Plant NB-LRR signaling: upstreams and downstreams

Plant disease resistance proteins commonly belong to the nucleotide binding-leucine rich repeat (NB-LRR) protein family. These specialized immune proteins mediate recognition of diverse pathogen-derived effector proteins and initiate potent defense responses. NB-LRRs exhibit a multidomain architecture and each domain appears to have discrete functions depending on the stage of NB-LRR signaling. Novel proteins that were found to interact with the core HSP90 chaperone complex regulate accumulation and activation of NB-LRR immune receptors. Recent studies have also advanced our understanding of how accessory proteins contribute to NB-LRR activation. The dynamic nature of NB-LRR localization to different subcellular compartments before and after activation suggests that NB-LRRs may activate immune responses in multiple parts of the cell. In this review we highlight recent advances in understanding NB-LRR function.     

Molecular determinants of glucagon receptor signaling

A 29-amino acid polypeptide hormone, glucagon has been one of the most prolific models in the study of hormone action. The key biologic function of glucagon is to counterbalance the actions of insulin and maintain a normal level of serum glucose. Diabetes mellitus can thus be considered a bihormonal disorder with an excess of glucagon contributing to the hyperglycemic state. The effects of glucagon are mediated by the glucagon receptor, which is itself a prototypical member of a distinct category called family B receptors within the G protein-coupled superfamily of seven-helical transmembrane receptors (GPCRs). At the structural level, the peptide ligands of family B receptors are highly homologous, in particular in the N-terminal region of the molecules. The mechanism by which highly homologous peptide ligands selectively recognize their receptors involves distinct molecular interactions that are gradually being elucidated. This review focuses on structural determinants of the glucagon receptor that are important for its activity with respect to interaction with its ligand and G proteins. Information about the glucagon receptor is presented within the context of what is known about other members of the family B GPCRs.     

Molecular and cellular constraints on vesicle traffic evolution

In eukaryotic cells, the budding and fusion of intracellular transport vesicles is carefully orchestrated in space and time. Locally, a vesicle's source compartment, its cargo, and its destination compartment are controlled by dynamic multi-protein specificity modules. Globally, vesicle constituents must be recycled to ensure homeostasis of compartment compositions. The emergence of a novel vesicle pathway therefore requires new specificity modules as well as new recycling routes. Here, we review recent research on local (molecular) constraints on gene module duplication and global (cellular) constraints on intracellular recycling. By studying the evolution of vesicle traffic, we may discover general principles of how complex traits arise via multiple intermediate steps.     

Molecular and biological constraints on ligand-binding affinity and specificity

Interactions between biological macromolecules have characteristic values of affinity and specificity that are set according to the biological function that is served by the interaction in the organism. Here we examine the molecular mechanisms that are used to achieve the required values of affinity and specificity in various biological systems. © 1997 John Wiley & Sons, Inc. 44: 181–198, 1997     

Molecular separation of two signaling pathways for the receptor, Notch

Notch is required for many aspects of cell fate specification and morphogenesis during development, including neurogenesis and axon guidance. We here provide genetic and biochemical evidence that Notch directs axon growth and guidance in Drosophila via a “non-canonical”, i.e. non-Su(H)-mediated, signaling pathway, characterized by association with the adaptor protein, Disabled, and Trio, an accessory factor of the Abl tyrosine kinase. We find that forms of Notch lacking the binding sites for its canonical effector, Su(H), are nearly inactive for the cell fate function of the receptor, but largely or fully active in axon patterning. Conversely, deletion from Notch of the binding site for Disabled impairs its action in axon patterning without disturbing cell fate control. Finally, we show by co-immunoprecipitation that Notch protein is physically associated in vivo with both Disabled and Trio. Together, these data provide evidence for an alternate Notch signaling pathway that mediates a postmitotic, morphogenetic function of the receptor.     

Molecular and Functional Analyses of a Maize Autoactive NB-LRR Protein Identify Precise Structural Requirements for Activity

Plant disease resistance is often mediated by nucleotide binding-leucine rich repeat (NLR) proteins which remain auto-inhibited until recognition of specific pathogen-derived molecules causes their activation, triggering a rapid, localized cell death called a hypersensitive response (HR). Three domains are recognized in one of the major classes of NLR proteins: a coiled-coil (CC), a nucleotide binding (NB-ARC) and a leucine rich repeat (LRR) domains. The maize NLR gene Rp1-D21 derives from an intergenic recombination event between two NLR genes, Rp1-D and Rp1-dp2 and confers an autoactive HR. We report systematic structural and functional analyses of Rp1 proteins in maize and N. benthamiana to characterize the molecular mechanism of NLR activation/auto-inhibition. We derive a model comprising the following three main features: Rp1 proteins appear to self-associate to become competent for activity. The CC domain is signaling-competent and is sufficient to induce HR. This can be suppressed by the NB-ARC domain through direct interaction. In autoactive proteins, the interaction of the LRR domain with the NB-ARC domain causes de-repression and thus disrupts the inhibition of HR. Further, we identify specific amino acids and combinations thereof that are important for the auto-inhibition/activity of Rp1 proteins. We also provide evidence for the function of MHD2, a previously uncharacterized, though widely conserved NLR motif. This work reports several novel insights into the precise structural requirement for NLR function and informs efforts towards utilizing these proteins for engineering disease resistance.     

Molecular insights into the biased signaling mechanism of the μ-opioid receptor

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Molecular identification of the human melanocortin-2 receptor responsible for ligand binding and signaling

The melanocortin-2 receptor (MC2R), also known as the adrenocorticotropic hormone (ACTH) receptor, plays an important role in regulating and maintaining adrenocortical function, specifically steroidogenesis. Mutations of the human MC2R (hMC2R) gene have also been identified in humans with familial glucocorticoid deficiency; however, the molecular basis responsible for hMC2R ligand binding and signaling remains unclear. In this study, both truncated ACTH peptides and site-directed mutagenesis studies were used to determine molecular mechanisms of hMC2R binding ACTH and signaling. Our results indicate that ACTH1-16 is the minimal peptide required for hMC2R binding and signaling. Mutations of common melanocortin receptor family amino acid residues E80 in transmembrane domain 2 (TM2), D107 in TM3, F178 in TM4, F235 and H238 in TM6, and F258 in TM7 significantly reduced ACTH-binding affinity and signaling. Furthermore, mutations of unique amino acids D104 and F108 in TM3 and F168 and F178 in TM4 significantly decreased ACTH binding and signaling. In conclusion, our results suggest that the residues in TM2, TM3, and TM6 of hMC2R share similar binding sites with other MCRs but the residues identified in TM4 and TM7 of hMC2R are unique and required for ACTH selectivity. Our study suggests that hMC2R may have a broad binding pocket in which both conserved and unique amino acid residues are required, which may be the reason why alpha-MSH was not able to bind hMC2R.     

Habitat constraints and spatial bias in seabird colony distributions

What governs the size and location of seabird colonies has long intrigued population ecologists. Previous analysis of the distribution of colonies of four European seabirds revealed a spatial bias - large colonies occurred farther apart than expected by chance alone - suggesting that intraspecific competition for food supplies during breeding may regulate colony size. Here we use computer intensive statistics to show that a similar spatial bias exists in three burrow-nesting seabirds - Cassin's auklet Ptychoramphus aleuticus , rhinoceros auklet Cerorhinca monoccrata , and ancient murrelet Synthliboramphus antiquns - nesting off the Pacific coast of Canada. Local habitat constraints explain much of the existing spatial bias: large colonies cannot fit on small islands, and large islands suitable for colonies tended to occur far apart. However, a residual spatial bias still remained for ancient murrelets and Cassin's auklets (but not rhinoceros auklets) after habitat constraints are built into the analysis, for which intraspecific food competition remains a plausible explanation.     

Molecular determinants of receptor binding and signaling by the CX3C chemokine fractalkine.

Fractalkine/CX3CL1 is a membrane-tethered chemokine that functions as a chemoattractant and adhesion protein by interacting with the receptor CX3CR1. To understand the molecular basis for the interaction, an extensive mutagenesis study of fractalkine's chemokine domain was undertaken. The results reveal a cluster of basic residues (Lys-8, Lys-15, Lys-37, Arg-45, and Arg-48) and one aromatic (Phe-50) that are critical for binding and/or signaling. The mutant R48A could bind but not induce chemotaxis, demonstrating that Arg-48 is a signaling trigger. This result also shows that signaling residues are not confined to chemokine N termini, as generally thought. F50A showed no detectable binding, underscoring its importance to the stability of the complex. K15A displayed unique signaling characteristics, eliciting a wild-type calcium flux but minimal chemotaxis, suggesting that this mutant can activate some, but not all, pathways required for migration. Fractalkine also binds the human cytomegalovirus receptor US28, and analysis of the mutants indicates that US28 recognizes many of the same epitopes of fractalkine as CX3CR1. Comparison of the binding surfaces of fractalkine and the CC chemokine MCP-1 reveals structural details that may account for their dual recognition by US28 and their selective recognition by host receptors.     

Combined spatial and enzymatic regulation of Csk by cAMP and protein kinase a inhibits T cell receptor signaling

Raft-associated Csk controls signaling through the T cell receptor (TCR) and was mainly anchored to Cbp/PAG (phosphoprotein associated with glycosphingolipid-enriched membrane domains). Treatment of cells with the cAMP-elevating agent prostaglandin E(2) (PGE(2)) augmented the level of Cbp/PAG phosphorylation with a concomitant increase in amounts of Csk bound to Cbp/PAG. While TCR-triggering resulted in transient dissociation of Csk from Cbp/PAG/rafts allowing TCR-induced tyrosine phosphorylation to occur, pretreatment with PGE(2) reduced Csk dissociation upon TCR triggering. This correlated with lowered TCR-induced phosphorylation of CD3 zeta-chain and linker for activation of T cells. Moreover, competition of endogenous Csk from lipid rafts abolished PGE(2)-mediated inhibition of TCR-induced zeta-chain phosphorylation and activation of the nuclear factor of activated T cells (NFAT) activator protein 1 (AP-1). Finally, raft-associated Csk already activated via Cbp/PAG binding, gained additional increase in phosphotransferase activity upon protein kinase A-mediated phosphorylation of Csk. We propose that cAMP regulates Csk via both spatial and enzymatic mechanisms, thereby inhibiting signaling through the TCR.     

Molecular mechanism for a role of SHP2 in epidermal growth factor receptor signaling

The Src homology 2-containing phosphotyrosine phosphatase (SHP2) is primarily a positive effector of receptor tyrosine kinase signaling. However, the molecular mechanism by which SHP2 effects its biological function is unknown. In this report, we provide evidence that defines the molecular mechanism and site of action of SHP2 in the epidermal growth factor-induced mitogenic pathway. We demonstrate that SHP2 acts upstream of Ras and functions by increasing the half-life of activated Ras (GTP-Ras) in the cell by interfering with the process of Ras inactivation catalyzed by Ras GTPase-activating protein (RasGAP). It does so by inhibition of tyrosine phosphorylation-dependent translocation of RasGAP to the plasma membrane, to its substrate (GTP-Ras) microdomain. Inhibition is achieved through the dephosphorylation of RasGAP binding sites at the level of the plasma membrane. We have identified Tyr992 of the epidermal growth factor receptor (EGFR) to be one such site, since its mutation to Phe renders the EGFR refractory to the effect of dominant-negative SHP2. To our knowledge, this is the first report to outline the site and molecular mechanism of action of SHP2 in EGFR signaling, which may also serve as a model to describe its role in other receptor tyrosine kinase signaling pathways.     

The tooth of perfection: functional and spatial constraints on mammalian tooth shape

This paper addresses the question of how close mammalian teeth are to ideal functional forms. An 'ideal' form is a morphology predicted to be the best functional shape according to information of the relationships between shape and function. Deviations from an ideal form are likely to indicate the presence of developmental or genetic constraints on form. Model tools were constructed to conform to functional principles from engineering and dental studies. The final model shapes are very similar to several mammalian tooth forms (carnassial teeth and tribosphenic-like cusps), suggesting that these tooth forms very closely approach ideal functional forms. Further evidence that these tooth forms are close to ideal comes from the conservation over 140 million years, the independent derivation and/or the occurrence over a size range of several orders of magnitude of these basic tooth forms. One of the main functional shapes derived here is the 'protoconoid', a fundamental design for double-bladed tools that fits a large number of functional parameters. This shape occurs in tooth forms such as tribosphenic, dilambdodont and zalambdodont. This study extends our understanding of constraints on tooth shape in terms of geometry (how space influences tooth shape) and function (how teeth divide food).  © 2003 The Linnean Society of London . Biological Journal of the Linnean Society , 2003, 78 , 173–191.