Molecular Basis Of Signal Transduction Research Articles

Molecular basis of signal transduction mediated by the human GIPR splice variants

Glucose-dependent insulinotropic polypeptide receptor (GIPR) is a potential drug target for metabolic disorders. It works with glucagon-like peptide-1 receptor and glucagon receptor in humans to maintain glucose homeostasis. Unlike the other two receptors, GIPR has at least 13 reported splice variants (SVs), more than half of which have sequence variations at either C or N terminus. To explore their roles in endogenous peptide-mediated GIPR signaling, we determined the cryoelectron microscopy (cryo-EM) structures of the two N terminus-altered SVs (referred as GIPR-202 and GIPR-209 in the Ensembl database, SV1 and SV2 here, respectively) and investigated the outcome of coexpressing each of them in question with GIPR in HEK293T cells with respect to ligand binding, receptor expression, cAMP (adenosine 3,5-cyclic monophosphate) accumulation, β-arrestin recruitment, and cell surface localization. It was found that while both N terminus-altered SVs of GIPR neither bound to the hormone nor elicited signal transduction per se, they suppressed ligand binding and cAMP accumulation of GIPR. Meanwhile, SV1 reduced GIPR-mediated β-arrestin 2 responses. The cryo-EM structures of SV1 and SV2 showed that they reorganized the extracellular halves of transmembrane helices 1, 6, and 7 and extracellular loops 2 and 3 to adopt a ligand-binding pocket-occupied conformation, thereby losing binding ability to the peptide. The results suggest a form of signal bias that is constitutive and ligand-independent, thus expanding our knowledge of biased signaling beyond pharmacological manipulation (i.e., ligand specific) as well as constitutive and ligand-independent (e.g., SV1 of the growth hormone-releasing hormone receptor).

Molecular and Biochemical Mechanisms of Elicitors in Pest Resistance.

Insect herbivores have a variety of life cycles and feeding habits, making them extremely diverse. With their host plants, they form close relationships and suppress their defense mechanisms. Molecular elicitors are the key bio-elements in the detection and recognition of attacking enemies in tissue consumption. Insect oral secretion, frass, and fluid of egg deposition contain biologically active molecules called herbivore-associated elicitors (HAEs) that are recognized by pattern-recognition receptors (PRRs). Many plants distinguish insect feeding from wounding by HAEs present in their oral secretions (OS) and induce local and/or systemic responses against arthropod feeding. PRRs perceive HAEs in the oral secretion of caterpillars in a species-specific manner to elicit exclusive defense responses. HAEs-PRRs interactions induce plant resistance by reprogramming plant metabolism and transcriptional machinery. Quantitative, timely, and coordinated plant response initiate early signaling events, including Ca2+, reactive oxygen species (ROS), and mitogen-activated protein kinases (MAPKs). However, in insect herbivory, little is known about the molecular basis of signal transduction and regulation of plant resistance. We discuss here how early signaling cascades converge into the accumulation of phytohormones that regulate downstream special metabolites against herbivores. In this review, we propose a hypothetical model of PPRs-HAEs-mediated-induced responses in plants and discuss how PRRs-HAEs interactions elicit short- and long-term induced defenses in plants. The understanding of PRRs-HAEs interactions will help to explore the fundamental molecular mechanisms of host manipulation and may generate prospects to develop novel pest-resistance strategies.

Kinases and pseudokinases online symposium

The Biochemical Society’s Kinases and pseudokinases online symposium took place on 26 May, with 110 participants joining from around the globe. The event was convened to celebrate the 30th anniversary of the publication of the first ever protein kinase crystal structure by Professor Susan Taylor. Professor Taylor joined us as a keynote speaker, along with Professor Stefan Knapp from Frankfurt. Professor Taylor presented a tour de force covering her career, explaining how structural and functional analyses of kinases, including neglected PKA isoforms within the ‘dark kinome’, are advancing knowledge of the molecular basis of signal transduction. Professor Knapp discussed new insights into technologies, tools and challenges in studying pseudokinase structure and function.The remainder of the program focused on 25 emerging researchers, who presented short and flash talks, with contributions from the USA, Australia, Europe, India and Japan. PhD students, post-docs and early career faculty gave engaging presentations spanning microbial, plant, yeast and mammalian kinases and pseudokinases, impinging on host defence, cell death, oncogenesis, chemical biology and beyond. These ‘taster’ presentations now set the scene for what promises to be an outstanding 88th Harden Conference on kinases and pseudokinases, scheduled for May 2022 in Warwickshire, UK. Our thanks go to our sponsors, the Biochemical Journal and SVI Phosphocellulose, for supporting our speaker prizes, which were awarded to Dr Florentine Rutaganira (UC Berkeley, USA) and Dr Chris Horne (WEHI, Australia).

Molecular Cloning and Abiotic Stress Expression Analysis of GTPase Era Gene in Mulberry (Morus alba L.)

Escherichia coli Ras-like protein (Era) forms a special GTPase family, which functions in the regulation of the cell cycle by coupling cell division with the growth rate. A cDNA sequence coding GTPase Era mRNA has been cloned from mulberry in the present report. Sequence analysis revealed that its ORF is 1275 bp in length, encoding 424 amino acid protein. The GTPase Era gene had an era domain that belonged to the FeoB_N superfamily. Phylogenetic analysis of the amino acid sequences encoded by the GTPase gene from different species reveals that Morus alba L. was closely connected to Morus notabilis, Ipomoea nill, Capsicum annuum, Solanum pennellii, Nicotiana sylvestris and Nicotiana tabacum. GTPase Era expression patterns treated under drought, low temperature, and salt stress treatment were investigated using quantitative PCR (RT-qPCR) in real-time. The expression level of mRNA showed a significant change in drought, cold, and salt stress treatment compared to the standard growth environment. The highest expression were observed on the third day for drought stress, on the sixth day for low temperature and the seventh day for the salt stress. These findings provide a more definite knowledge on the molecular basis of signal transduction in mulberry trees during stress responses.

Directed evolution reveals the mechanism of HitRS signaling transduction in Bacillus anthracis.

Two component systems (TCSs) are a primary mechanism of signal sensing and response in bacteria. Systematic characterization of an entire TCS could provide a mechanistic understanding of these important signal transduction systems. Here, genetic selections were employed to dissect the molecular basis of signal transduction by the HitRS system that detects cell envelope stress in the pathogen Bacillus anthracis. Numerous point mutations were isolated within HitRS, 17 of which were in a 50-residue HAMP domain. Mutational analysis revealed the importance of hydrophobic interactions within the HAMP domain and highlighted its essentiality in TCS signaling. In addition, these data defined residues critical for activities intrinsic to HitRS, uncovered specific interactions among individual domains and between the two signaling proteins, and revealed that phosphotransfer is the rate-limiting step for signal transduction. Furthermore, this study establishes the use of unbiased genetic selections to study TCS signaling and provides a comprehensive mechanistic understanding of an entire TCS.

Molecular Cloning and Abiotic Stress Expression Analysis of Gtpase Era Gene in Mulberry (Morus Alba L.)

Era (E. coli RAS-like protein) forms a unique family of GTPase, its functions in cell cycle control by coupling cell division with growth rate. In the present paper, a cDNA sequence coding GTPase Era mRNA was cloned from mulberry. Sequence analysis showed that its open reading frame (ORF) is 1275 bp in length encoding a protein of 424 amino acids. The GTPase Era gene had era domain and belonged to the FeoB_N superfamily. Phylogenetic analysis based on the amino acid sequences encoded by the GTPase gene from various species showed that the mulberry was closely related to Morus notabilis, Ipomoea nil, Capsicum annuum, Solanum pennellii, Nicotiana sylvestris and Nicotiana tubacum. The expression patterns of GTPase Era treated under drought, low temperature and salt stresses were examined using real-time quantitative PCR (RT-qPCR). The expression level of the mRNA had a significant change under drought, cold and salt stress treatments compared to the normal growth environment with the maximum expression on third day for drought stress, the sixth day for the low temperature stress and the seventh day for the salt stress. These data provide a better understanding of the molecular basis of signal transduction during stress responses in mulberry trees

Ciliary Proteins: Filling the Gaps. Recent Advances in Deciphering the Protein Composition of Motile Ciliary Complexes.

Cilia are highly evolutionarily conserved, microtubule-based cell protrusions present in eukaryotic organisms from protists to humans, with the exception of fungi and higher plants. Cilia can be broadly divided into non-motile sensory cilia, called primary cilia, and motile cilia, which are locomotory organelles. The skeleton (axoneme) of primary cilia is formed by nine outer doublet microtubules distributed on the cilium circumference. In contrast, the skeleton of motile cilia is more complex: in addition to outer doublets, it is composed of two central microtubules and several diverse multi-protein complexes that are distributed periodically along both types of microtubules. For many years, researchers have endeavored to fully characterize the protein composition of ciliary macro-complexes and the molecular basis of signal transduction between these complexes. Genetic and biochemical analyses have suggested that several hundreds of proteins could be involved in the assembly and function of motile cilia. Within the last several years, the combined efforts of researchers using cryo-electron tomography, genetic and biochemical approaches, and diverse model organisms have significantly advanced our knowledge of the ciliary structure and protein composition. Here, we summarize the recent progress in the identification of the subunits of ciliary complexes, their precise intraciliary localization determined by cryo-electron tomography data, and the role of newly identified proteins in cilia.

Cloning and sequence analysis of the Δ1-pyrroline-5-carboxylate synthase gene (MP5CS) from mulberry (Morus alba) and patterns of MP5CS gene expression under abiotic stress conditions

ABSTRACTDrought is an important plant growth-limiting factor. ∆1-Pyrroline-5-carboxylate synthase (P5CS) is a key enzyme closely related to proline biosynthesis under drought stress. A full-length cDNA sequence from the P5CS gene in mulberry (Morus alba), designated MP5CS (GenBank Accession Number KC202259), was cloned. MP5CS was 2190 bp in length, contained a 47-bp 5ʹ-untranslated region (5ʹ-UTR) and a 648-bp 3ʹ-UTR, and encoded 497 amino acids with a predicted molecular weight of 53.92 kDa and an isoelectric point of 9.35. Phylogenetic analysis based on P5CS gene sequences from various species showed that mulberry was closely related to Boehmeria nivea, Malus hupehensis, Gossypium arboreum, and Euonymus japonicas. MP5CS gene expression under drought or salt stress conditions was quantified by quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR). The results showed that both abiotic stresses caused a significant upregulation in the expression of MP5CS in leaves compared to a normal growth environment. These data provide a better understanding of the molecular basis of signal transduction during stress responses in mulberry trees.

Application of computational approaches to study signalling networks of nuclear and Tyrosine kinase receptors

BackgroundNuclear receptors (NRs) and Receptor tyrosine kinases (RTKs) are essential proteins in many cellular processes and sequence variations in their genes have been reported to be involved in many diseases including cancer. Although crosstalk between RTK and NR signalling and their contribution to the development of endocrine regulated cancers have been areas of intense investigation, the direct coupling of their signalling pathways remains elusive. In our understanding of the role and function of nuclear receptors on the cell membrane the interactions between nuclear receptors and tyrosine kinase receptors deserve further attention.ResultsWe constructed a human signalling network containing nuclear receptors and tyrosine kinase receptors that identified a network topology involving eleven highly connected hubs.We further developed an integrated knowledge database, denominated NR-RTK database dedicated to human RTKs and NRs and their vertebrate orthologs and their interactions. These interactions were inferred using computational tools and those supported by literature evidence are indicated. NR-RTK database contains links to other relevant resources and includes data on receptor ligands. It aims to provide a comprehensive interaction map that identifies complex dynamics and potential crosstalk involved.Availability: NR-RTK database is accessible at http://www.bioinfo-cbs.org/NR-RTK/ConclusionsWe infer that the NR-RTK interaction network is scale-free topology. We also uncovered the key receptors mediating the signal transduction between these two types of receptors. Furthermore, NR-RTK database is expected to be useful for researchers working on various aspects of the molecular basis of signal transduction by RTKs and NRs.ReviewersThis article was reviewed by Professor Paul Harrison (nominated by Dr. Mark Gerstein), Dr. Arcady Mushegian and Dr. Anthony Almudevar.

Receptor tyrosine kinase signaling: a view from quantitative proteomics

Growth factor receptor signaling via receptor tyrosine kinases (RTKs) is one of the basic cellular communication principals found in all metazoans. Extracellular signals are transferred via membrane spanning receptors into the cytoplasm, reversible tyrosine phosphorylation being the hallmark of all RTKs. In recent years proteomic approaches have yielded detailed descriptions of cellular signaling events. Quantitative proteomics is able to characterize the exact position and strength of post-translational modifications (PTMs) providing essential information for understanding the molecular basis of signal transduction. Numerous new post-translational modification sites have been identified by quantitative mass spectrometry-based proteomics. In addition, plentiful new players in signal transduction have been identified underlining the complexity and the modular architecture of most signaling networks. In this review, we outline the principles of signal transduction via RTKs and highlight some of the new insights obtained from proteomic approaches such as protein microarrays and quantitative mass spectrometry.

Conformational Changes in the Integrin औA Domain Provide a Mechanism for Signal Transduction via Hybrid Domain Movement

The ligand-binding head region of integrin beta subunits contains a von Willebrand factor type A domain (betaA). Ligand binding activity is regulated through conformational changes in betaA, and ligand recognition also causes conformational changes that are transduced from this domain. The molecular basis of signal transduction to and from betaA is uncertain. The epitopes of mAbs 15/7 and HUTS-4 lie in the beta(1) subunit hybrid domain, which is connected to the lower face of betaA. Changes in the expression of these epitopes are induced by conformational changes in betaA caused by divalent cations, function perturbing mAbs, or ligand recognition. Recombinant truncated alpha(5)beta(1) with a mutation L358A in the alpha7 helix of betaA has constitutively high expression of the 15/7 and HUTS-4 epitopes, mimics the conformation of the ligand-occupied receptor, and has high constitutive ligand binding activity. The epitopes of 15/7 and HUTS-4 map to a region of the hybrid domain that lies close to an interface with the alpha subunit. Taken together, these data suggest that the transduction of conformational changes through betaA involves shape shifting in the alpha7 helix region, which is linked to a swing of the hybrid domain away from the alpha subunit.

The inflammatory aspect of the microcirculation in hypertension: oxidative stress, leukocytes/endothelial interaction, apoptosis.

Evidence is increasing in hypertensive models for an inflammatory reaction in the microcirculation with abnormal leukocyte counts and adhesion to the endothelium, enhanced arteriolar tone, and microvascular and tissue apoptosis. The spontaneous form of hypertension (SHR) is accompanied by a glucocorticoid-dependent increase in circulating leukocyte count with elevated levels of activation and at the same time depressed leukocyte-endothelial interaction and endothelial P-selectin function. The SHR exhibits immune suppression with lymphocyte apoptosis in the thymus. Generation of reactive oxygen species (ROS) in and around microvascular endothelial cells may regulate signal transduction pathways responsible for controlling gene expression and protein modification and thereby cause an elevation of vascular tone and, in excess, may form an injury mechanism for cells and tissue. A series of enzyme systems such as xanthine oxidase, reduced nicotinamide adenine dinucleotide phosphate/reduced nicotinamide adenine dinucleotide oxidase, and cytochrome P450 monooxygenases in conjunction with suppression of ROS scavengers seem to be involved in the oxidative stress responses in hypertension. The increase in ROS generation contributes to vascular remodeling, apoptosis, and proliferation of vascular smooth muscle, whereas gaseous monoxides such as nitric oxide and carbon monoxide have the ability to modulate elevated vascular tone and proliferative cell responses. Such biological actions of gases not only regulate activation of soluble guanylate cyclase but could also be attributable to inhibition of cytochrome P450 monooxygenases. We examine here the molecular basis of signal transduction by ROS, NO, and CO and functional alterations in their sensor molecules. An inflammatory reaction may underlie the pathogenesis of hypertension and its associated lesion formation and organ dysfunction.

Preface to The Molecular basis of signal transduction in plants. A Discussion Meeting held at the Royal Society of London on 18 and 19 February 1998.

Preface to The Molecular basis of signal transduction in plants. A Discussion Meeting held at the Royal Society of London on 18 and 19 February 1998. Organized and edited by N.-H. Chua, A. Hetherington, R. Hooley and R. F. Irvine.

Determination of photoreceptor cell fate in the Drosophila retina

Cell-cell communication directs the development of photoreceptor cells in the Drosophila retina. A recent set of studies has provided a genetic dissection of one of these interactions, the induction of the R7 photoreceptor cell by the neighboring R8 photoreceptor cell. The results from these experiments shape our understanding of both the molecular basis of signal transduction mediated through receptor tyrosine kinases, as well as the developmental strategies used to generate different cell types in neuronal systems.

Stimulation of cAMP and phosphomonoester production by melanotropin in melanoma cells: 31P NMR studies.

A major part of the present understanding of the molecular basis of signal transduction has been gained from in vitro studies using classical biochemical methods. In this study, we used 31P NMR spectroscopy to investigate the response of live M2R mouse melanoma cells to stimulation by melanocyte-stimulating hormone (MSH; melanotropin). In the presence of 3-isobutyl-1-methylxanthine and a synergistic dose of forskolin (1.67 microM), MSH induced a transient (approximately 60-min) rise in the cellular concentration of 3',5'-cyclic adenosine monophosphate (cAMP), which coincided in time with an equivalent decrease (approximately 40%) in ATP. However, no detectable change in phosphocreatine concentration was observed. Concomitantly, MSH induced a striking and unexpected increase in the concentration of three phosphomonoester (PME) metabolites (approximately 2-fold increase in total PME signal area); one signal has been assigned to phosphoethanolamine. The levels of the PMEs remained high for 2-4 hr and declined slowly (approximately 10 hr) to basal level, following perfusion with fresh culture medium. The increase in PME was also observed after stimulation with MSH alone. In contrast, stimulation with a high dose of forskolin (50 microM) and isobutylmethylxanthine (0.2 mM), although effective in stimulating the production of cAMP, did not induce the PME response. Evaluation of the cells' energetics indicated that the enhanced production of phosphoethanolamine is probably not due to ethanolamine phosphorylation. Therefore, it is likely to result from hydrolysis of phosphatidylethanolamine by a specific phospholipase C. The response of the PMEs appears to be regulated by a cAMP-independent process, suggesting the existence of an alternative transduction pathway controlled by MSH.

Reconstitution of functional receptor for human interleukin-2 in mouse cells

Interleukin-2 (IL-2) has a key role in the antigen-specific clonal growth of T lymphocytes, by virtue of its interaction with a specific cell-surface receptor (IL-2R). The growth signal seems to be delivered by IL-2 bound to the high-affinity, but not the low-affinity, receptor. Genes encoding IL-2 and its receptor (that is, Tac-antigen) have been cloned and analysed in detail. We have now achieved cell-type-specific reconstitution of the high-affinity human IL-2R by expressing the complementary DNA cloned from normal lymphocytes. A mouse T-lymphocytic line, EL-4, expressed human IL-2R with high (dissociation constant (Kd) = 160-220 pM) and low (Kd = 2.1-2.2 nM) affinity for recombinant human IL-2, while mouse L929 cells expressed only a single class of the IL-2R with lower affinity (Kd = 34.5 nM) for the ligand. We also show that the human IL-2R expressed in EL-4 cells responds to IL-2 and mediates reversed signal transduction: growth of the EL-4 cells harbouring the IL-2R is inhibited specifically by human recombinant IL-2. The approach described here may provide a general experimental framework for elucidating the molecular basis of signal transduction mediated by specific receptor-ligand interaction.