Cellular & Intracellular Communications PDF

Cellular & Intracellular Communications Dr. Halil Resmi Introduction Bacteria and unicellular eukaryotes respond to environmental signals, and to signaling molecules secreted by other cells—for proliferation and other communication. In multicellular orga...

Cellular & Intracellular Communications Dr. Halil Resmi Introduction Bacteria and unicellular eukaryotes respond to environmental signals, and to signaling molecules secreted by other cells—for proliferation and other communication. In multicellular organisms, cell-cell communication is highly sophisticated. Each cell must be carefully regulated to meet the needs of the organism as a whole. Introduction Communication is accomplished by signaling molecules from one cell that bind to receptors on other cells. This initiates a series of reactions that regulate virtually all aspects of cell behavior. Signaling Molecules Signaling molecules range in complexity from simple gases to proteins. Some carry signals over long distances; others act locally. Signaling molecules also differ in their mode of action on their target cells. Some can cross the plasma membrane and bind to intracellular receptors. Modes of cellular communication Modes of cell signaling include: Direct cell-cell signaling—direct interaction of a cell with its neighbor. Signaling by secreted molecules— three categories are based on the distance over which signals are transmitted. Figure 15.1 Modes of cell-cell signaling Signaling Molecules and Their Receptors Endocrine signaling—signaling molecules (hormones) are secreted by specialized endocrine cells and carried through the circulation to target cells at distant body sites. Example: estrogen. Paracrine signaling—molecules released by one cell act on neighboring target cells, e.g., neurotransmitters. Signaling Molecules Autocrine signaling—cells respond to signaling molecules that they themselves produce. Example: T lymphocytes respond to antigens by making a growth factor that drives their own proliferation, thereby amplifying the immune response. Signaling Molecules and Their Receptors Receptors may be located on the cell surface or intracellularly. Intracellular receptors respond to small hydrophobic signaling molecules that can diffuse across the plasma membrane. Steroid hormones, thyroid hormone, vitamin D3, and retinoic acid are in this class of signaling molecules. Structure of steroid hormones, thyroid hormone, vitamin D3, and retinoic acid Steroid hormones Steroid hormones are synthesized from cholesterol: Testosterone, estrogen, and progesterone are the sex steroids, produced by the gonads. Corticosteroids from the adrenal gland: Glucocorticoids—stimulate production of glucose, Mineralocorticoids—act on the kidney to regulate salt and water balance. Thyroid hormone, Vitamin D3 & Retinoic acid Thyroid hormone is synthesized from tyrosine in the thyroid gland; it is important in development and metabolism. Vitamin D3 regulates Ca2+ metabolism and bone growth. Retinoic acid and related compounds (retinoids) synthesized from vitamin A play important roles in human’s body development. Nuclear receptor Receptors for these molecules are members of the nuclear receptor superfamily. They are transcription factors that have domains for ligand binding, DNA binding, and transcriptional activation. Ligand binding regulates their function as activators or repressors of genes. Glucocorticoid receptor Ligand binding has different effects on different receptors. Some nuclear receptors are inactive in the absence of hormone: Glucocorticoid receptor is bound to Hsp90 chaperones in the absence of hormone. Glucocorticoid binding displaces Hsp90 and leads to binding of regulatory DNA sequences. Glucocorticoid action Thyroid hormone receptor Hormone binding may alter the activity of the receptor: In the absence of hormone, thyroid hormone receptor is associated with a corepressor complex and represses transcription of target genes. Hormone binding results in activation of transcription. Gene regulation by the thyroid hormone receptor HDAC vs. HAT HDAC inhibitors are currently used to treat multiple myeloma. Nitric oxide (NO) Nitric oxide (NO) is a paracrine signaling molecule in the nervous, immune, and circulatory systems, which alters the activity of enzymes. NO is synthesized from arginine by nitric oxide synthase. Its action is restricted to local effects because it is extremely unstable, with a half-life of only a few seconds. Synthesis of nitric oxide Nitric Oxide NO is involved in dilation of blood vessels. Neurotransmitters released from nerve cells in the blood vessel wall stimulate NO synthesis by endothelial cells. NO diffuses to smooth muscle cells, and induces muscle cell relaxation and blood vessel dilation. Carbon monoxide Carbon monoxide (CO), also functions as a signaling molecule in the nervous system. It acts similarly to NO as a neurotransmitter and mediator of blood vessel dilation. Neurotransmitters Neurotransmitters carry signals between neurons or from neurons to other types of target cells. Release of neurotransmitters is signaled by arrival of an action potential at the end of a neuron. The neurotransmitters then diffuse across the synaptic cleft and bind to receptors on the target cell surface. The action of acetylcholine Structure of representative neurotransmitters Neurotransmitter Receptors Because neurotransmitters are hydrophilic, they can’t cross the plasma membrane of target cells and must bind to cell surface receptors. Many neurotransmitter receptors are ligand-gated ion channels. Neurotranmitter binding opens the channels. Figure 15-5b Molecular Biology of the Cell (© Garland Science 2008) Figure 15-16a Molecular Biology of the Cell (© Garland Science 2008) Peptide hormones Peptide signaling molecules include peptide hormones, neuropeptides, and polypeptide growth factors. Peptide hormones include insulin, glucagon, and pituitary gland hormones (growth hormone, follicle-stimulating hormone, prolactin, etc.). Neuropeptides & Enkephalins Neuropeptides are secreted by some neurons instead of small-molecule neurotransmitters. Enkephalins and endorphins act as neurotransmitters at synapses, and as neurohormones. They are natural analgesics that decrease pain responses; they bind to the same receptors on brain cells as morphine does. Growth factors Nerve growth factor (NGF) is a member of the neurotrophin family that regulate the development and survival of neurons. Epidermal growth factor (EGF) stimulates cell proliferation. Structure of epidermal growth factor (EGF) Platelet-derived growth factor Platelet-derived growth factor (PDGF) is stored in blood platelets and released during blood clotting at the site of a wound. It stimulates proliferation of fibroblasts, contributing to regrowth of the damaged tissue. Cytokines Cytokines regulate development and differentiation of blood cells and activities of lymphocytes during the immune response. Membrane-anchored growth factors remain associated with the plasma membrane and function as signaling molecules in direct cell-cell interactions. Eicosanoids … Eicosanoids are lipid signaling molecules which include prostaglandins, prostacyclin, thromboxanes, and leukotrienes. They act in autocrine or paracrine pathways, and then break down rapidly. Synthesis and structure of eicosanoids (Part 1) Synthesis and structure of eicosanoids (Part 2) Synthesis of eicosanoids Eicosanoids are synthesized from arachidonic acid, which is formed from phospholipids. Arachidonic acid is converted to prostaglandin H2, catalyzed by cyclooxygenase. This enzyme is the target of aspirin and other nonsteroidal anti-inflammatory drugs (NSAIDs). Inhibiting synthesis of prostaglandins Inhibiting synthesis of the prostaglandins reduces inflammation and pain. By inhibiting synthesis of thromboxane, aspirin reduces platelet aggregation and blood clotting; thus small daily doses of aspirin are often prescribed for prevention of strokes. Cell Surface Receptors Most ligands responsible for cell-cell signaling bind to surface receptors on their target cells. This initiates a chain of intracellular reactions, ultimately reaching the nucleus and resulting in programmed changes in gene expression. G-protein-coupled receptors G-protein-coupled receptors are the largest family of cell surface receptors. Signals are transmitted via guanine nucleotide-binding proteins (G-proteins). The receptors have seven membrane- spanning α helices. Structure of a G-protein-coupled receptor G protein mediated signalling Binding of ligands induces a conformational change that allows the cytosolic domain to activate a G-protein on the inner face of the plasma membrane. The activated G-protein then dissociates from the receptor and carries the signal to an intracellular target. Adenylyl cyclase activation A G-protein is an intermediary in adenylyl cyclase activation, which synthesizes cAMP. Hormonal activation of adenylyl cyclase Heterotrimeric G-proteins G-proteins have three subunits named α, β, and γ. They are also called heterotrimeric G-proteins to distinguish them from other guanine nucleotide-binding proteins, (such as the Ras proteins). Regulation of G-proteins The α subunit binds guanine nucleotides, which regulate G-protein activity. In the inactive state, α is bound to GDP in a complex with β, and γ. Hormone binding to the receptor causes exchange of GTP for GDP. The α and βγ complex then dissociate from the receptor and interact with their targets. Regulation of G-proteins G-proteins A large array of G-proteins connect receptors to distinct targets. In addition to enzyme regulation, G-proteins can also regulate ion channels. G protein & heart muscle contraction Heart muscle cells have a different acetylcholine receptor than nerve and skeletal muscle cells, which is G-protein-coupled. The α subunit of this G-protein (Gi) inhibits adenylyl cyclase. The Gi βγ subunits open K+ channels in the plasma membrane, which slows heart muscle contraction. Receptor protein-tyrosine kinases Other cell surface receptors are directly linked to intracellular enzymes. The largest family of these is the receptor protein-tyrosine kinases, which phosphorylate their substrate proteins on tyrosine residues. The family includes receptors for most polypeptide growth factors. Receptor protein-tyrosine kinases The human genome encodes 59 receptor protein-tyrosine kinases, including receptors for EGF, NGF, PDGF, insulin, and other growth factors. All have an N-terminal extracellular ligand-binding domain, a single transmembrane α-helix, and a cytosolic C-terminal domain with protein-tyrosine kinase activity. Organization of receptor protein-tyrosine kinases Phosphorylation of receptors/intracellular target proteins Binding of ligands such as growth factors to the extracellular domains activates their cytosolic kinase domains, resulting in phosphorylation of both the receptors and intracellular target proteins that propagate the signal. Autophosphorylation of receptor The first step is ligand-induced receptor dimerization. This results in receptor autophosphorylation as the two polypeptide chains cross-phosphorylate one another. Dimerization and autophosphorylation of receptor protein-tyrosine kinases Autophosphorylation Autophosphorylation has two roles: Phosphorylation of tyrosine in the catalytic domain increases protein kinase activity. Phosphorylation of tyrosine outside the catalytic domain creates binding sites for other proteins that transmit signals downstream of the activated receptors. Association of downstream signaling molecules with receptor protein- tyrosine kinases SH2 domains The downstream signaling molecules have domains that bind to specific phosphotyrosine-containing peptides. SH2 domains are the first domain that recognizes phosphorylated protein- tyrosine kinases Complex between an SH2 domain and a phosphotyrosine peptide PTB domains Other proteins bind to phosphotyrosine- containing peptides via PTB domains (phosphotyrosine-binding). Cytokine receptor superfamily The cytokine receptor superfamily includes receptors for most cytokines (interleukin-2 and erythropoietin) and for some polypeptide hormones. Their structure is similar to receptor protein-tyrosine kinases, but the cytosolic domains have no catalytic activity. Nonreceptor protein-tyrosine kinases Cytokine receptors function in association with nonreceptor protein- tyrosine kinases. Ligand binding induces dimerization of receptors, and cross-phosphorylation of associated nonreceptor protein-tyrosine kinases. Nonreceptor protein-tyrosine kinases The activated kinases then phosphorylate the receptor, providing phosphotyrosine-binding sites for recruitment of downstream signaling molecules with SH2 domains. Signaling from cytokine receptors Janus kinase (or JAK) family The kinases associated with cytokine receptors belong to the Janus kinase (or JAK) family. Members of the JAK family appear to be universally required for signaling from cytokine receptors. Src family Additional nonreceptor protein-tyrosine kinases belong to the Src family. The Src family plays key roles in signaling downstream of cytokine receptors, receptor protein-tyrosine kinases, antigen receptors on B and T lymphocytes, and from integrins at sites of cell attachment to the extracellular matrix. Functions of Cell Surface Receptors Some enzyme-linked receptors are associated with other enzymatic activities: Protein-tyrosine phosphatases remove phosphate groups from phosphotyrosine, counterbalancing the effects of protein-tyrosine kinases. Receptors for transforming growth factor β Receptors for transforming growth factor β (TGF- β) and related polypeptides are protein- serine/threonine kinases. These growth factors control cell proliferation and differentiation. Receptor guanylyl cyclases Receptor guanylyl cyclases have a cytosolic domain which catalyzes formation of cyclic GMP, a second messenger. Intracellular signal transduction Intracellular signal transduction is a chain of reactions that transmits signals from the cell surface to intracellular targets. The targets often include transcription factors that regulate gene expression. cyclic AMP is a second messenger Intracellular signaling was first studied in hormones such as epinephrine, which signals the breakdown of glycogen to glucose. In 1958 Sutherland discovered that the action of epinephrine was mediated by an increase in cyclic AMP (cAMP), leading to the concept of cAMP as a second messenger. Formation of AMP cAMP is formed from ATP by adenylyl cyclase and degraded to AMP by cAMP phosphodiesterase. The epinephrine receptor is coupled to adenylyl cyclase via a G-protein that stimulates enzymatic activity, increasing the concentration of cAMP. Synthesis and degradation of cAMP Protein kinase A Effects of cAMP in animal cells are mediated by cAMP-dependent protein kinase, or protein kinase A. The inactive form has two regulatory and two catalytic subunits. cAMP binds to the regulatory subunits, which then dissociate. The free catalytic subunits can then phosphorylate serine on target proteins. Regulation of protein kinase A Intracellular Signals in glycogen metabolism In glycogen metabolism, protein kinase A phosphorylates two enzymes: Phosphorylase kinase is activated, and in turn activates glycogen phosphorylase. Glycogen synthase is inactivated. So, glycogen breakdown is stimulated and glycogen synthesis is blocked. Regulation of glycogen metabolism by protein kinase A Signal amplification Signal amplification: each molecule of epinephrine activates one receptor. Each receptor may activate up to 100 molecules of Gs, which then stimulates the adenylyl cyclase, which can catalyze the synthesis of many cAMP. Each molecule of protein kinase A phosphorylates many molecules of phosphorylase kinase, and so forth. Signal amplification Hormones are present in blood very small concentrations yet they produce profound biological effects. The action of a hormon occurs through a cascade of events in which the signal is amplified at a number of stages. Signal amplification Pathways of Intracellular Signal Transduction Increased cAMP can activate transcription of genes that contain a regulatory sequence—the cAMP response element, or CRE. The free catalytic subunit of protein kinase A goes to the nucleus and phosphorylates the transcription factor CREB (CRE- binding protein). This leads to expression of cAMP-inducible genes. Cyclic AMP-inducible gene expression Protein phosphorylation/de phosphorylation Protein phosphorylation is rapidly reversed by the action of protein phosphatases, which terminates responses initiated by receptor activation of protein kinases. Regulation of protein phosphorylation by protein kinase A and protein phosphatase 1 Diacylglycerol & Inositol 1,4,5-trisphosphate Signaling Two major pathways of intracellular signaling use second messengers derived from the membrane phospholipid phosphatidylinositol 4,5- bisphosphate (PIP2). Hydrolysis of PIP2 by phospholipase C produces two second messengers: diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). Hydrolysis of PIP2 Two forms of phospholipase C There are two forms of phospholipase C: PLC-β is stimulated by G proteins. PLC-γ has SH2 domains that associate with receptor protein-tyrosine kinases. Tyrosine phosphorylation increases PLC-γ activity, stimulating hydrolysis of PIP2. Activation of phospholipase C by protein-tyrosine kinases DAG & IP3 Signalling DAG remains associated with the plasma membrane and activates protein- serine/threonine kinases of the protein kinase C family. IP3 is a small polar molecule that is released to the cytosol, where it signals release of Ca2+ from the ER. Ca2+ mobilization by IP3 IP3 stimulates release Ca2+ Cytosol concentration of Ca2+ is maintained at an extremely low level by Ca2+ pumps. IP3 stimulates release Ca2+ from the ER by binding to receptors that are ligand- gated Ca2+ channels. Increased Ca2+ affects activity of several proteins, including protein kinases and phosphatases. Ca2+/calmodulin Calmodulin is activated when Ca2+ concentration increases. Ca2+/calmodulin then binds to target proteins, including protein kinases. Function of calmodulin CaM kinase family The CaM kinase family are activated by Ca2+/calmodulin; they phosphorylate metabolic enzymes, ion channels, and transcription factors. One form of CaM kinase regulates synthesis and release of neurotransmitters. Phosphatidylinositol 3,4,5-trisphosphate (PIP3) PIP2 is also the start of another signaling pathway. PIP2 is phosphorylated by phosphatidylinositide (PI) 3-kinase. This yields the second messenger phosphatidylinositol 3,4,5- trisphosphate (PIP3). Activity of PI 3-kinase Activation of Akt PIP3 targets a protein-serine/threonine kinase called Akt and also binds protein kinase PDK1. Activation of Akt also requires protein kinase mTOR (in a complex called mTORC2) which is also stimulated by growth factors. The PI 3-kinase/Akt pathway Akt (PKB) & Glycogen synthesis Akt phosphorylates several targets Akt phosphorylates several target proteins, transcription factors, and other protein kinases. Transcription factors include members of the Forkhead or FOXO family. Akt phosphorylation of FOXO sequesters it in inactive form. FOXO induce cell death If growth factors are not present, Akt is not active, and FOXO travels to the nucleus where it stimulates transcription of genes that inhibit cell proliferation, or induce cell death. Regulation of FOXO (Part 1) Regulation of FOXO (Part 2) Pathways of Intracellular Signal Transduction Protein kinase GSK-3 is also inhibited by Akt phosphorylation. GSK-3 targets include the translation initiation factor eIF2B. Phosphorylation of eIF2B leads to a global downregulation of translation initiation. Integrins as receptors Integrins are the main receptors for the attachment of cells to the extracellular matrix and also interact with the cytoskeleton. Integrins also serve as receptors that activate intracellular signaling pathways. FAK ( focal adhesion kinase), In one pathway, binding of integrins to the extracellular matrix leads to activation of FAK ( focal adhesion kinase), a nonreceptor protein-tyrosine kinase. Phosphorylation of FAK provides binding sites for several signaling molecules and leads to activation of PI 3-kinase and phospholipase C-γ. Integrin signaling (Part 1) Integrin signaling (Part 2) Integrin signaling (Part 3) Thank you for attending my lecture.