Structural basis for cytokinin receptor signaling: an evolutionary approach

Cytokinins are ubiquitous plant hormones; their signal is perceived by sensor histidine kinases—cytokinin receptors. This review focuses on recent advances on cytokinin receptor structure, in particular sensing module and adjacent domains which play an important role in hormone recognition, signal transduction and receptor subcellular localization. Principles of cytokinin binding site organization and point mutations affecting signaling are discussed. To date, more than 100 putative cytokinin receptor genes from different plant species were revealed due to the total genome sequencing. This allowed us to employ an evolutionary and bioinformatics approaches to clarify some new aspects of receptor structure and function. Non-transmembrane areas adjacent to the ligand-binding CHASE domain were characterized in detail and new conserved protein motifs were recovered. Putative mechanisms for cytokinin-triggered receptor activation were suggested.     

EGF receptor signalling: roles of star and rhomboid revealed.

Recent studies have clarified how the active form of the Drosophila EGF receptor ligand Spitz is produced: Star chaperones Spitz in the ER and mediates its transport to the Golgi, where the intramembrane serine protease Rhomboid cleaves the Spitz proprotein to initiate secretion.     

Negative receptor signalling

Binding of external factors to cell membrane receptors triggers intracellular signalling pathways that ultimately determine if the cell proliferates, differentiates or undergoes apoptosis. Activated receptors also initiate a cascade of events, called negative receptor signalling, that decreases the amplitude of positive signals and modulates the level of cell stimulation. Recent studies have revealed that negative signalling by receptor tyrosine kinases involves coordinated action of ubiquitin ligases (i.e. Cbl), adaptor proteins (i.e. Grb2 and CIN85), inhibitory molecules (i.e. Sprouty), cytoplasmic kinases (i.e. activated Cdc42-associated kinase) and phosphoinositol metabolites. These inhibitory signals are essential for normal cell functioning, and their deregulation often results in human diseases.     

Exogenous brassinosteroids activate cytokinin signalling pathway gene expression in transgenic Arabidopsis thaliana

Transgenic Arabidopsis thaliana plants carrying the GUS reporter gene fused to the promoter of the gene of primary response to cytokinins (CKs), ARR5, were used to estimate the influence of several brassinosteroids (BRs): brassinolide (BL), epibrassinolide (EBL), homobrassinolide (HBL), and 6-o-carboxymethyloxohomocastasterone (CHC) on the expression of CK signalling genes. BRs tested differed in their ability to activate the ARR5 gene promoter in 4-day-old seedlings and 3-week-old plants. BL caused the most prominent effect, yet it was considerably less than that of 6-benzylaminopurine (BA). An increase in GUS activity was observed in both dark and light conditions; however, the rate of elevation was higher in dark conditions. The activation of the P _ARR5 :GUS fusion was accompanied by a moderate induction of the P _AHK :GUS constructs, in which the reporter GUS gene was fused to the promoter of one of the CK receptor histidine kinases. The effects of BL on the AHK gene promoters were organ specific and correlated with the ability of a particular AHK gene to respond to BA treatment. BL activated the AHK3 promoter in 4-day-old seedlings and in shoots and roots of 3-week-old plants without any effect in detached leaves. The AHK2 gene promoter was activated by BA and BL only in seedlings, whereas the AHK4 gene promoter was activated only in roots. BL treatment caused the coordinate elevation of the CK levels in leaves to the same degree as the activation of the P _AHK :GUS construct, suggesting that the accumulation of CKs was the reason for the activation by BRs of the CK signalling genes. The data obtained provide the evidence for the involvement of BRs in the regulation of the genes of the CK signalling pathway through an increase in the CK levels. However, the exact molecular mechanisms underlying BR-induced elevation of the CK content are unclear and warrant identification in the future.     

Thromboxane receptor signalling in human myometrial cells.

We measured the effects of stable thromboxane A2 (TXA2) analogues on signalling in cultured human myometrial cells. U46619 and/or IBOP stimulated total inositol phosphates (IPs) and cAMP production, RhoA-associated protein kinase (ROK) activity and elevated intracellular calcium [Ca2+]i. Pretreatment of the cells with pertussis toxin did not inhibit IPs or [Ca2+]i production but the thromboxane receptor (TP) antagonist SQ-29548 did inhibit IPs and cAMP production, the elevation of [Ca2+]i, and the increase in ROK activity. Pretreatment with thapsigargin inhibited [Ca2+]i elevation. TP receptor-stimulated ROK activity was inhibited by the ROK inhibitor Y27632 while ROK activity was enhanced by the caspase 3 inhibitor, Z-DEVD-FMK. TP receptor-stimulated IPs production is additive to prostaglandin F2alpha (FP) or prostaglandin E (EP) receptor-stimulated IPs production and neither FP nor EP receptor-stimulated IPs production is inhibited by SQ29548. Thus cultured human myometrial cells express at least two functional TP receptor subtypes; TPalpha-like (cAMP-stimulating) and TPbeta-like (IPs, [Ca2+] and ROK-stimulating).     

Review: Negative regulation of leptin receptor signalling

Leptin was discovered as an adipostat, regulating body weight by balancing food intake and energy expenditure. Recently, leptin emerged as a pleiotropic cytokine. It plays a substantial role in a wide spectrum of other functions including immune regulation, bone formation and fertility. Leptin signalling is under tight control. Aberrations of this stringent control system may be implicated in a variety of pathologies. Here, we review the various mechanisms that control cellular leptin receptor signalling.     

EGF receptor signalling: the importance of presentation

Activation of the Drosophila EGF receptor requires the transmembrane TGF-alpha-like ligand Spitz. Recent studies have shed new light on the role of two transmembrane proteins, Star and Rhomboid, in the presentation and subsequent proteolytic processing of Spitz.     

MicroRNAs: the fine-tuners of Toll-like receptor signalling

Toll-like receptor (TLR) signalling must be tightly regulated to avoid excessive inflammation and to allow for tissue repair and the return to homeostasis after infection and tissue injury. MicroRNAs (miRNAs) have emerged as important controllers of TLR signalling. Several miRNAs are induced by TLR activation in innate immune cells and these and other miRNAs target the 3' untranslated regions of mRNAs encoding components of the TLR signalling system. miRNAs are also proving to be an important link between the innate and adaptive immune systems, and their dysregulation might have a role in the pathogenesis of inflammatory diseases.     

Activation of signalling by the activin receptor complex.

Activin exerts its effects by simultaneously binding to two types of p rotein serine/threonine kinase receptors, each type existing in various isoforms. Using the ActR-IB and ActR-IIB receptor isoforms, we have investigated the mechanism of activin receptor activation. ActR-IIB are phosphoproteins with demonstrable affinity for each other. However, activin addition strongly promotes an interaction between these two proteins. Activin binds directly to ActR-IIB, and this complex associates with ActR-IB, which does not bind ligand on its own. In the resulting complex, ActR-IB becomes hyperphosphorylated, and this requires the kinase activity of ActR-IIB. Mutation of conserved serines and threonines in the GS domain, a region just upstream of the kinase domain in ActR-IB, abrogates both phosphorylation and signal propagation, suggesting that this domain contains phosphorylation sites required for signalling. ActR-IB activation can be mimicked by mutation of Thr-206 to aspartic acid, which yields a construct, ActR-IB(T206D), that signals in the absence of ligand. Furthermore, the signalling activity of this mutant construct is undisturbed by overexpression of a dominant negative kinase-defective ActR-IIB construct, indicating that ActR-IB(T206D) can signal independently of ActR-IIB. The evidence suggests that ActR-IIB acts as a primary activin receptor and ActR-IB acts as a downstream transducer of activin signals.     

Ligand-specific receptor states: implications for opiate receptor signalling and regulation

Opiate drugs produce their effects by acting upon G protein coupled receptors (GPCRs) and although they are among the most effective analgesics available, their clinical use is restricted by unwanted side effects such as tolerance, physical dependence, respiratory depression, nausea and constipation. As a class, opiates share a common profile of unwanted effects but there are also significant differences in ligand liability for producing these actions. A growing number of studies show that GPCRs may exist in multiple active states that differ in their signalling and regulatory properties and which may distinctively bind different agonists. In this review we summarize evidence supporting the existence of multiple active conformations for MORs and DORs, analyze information favouring the existence of ligand-specific receptor states and assess how ligand-selective efficacy may contribute to the production of longer lasting, better tolerated opiate analgesics.     

Src-related protein tyrosine kinases and T-cell receptor signalling.

Upon antigen stimulation, the T-cell receptor for antigen transduces an intracellular protein tyrosine phosphorylation signal that is critical for subsequent T-lymphocyte activation. As the antigen receptor does not possess an intrinsic protein tyrosine kinase activity, the mechanism by which it regulates protein tyrosine phosphorylation is unconventional. Evidence is increasing that the Src-related protein tyrosine kinases P56lck and p59fyn, as well as the protein tyrosine phosphatase CD45, are involved in this process.     

Alternate signalling pathways from the interleukin-2 receptor.

Interleukin-2 (IL-2) plays a major role in the proliferation of cell populations during an immune reaction. The beta(c) and gamma(c) subunits of the IL-2 receptor (IL-2R) are sufficient and necessary for signal transduction. Despite lacking known catalytic domains, receptor engagement leads to the activation of a diverse array protein tyrosine kinases (PTKs). In resting or anergised T cells, Jak3 is not activated. Signals arising from the PROX domain of the gamma(c) subunit activate p56(lck) (lck) leading to the induction of anti-apoptotic mechanisms. When Jak3 is activated, in primed T cells, other PTKs predominantly mediate the induction of anti-apoptotic mechanisms and drive cellular proliferation. This review intends to suggest a role for these differences within the context of the immune system.     

Dectin-1: a signalling non-TLR pattern-recognition receptor

Dectin-1 is a natural killer (NK)-cell-receptor-like C-type lectin that is thought to be involved in innate immune responses to fungal pathogens. This transmembrane signalling receptor mediates various cellular functions, from fungal binding, uptake and killing, to inducing the production of cytokines and chemokines. These activities could influence the resultant immune response and can, in certain circumstances, lead to autoimmunity and disease. As I discuss here, understanding the molecular mechanisms behind these functions has revealed new concepts, including collaborative signalling with the Toll-like receptors (TLRs) and the use of spleen tyrosine kinase (SYK), that have implications for the role of other non-TLR pattern-recognition receptors in immunity.     

Early cytokinin response proteins and phosphoproteins of Arabidopsis thaliana identified by proteome and phosphoproteome profiling

Cytokinins are plant hormones involved in regulation of diverse developmental and physiological processes in plants whose molecular mechanisms of action are being intensely researched. However, most rapid responses to cytokinin signals at the proteomic and phosphoproteomic levels are unknown. Early cytokinin responses were investigated through proteome-wide expression profiling based on image and mass spectrometric analysis of two-dimensionally separated proteins and phosphoproteins. The effects of 15 min treatments of 7-day-old Arabidopsis thaliana seedlings with four main cytokinins representing hydroxyisopentenyl, isopentenyl, aromatic, and urea-derived type cytokinins were compared to help elucidate their common and specific function(s) in regulating plant development. In proteome and phosphoproteome maps, significant differences were reproducibly observed for 53 and 31 protein spots, respectively. In these spots, 96 proteins were identified by matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS), providing a snapshot of early links in cytokinin-regulated signalling circuits and cellular processes, including light signalling and photosynthesis, nitrogen metabolism, the CLAVATA pathway, and protein and gene expression regulation, in accordance with previously described cytokinin functions. Furthermore, they indicate novel links between temperature and cytokinin signalling, and an involvement of calcium ions in cytokinin signalling. Most of the differentially regulated proteins and phosphoproteins are located in chloroplasts, suggesting an as yet uncharacterized direct signalling chain responsible for cytokinin action in chloroplasts. Finally, first insights into the degree of specificity of cytokinin receptors on phosphoproteomic effects were obtained from analyses of cytokinin action in a set of cytokinin receptor double mutants.     

Insulin receptor internalization and signalling

The insulin receptor kinase (IRK) is a tyrosine kinase whose activation, subsequent to insulin binding, is essential for insulin-signalling in target tissues. Insulin binding to its cell surface receptor is rapidly followed by internalization of insulin-IRK complexes into the endosomal apparatus (EN) of the cell. Internalization of insulin into target organs, especially liver, is implicated in effecting insulin clearance from the circulation. Internalization mediates IRK downregulation and hence attenuation of insulin sensitivity although most internalized IRKs readily recycle to the plasma membrane at physiological levels of insulin. A role for internalization in insulin signalling is indicated by the accumulation of activated IRKs in ENs. Furthermore, the maximal level of IRK activation has been shown to exceed that attained at the cell surface. Using an in vivo rat liver model in which endosomal IRKs are exclusively activated has revealed that IRKs at this intracellular locus are able by themselves to promote IRS-1 tyrosine phosphorylation and induce hypoglycemia. Furthermore, studies with isolated rat adipocytes reveal the EN to be the principle site of insulin-stimulated IRS-1 tyrosine phosphorylation and associated PI3K activation. Key steps in the termination of the insulin signal are also operative in ENs. Thus, an endosomal acidic insulinase has been identified which limits the extent of IRK activation. Furthermore, IRK dephosphorylation is effected in ENs by an intimately associated phosphotyrosine phosphatase(s) which, in rat liver, appears to regulate IRK activity in both a positive and negative fashion. Thus, insulin-mediated internalization of IRKs into ENs plays a crucial role in effecting and regulating signal transduction in addition to modulating the levels of circulating insulin and the cellular concentration of IRK in target tissues.     

Membrane signalling complexes: implications for development of functionally selective ligands modulating heptahelical receptor signalling

Technological development has considerably changed the way in which we evaluate drug efficacy and has led to a conceptual revolution in pharmacological theory. In particular, molecular resolution assays have revealed that heptahelical receptors may adopt multiple active conformations with unique signalling properties. It is therefore becoming widely accepted that ligand ability to stabilize receptor conformations with distinct signalling profiles may allow to direct the stimulus generated by an activated receptor towards a specific signalling pathway. This capacity to induce only a subset of the ensemble of responses regulated by a given receptor has been termed "functional selectivity" (or "stimulus trafficking"), and provides the bases for a highly specific regulation of receptor signalling. Concomitant with these observations, heptahelical receptors have been shown to associate with G proteins and effectors to form multimeric arrays. These complexes are constitutively formed during protein synthesis and are targeted to the cell surface as integral signalling units. Herein we summarize evidence supporting the existence of such constitutive signalling arrays and analyze the possibility that they may constitute viable targets for developing ligands with "functional selectivity".     

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.