BL 424 Chapter 15: Cell Signaling; Signal Transduction All cells receive and respond to signals from their environments. The behavior of each individual cell in multicellular plants and animals must be carefully regulated to meet the needs of the organism. This is accomplished by a variety of signaling molecules that are secreted or expressed on the surface of one cell and bind to receptors expressed on other cells, thereby integrating and coordinating the functions of cells. The binding of most signaling molecules to their (cell surface) receptors initiates a series of intracellular reactions that regulate all aspects of cell behavior: metabolism, movement, proliferation, differentiation. The study of these processes remains a major area of research, in large part because breakdown of signaling paths results in cancer. Student Learning Outcomes: Transmembrane proteins outside the cell communicate signals to the inside 1*. to explain the various signaling molecules and their cell surface receptors: how cells receive information from outside. 2*. to explain the several general ways signals to receptors are transmitted through the cell to regulate processes (second messenger pathways) 3. to describe examples of signal transduction in development and differentiation, 4. to describe the involvement of the cytoskeleton in signal transduction. Important Figures: 1*, 3*, 4*, 5, 6, 11, 12*, 13*, 14, 15*, 16*, 19, 20, 21*, 22, 25, 26*, 27, 28,* 29, 30, 31, 32, 34*, 35, 41, 42, 43*, 44, 45, 46*; Table: 1* 15.1. Signaling molecules and their receptors. Signaling can involve direct cell-cell contact (cell-matrix) including integrins and cadherins A common mode of cell-cell signaling involves secreted molecules that bind to receptors on target cells. Signaling is categorized according to distance signals are transmitted: (Fig. 15.1) Endocrine distant - many hormones (e.g., estrogen) Paracrine local many neurotransmitters Autocrine self ex. T lymphocytes respond to antigen by synthesizing growth factors to stimulate proliferation **a. Steroid hormones and nuclear receptor superfamily. Hormones are hydrophobic (Fig. 15.2); diffuse across plasma membrane to bind intracellular receptors. Nuclear receptor family are transcription factors that bind specific DNA targets and activate transcription when hormone is bound to them (Figs. 15.3, 4). Ex. estrogen, progesterone, thyroid hormone, glucocorticoid Retinoic acid, vitamin D receptors GR bound to Hsp90 is inactive; Glucocorticoid makes GR active (binds coactivator HAT, transcription) TR normally repressing complex; T makes it active (binds coactivator HAT)
b. Nitric oxide (and carbon monoxide) Paracrine signaling in nervous system, immune, circulatory Ex. dilation of blood vessels (Fig. 15.5) Nitric oxide synthase: arg + O 2 -> citrulline + NO NO diffuses across membranes, affects enzymes Ex. guanylyl cyclase makes cyclic GMP (2 nd messenger) **c. Neurotransmitters are small hydrophilic molecules that carry signals between neurons, or between neurons and other target cells at synapses. (Fig. 15.6) Ex. acetylcholine, dopamine, glutamate Many bind to cell-surface ligand-gated ion channels (Figs. 13.24, 25) Others bind cell-surface receptors, and are coupled to G proteins to indirectly regulate an ion channel. Some neurotransmitters are also hormones: Ex. epinephrine (adrenaline) signals glycogen breakdown in muscles **d. Peptide hormones and growth factors These molecules have diverse sizes (5-230 aa) and roles: Ex. insulin, endorphin, nerve growth factor (NGF), epidermal growth factor (EGF) (Table 1). Signal does not enter cell, but binds cell-surface Transmembrane receptor Neuropeptides are secreted by some neurons; Some act as hormones on distant cells (enkephalins, endorphins). Growth factors are polypeptides and regulate cell growth: NGF affects nerve cells. EGF (53-aa) stimulates cell proliferation (fig. 15.7). e. Eicosanoids are lipids that function in paracrine or autocrine signaling: ex. prostaglandin, leukotriene (Fig.15. 8) They are synthesized from arachidonic acid (which is derived from phospholipids). Aspirin (NSAID) reduces inflammation & pain by inhibiting synthesis of prostaglandins (reduces platelet aggregation, blood clotting) f. Plant hormones are small molecules that regulate plant growth: (Fig. 15.9) auxin, gibberrellin, ethylene Some activate protein kinases Auxin also stimulates ubiquitination And degradation of a repressor of ARF (auxin response factor)
**15.2 Functions of cell-surface receptors Most ligands bind receptors on surface of target cell; Signal is transmitted inside. Some receptors are ligand-gated ion channels that directly control activity (Ch. 13) Other receptors regulate activity of intracellular proteins, Which transmits signal to intracellular target (often transcription factors). a. G-protein coupled receptors are the largest family: (1000); transmembrane proteins (7 times) (Fig. 11) Includes receptors for many hormones, eicosanoids, neurotransmitters, neuropeptides, also smell, taste, sight. Hormone causes receptor to transmit signals to intracellular targets via intermediary action of heterotrimeric G proteins (Fig. 13). Hormone binding to extracellular part of receptor causes shape change in cytoplasmic part, which then binds G protein. The α subunit binds GTP, is activated and dissociates from βγ. α subunit binds to enzyme or ion channel protein to transmit signal. (βγ can also transmit signals) Ex. epinephrine receptor regulates target enzyme of adenylyl cyclase (Fig. 12): ATP -> camp ex. Acetylcholine regulates ion channels via receptor which is ligand-gated ion channel. Acetylcholine and different receptor can regulate Heart muscle via G proteins Mammals have 20 a, 5 β and 12 γ, so different combinations for different receptors and can inhibit or stimulate when activated b. Receptor protein-tyrosine kinases (RTK) Receptors are directly linked to intracellular enzyme activity; includes receptors for most growth factors (Fig. 14). ex. insulin, EGF, NGF receptors Receptors have N-terminal extracellular portion, C-terminal tyr kinase; Binding of hormone causes receptor dimerization and autophosphorylation (Fig. 15.15*), Other proteins bind the Phospho-peptide of RTK; these proteins localize and get phoshorylated, and then are stimulated and transmit signal. They bind via SH2 domain (Src homology 2, RSV oncogene) or PTB domain (phospho-tyrosine binding) (Fig. 15.16).
c. Cytokine receptors often act associated non-covalent with cytoplasmic nonreceptor protein-tyrosine kinases. (Fig. 15.18) Ex. interleukins, erythropoietin, Some polypeptide hormones (growth hormones) Receptors have N-terminal extracell ligand-binding domain, C-terminal cytosolic (no enzyme activity). Ligand binding stimulates cross-phosphorylation of associated protein tyr kinases: Janus kinase (JAK) family Src kinase family (has SH2 domains) d. Receptors linked to other enzymatic activities: Protein-tyrosine phosphatases removes phosphates often terminates signaling, some are cell surface CD45 on B and T cells dephosphorylates a phospho-tyr residue that was inhibiting a Src kinase and therefore stimulates. Protein-serine/threonine kinases TGF-β receptor for transforming growth factor β Binding of polypeptide growth factor -> receptors dimerize, phosphorylate self and bind, PO4 Smads, which stimulate gene expression Guanylyl cyclases Receptor directly linked to cgmp activity Protease-associated Apoptosis TNF (Ch. 16) Tumor necrosis factor signals proteolysis **15.3 Pathways of intracellular signal transduction. Binding of signal to cell surface receptor stimulates intracellular targets, including enzymes or transcription; directly or indirectly via proteins coupled to receptors. Hormone outside the cell is the first signal; intracellular second messengers are small molecules and enzymes. Enzymes amplify and propagate ligand signal; often consist of cascades of reactions that affect gene expression or cell metabolism. A*. camp pathway and protein phosphorylation Many hormones and odorants use cyclic AMP (camp) as second message in animal cells. Ex. Epinephrine signals glycogen breakdown to glucose in muscle cells. (Fig. 15.21) G-protein coupled receptors stimulate adenylyl cyclase to make camp from ATP. Phosphodiesterase degrades camp to AMP to remove signal.
Most actions of camp are mediated by protein kinase A (PKA) (Fig. 19, 20); Binding of camp dissociates regulatory subunit. Active PKA adds phosphate to ser/thr of enzymes; PO 4 of phosphorylase kinase makes it active; PO 4 of glycogen synthase makes it inactive. PO 4 Of transcription factor CREB (camp response element binding factor) (Fig. 22) stimulates transcription. Phosphatases (protein phosphatase 1) stop signal; Odorant hormones stimulate via camp to open a Na+ channel. b. cyclic GMP (cgmp) is also a second messenger. cgmp is formed by guanylyl cyclases; is degraded by phosphodiesterase ex. visual reception in vertebrate eye: light activates via G protein transducin (Fig. 15.24) which stimulates cgmp phosphodieseterase; decreased cgmp stimulates nerve. C*. Phospholipids and Ca++ are common second messengers. Phosphatidyl inositol 4,5 bisphosphate (PIP2) (Fig. 15.25) Is found in inner leaflet of plasma membrane. Hormones and growth factors stimulate hydrolysis Of PIP2 by Phospholipase C (PLC) to form: Diacylglycerol (DAG) in the membrane Iinositol triphosphate (IP3) in cytosol. These compounds activate two signaling paths. DAG activates protein kinase C (PKC) and signals cell growth. IP3 binds IP3 receptor on ER, and mobilizes Ca++ from intracellular stores (ER lumen). Increased Ca++ activates targets, often mediated by Calmodulin binding Ca++, and then binding to Calmodulin-dependent protein kinases which add Phosphates to other enzymes (Fig. 15.28) Ex. myosin light chain kinase (Ch. 13). In electrically excitable cells of nerve and muscle, Ca++ is increased by opening of voltage-gated Ca++ channels in plasma membrane, and ryanodine receptors in the ER and sarcoplasmic reticulum (Fig. 15.29). Increased Ca++ triggers release of neurotransmitter, muscle contraction.
PI3-kinase/AKT and mtor path PIP2 also gets phosphorylated to second messenger PIP3: By enzyme PI3-kinase (Fig. 15.30). Binds protein ser/tr kinase AKT (Fig. 15.31) which then gets phosphorylated by two protein kinases. Active AKT phosphorylates target proteins for cell survival (Ch. 16). One target is mtor, central regulator of cell growth. D*. Ras, Raf and MAP kinase pathway is conserved chain of protein kinases which convey downstram signals. (Fig. 15.34*). MAP kinases (Mitogen-activated protein kinases) are ser/thr kinases that respond to growth factors (figs. 15.38,39). In animal cells, best-characterized pathway is growth factor receptors and small GTP binding protein Ras, which initates kinase cascade via Raf kinases (ser/thr) to MEK (thr, tyr) to the MAP kinase (ERK, extracell signal regulated kinase) Activated ERK adds PO 4 to cytosolic and nuclear proteins, including transcription factor Elk-1 (fig. 15.37). Binding of growth factor to RTK, and its phosphorylation recruited GRB2 and SOS (GEF) to give Ras GTP (Fig. 15.36). Ras is normal protein, required for cell function (proto-oncogene). Ras protein was first found as mutated version: oncogene carried by virus (Rat sarcoma virus); mutated Ras binds GTP and is always active. Normally, monomer Ras is activated by GTP binding. Ras and relatives are ½ size of the G protein Gα, Ran (nuclear transport), Rab (vesicle fusion), Rho (cytoskeleton). e. JAK/STAT pathway and TGFβ/Smad. (Fig. 40, 41) STAT proteins are transcription proteins containing SH2 domains. They are activated directly by JAK protein-tyr kinases associated with cytokine and growth factor receptors. STAT (signal transducers & activators of transcription). NFKβ family of transcription factors (Fig. 42). Are important in immune system. Signal outside -> inhibitory IKβ gets PO 4 by IKβ kinase -> NFKβ goes to nucleus to stimulate transcription.
f. Hedgehog, Wnt and Notch signaling. These signals, discovered in Drosophila, Are critical for development, patterning organism. Transmembrane receptor, secreted signals (Figs. 15.43, 44, 45) Hedgehog and Wnt pathways act to prevent degradation of transcription factors that are held in complexes in cytoplasm. Notch is receptor in membrane upon receiving signal from neighboring cell, cytoplasmic fragment is cleaved and goes to nucleus. 15.4 Signal transduction and the cytoskeleton a. Integrins and signal transduction. Binding of integrins to extracellular matrix stimulates nonreceptor protein-tyr kinases FAK (Focal Adhesion kinase) and Src -> Activation of Phospholipase C (PLC), PI3-kinase and Ras/Raf/ERK paths (Figs.15. 46) b. Regulation of actin cytoskeleton (Figs. 15.47-49) Occurs through growth factors that induce alterations Mediated by members of Rho subfamily of small GTP-binding proteins (Rho, Rac, Cdc42). Myosin light chain kinase 15.5 Signaling networks Include feedback for NFKβ (Φιγσ. 15.50) Cross-talk ERK path & G-coupled receptors & B-arrestin (Fig. 15.51) Assemble networks different ways positive or negative (fig. 15.52) Questions for review: Questions, all but #9. Diagram a plasma membrane with the two major signaling paths. Consider effects of mutations of the receptors, enzymes Prepare list of major types of signals, of enzymes and what they do.