Chapter 9b-cellular Signaling.ppt

An important basic and advanced course of life science

Cellular and Molecular Biology: Concepts and Experiments Department of Biotechnology Zhi Huang (黄峙), Ph.D & Prof. Email: [email protected], Ph: 13678988403, qq:1547461148

Chapter 9

CELLULAR SIGNALING Learning Objectives: 1. Basic characteristics of cell signaling systems 2. The types of signal molecules, receptors, molecular switches and effectors; 3. The different signal transduction pathways; 4. The convergence, divergence, and crosstalk between different signaling pathways.

9.3 Cell signaling pathways

G protein coupled receptors (GPCR) mediated signaling pathways Receptor tyrosine kinases: a second major type of signaling pathway Intracellular receptor signaling pathways

Other signaling pathways

9.3.1 GPCR SIGNALING PATHWAYS G protein coupled receptors (GPCRs) constitute a large protein family of receptors that sense molecules outside the cell and activate inside signal transduction pathways and, ultimately, cellular responses. They are also called seventransmembrane receptors, 7TM receptors, because they pass through the cell membrane seven times.

 GPCRs are found only in eukaryotes, nearly 800 different human GPCRs genes (or ≈4% of the entire protein-coding genome) have been predicted from genome sequence analysis.  GPCRs’ ligands include light-sensitive compounds, odors, pheromones, hormones, and neurotransmitters, and vary in size from small molecules to peptides to large proteins.  GPCRs are involved in many diseases, and are also the target of approximately 40% of all modern medicinal drugs.  2012 Nobel prize in chemistry was awarded to Brian Kobilka and Robert Lefkowitz for their work that was "crucial for understanding how G-protein–coupled receptors function.

GPCR signaling and G protein cycle

Three types of GPCR Signaling Pathway

GPCRs associated with G protein-gated ion channels The cAMP signal pathway The phosphatidylinositol signal pathway

GPCRs associated with G protein-gated ion channels

 Potassium channels: muscarinic acetylcholine receptors (http://en.wikipedia.org/wiki/Muscarinic), or mAChRs, are acetylcholine receptors that form G protein-receptor complexes in the cell membranes of certain neurons and other cells. They play several roles, including acting as the main end-receptor stimulated by acetylcholine released from postganglionic fibers in the parasympathetic nervous system.

   

Calcium channels Sodium channels Chloride channels Visual phototransduction mediated by opsin GPCR: Opsins are a group of light-sensitive 35–55 kDa membrane-bound G proteincoupled receptors of the retinylidene protein family found in photoreceptor cells of the retina. Five classical groups of opsins are involved in vision, mediating the conversion of a photon of light into an electrochemical signal, the first step in the visual transduction cascade. Another opsin found in the mammalian retina, melanopsin, is involved in circadian rhythms and pupillary reflex but not in image-forming. (http://en.wikipedia.org/wiki/Opsin)

Three-dimensional structure of bovine rhodopsin. The seven transmembrane domains are shown in varying colors. The chromophoreis shown in red.

rhodopsin signal transduction

The cAMP dependent signal pathway

cAMP production

 The inactive form of protein kinase A (PKA) consists of two regulatory (R) and two catalytic (C) subunits.  Cyclic AMP binds to the R subunits of PKA, leading to their dissociation from the C subunits. The free C subunits are then enzymatically active and able to phosphorylate serine residues on their target proteins.  PKA phosphorylates key target enzymes  In many animal cells, increases in cAMP activate the transcription of specific target genes that contain a regulatory sequence called the cAMP response element, or CRE .

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CREB (cAMP response element-binding protein

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Relay of the signal from G protein to effectors

The free catalytic subunit of protein kinase A translocates to the nucleus and phosphorylates the transcription factor CREB (CRE-binding protein), leading to expression of cAMPinducible genes.

The phosphatidylinositol signal pathway

 Lipid-derived second messengers: phospholipid-based second messengers

 Generation of second messengers by PLC

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 IP3 signaling and Calcium flux  IP3 molecules formed at the membrane diffuse into the cytosol and bind to a specific IP3 receptor located at the surface of the smooth endoplasmic reticulum .  The IP3 receptor does more than bind a ligand; it is also a tetrameric Ca2+ channel. Binding of IP3 opens the channel, allowing Ca2+ ions to diffuse into the cytoplasm .  Calcium ions can also be considered as intracellular messengers because they bind to various target molecules, triggering specific responses. 20

PKC activity ◆Protein kinase C is a multifunctional serine and threonine kinase that phosphorylates a wide variety of proteins. ◆Protein kinase C has a number of important roles in cellular growth and differentiation, cellular metabolism, and transcriptional activation.

 IP3-Ca2+ pathway and DG-PKC pathway SignalsGPCR GP PLC IP3 and DAG (twin signals). IP3 IP3 receptor(Ca2+ channel, located at the surface of sER)  Elevation of cytosolic Ca2+; DAG activates PKC to phosphoralate Ser and Thr on target proteins. Calcium binds to calciumbinding proteins(CaM) which affects other proteins. Elevation of cytosolic Ca2+ via the IP signaling pathway

◆Calcium ions play a significant role in a remarkable variety of cellular activities, including muscle contraction, cell division, secretion, endocytosis, fertilization, synaptic transmission, metabolism, and cell movement. ◆Calmodulin activity Each molecule of calmodulin contains four binding sites for calcium. Calmodulin does not have sufficient affinity for Ca2+ to bind the ion in a non-stimulated cell. If, however, the Ca2+ concentration rises in response to a stimulus, the ions bind to calmodulin, changing the conformation of the protein and increasing its affinity for a variety of effectors.

Calmodulin

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Structure of calmodulin, a cytosolic protein of 148 amino acids that bind Ca2+ ions Ca2+/CaM dep. protein kinase (CaMkinase) mediate many of the actions of Ca2+ in animal cells.

The functions of increase the levels of cytosolic calcium-CaM : start-up embryo development after the fecundation. excitating contract of muscle cells;  excitating secretion of endocrine and nerve cells. Regulating calcium concentrations in plant cells Cytosolic calcium changes in response to several stimuli, including light, pressure, gravity, and hormones. Calcium signaling aids in decreasing turgor pressure in guard cells.

Ending the response Dropping in concentration of ligands ? Desensitization takes place in two steps. In the first step, the cytoplasmic domain of the activated GPCR is phosphorylated by a specific type of kinase called a G protein-coupled receptor kinase (GRK). Phosphorylation of the GPCR sets the stage for the second step, which is the binding of a protein, called arrestin, that inhibits the receptor’s ability to activate additional G proteins. This action is termed desensitization because the cell stops responding to the stimulus, even while that stimulus is still acting on the outer surface of the cell. Desensitization is one of the mechanisms that allows a cell to respond to a change in its environment, rather than continuing to “fire” endlessly in the presence of an unchanging environment. Signaling by the activated Gα subunit is terminated by a very different mechanism.

G-protein-linked receptor desensitization depends on receptor phosphorylation by PKA,PKC, CaMK2 or G-protein-linked receptor kinases(GRKs)

The target cells can become desensitized to a signal molecule by five ways.

Sequestration; down-regulation; protein

inactivation;

inactivation;

Three general ways of the desensitization: 1. Receptor inactivation by alteration; 2. Receptor sequestration by internalization; 3. Receptor down-regulation by destroying in Ls.

inhibitory

9.3.2 Receptor tyrosine kinase(RTK) and RTK-Ras signaling pathway  RTK are the second major type of cell-surface receptors  Signaling ligands to RTKs:

1.Nerve growth factor (NGF)  Six classes of enzyme-linked receptors have thus far been identified: 2.Platelet-derived growth factor Receptor tyrosine kinase(RTK); (PDGF) Tyrosine-kinase-associated receptor; 3.Fibroblast growth factor (FGF) Receptorlike tyrosine phosphatases; 4.Epidermal growth factor (EGF) Receptor serine/threonine kinase; 5.insulin and insulin-like GF(IGF-1) Receptor guanylyl cyclases; Histidine-kinase-associated receptor 6.ephrins(Eph)

7.vascular endothelial factor(VEGF)

 Ligand binding leads to RTK autophosphorylation RTK activity stimulated by cross-phosphorylation.

Phsphorylated Tyrosine Serve as docking sites for protein with SH2 domains (Src homology region). Other protein modules such as SH3 binds to proline-rich motifs in intracellular proteins.

dimerization

Steps in activation of Ras by RTKs Phosphotyrosines of RTK act as binding sites for a specific SH2 protein called GRB2 (Growth factor receptor binding protein in mammalian). GRB2 is not a protein with catalytic activity, but one that functions solely as an adapter molecule that links other proteins into a complex. Sos(son of sevenless) is a guanine nucleotide exchange factor for Ras (Ras-GEF) When a ligand binds to the RTK and recruits the Grb2-Sos to the inner surface of the membrane, the Sos protein binds to Ras causing GDP/GTP exchange, thus activating Ras.

In the cell, Ras activity is regulated by GAPs( GTPase – Activating proteins)—100 000-fold

 MAP-kinase serine/threonine phosphorylation Pathway activated by Ras

Ras-activated phosphorylation cascade

MAP kinase=mitogen-activated protein kinase; MAP-KKK=Raf (Ser/Thr-PK)

RTK-Ras signaling pathway

9.3.3 Other pathways: Jak-STAT signaling pathway Jaks: A class of cytoplasmic tyrosine kinases, including Jak1, Jak2, Jak3, and Tyk2, and each is associated with particular cytokine receptors; STATs: Jaks then phosphorylate and activate a set of latent gene regulatory proteins called STATs(signal transducers and activators of transcription,which have an SH2 domain),which move into the nucleus and stimulate the transcription of specific genes.

Signaling ligands and Cytokine receptors: Ligands: more than 30 cytokines and hormones activate the Jak-STAT pathway by binding to cytokine receptors. Cytokine receptors: they are composed of two or more poly peptide chains. All receptor are associated with one or more Jaks.

MBC 885: 15-63

The Jak-STAT signaling pathway avtivated by -interferon. Providing a fast track to the nucleus.

PI3K-PKB (Akt) signaling pathway http://cshperspectives.cshlp.org/content/4/9/a011189.full

Mechanisms of TGF-beta signaling from cell membrane to the nucleus http://genetica.umh.es/Doctorado/AvancesenGen%E9tica/2005/IO/Mechanisms% 20of%20TGF%20Signaling%20from%20Cell%20Membrane%20to%20the%20Nucleus.pdf

http://www.ncbi.nlm.nih.gov/pubmed/12809600

Wnt-beta Catenin signaling pathway Hedgehog signaling pathway

INTRACELLULAR RECEPTOR SIGNALING Intracellular receptor superfamily: Intracellular receptors are receptors located inside the cell rather than on its cell membrane. Examples are the class of nuclear receptors located in the cell nucleus and cytoplasm and the IP3 receptor located on the endoplasmic reticulum. The ligands that bind to them are usually intracellular second messengers like inositol trisphosphate (IP3) and extracellular lipophilic hormones like steroid hormones. Some intracrine peptide hormones also have intracellular receptors.

Nuclear receptors Nuclear receptors are a class of proteins found within cells that are responsible for sensing steroid, retinoic acid, vitamin D and thyroid hormones and certain other molecules. In response, these receptors work with other proteins to regulate the expression of specific genes, thereby controlling the development, homeostasis, and metabolism of the organism. Nuclear receptors have the ability to directly bind to DNA and regulate the expression of adjacent genes, hence these receptors are classified as transcription factors.

 Nuclear receptors are modular in structure and contain the following basic domains:  N-terminal regulatory domain: Contains the activation function 1 (AF-1) whose action is independent of the presence of ligand.  DNA-binding domain (DBD): Highly conserved domain containing two zinc fingers that binds to specific sequences of DNA called hormone response elemen  Ligand binding domain (LBD): Moderately conserved in sequence and highly conserved in structure between the various nuclear receptors. The LBD also contains the activation function 2 (AF-2) whose action is dependent on the presence of bound ligand.

 Action Mechanism of class I NRs

 Action Mechanism of Class II NRs

NO acts through the stimulation of the soluble guanylate cyclase

9.4 Convergence, divergence, and crosstalk among different signaling pathway

A. Convergence: Signals from a variety of unrelated receptors can converge to activate a common effecter.

B. Divergence: Signals from the same ligand can diverge to activate a variety of different effectors.

C. Crosstalk: Signals can be passed back and forth between different pathways

In actual fact, signaling pathways in the cell are much more complex.