of methyl alpha-D-mannopyranoside into ConA as revealed by the 2.9 A resolution of the crystalline complex (Derewenda et al., 1989). Some implications of the present modelling study with respect to the molecular basis of the specificity of the interaction of lectins with various monosaccharides are presented.     

Calcium signatures and signal transduction schemes during microbe interactions in Arabidopsis thaliana

Journal of Plant Biochemistry and Biotechnology - Calcium (Ca2+) is considered as crucial second messenger in all living organisms. Ca2+ signaling regulates a diverse array of different biological...