Organization of lectures: Cell Signaling I: Sex, Drugs and Violence. Cell signaling is central to modern medicine. Forms of Cell Signaling

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1 Cell Signaling I: Sex, Drugs and Violence Joe W. Ramos Organization of lectures: General Principles of signaling cascades Hormone Signaling Signaling via G-protein G coupled receptors: camp Signaling via G-protein G coupled receptors: IP3 &DAG Signaling via enzyme-linked receptors Signal integration Apoptosis A Metaphor for Cell Signaling: The milkman is the signal. From Genes and the Biology of Cancer, Varmus and Weinberg, 1993 What is signaling? Cell signaling touches all aspects of cell biology: A Neutrophil Chasing a Bacterium Courtesy Peter Devreotes Cell Signaling pathways regulate: the cell cycle- proliferation the cytoskeleton- migration transcription- differentiation membrane traffic- exocytosis cell survival and death- apoptosis Development- body plan Cell signaling is central to modern medicine Forms of Cell Signaling Cancer Diabetes Steroids AIDS Cholera Viagra Angina Alzheimer s Whooping cough.among others Adrenaline, Estradiol Acetylcholine Epidermal Growth Factor, NO Delta, FasL 1

2 Cell matrix proteins may also serve as signals Same signaling molecule elicits different responses in different cells (e) Extracellular matrix-dependent Target Cells Different Receptorligand complexes can also activate the SAME response: ECM protein (fibronectin) epinephrin or glucagon can activate glycogen breakdown and release of glucose into the blood. Cells depend on multiple signals Cell-to to-cell communication by extracellular signaling usually involves six steps (1) synthesis of the signaling molecule by the signaling cell (2) release of the signaling molecule by the signaling cell (3) transport of the signal to the target cell (4) detection of the signal by a specific receptor protein (5) a change in cellular metabolism, function, or development triggered by the receptor-signal complex (6) removal of the signal, which usually terminates the cellular response An intracellular signaling cascade Phosphorylation Recall: Produced by cellular respiration

3 Intracellular signaling proteins that act as molecular switches Other signaling modifications Acetylation, methylation Ubiquitination, Sumoylation Proteolysis (caspases) Second messenger Binding (camp, cgmp,, DAG, IP3, Ca ) Fast and slow responses to signals Turning off the signal Figure from Molecular Biology of the Cell, 4th edn. Signaling Molecules: Hormones 1. Steroids (Estradiol, Testosterone) 2. Amino acid derivatives (Histamine) =cholesterol derivatives (steroids) 3. Gases (NO, CO) (Adrenal gland) (Ovary) (Testis) 4. Peptides and Proteins (Insulin,PDGF) (Thyroid gland) Small hydrophobic molecules: -can diffuse across membrane =tyrosine derivative (as is adrenaline)

4 Androgen Insensitivity Syndrome (AIS) Some very rare individuals are born as genetic males but lack the testosterone (androgen) receptor. They make the hormone, but their cells cannot respond to it. They therefore develop outwardly as females Female genitalia However, have non-descended testis No secondary female characteristics (no estrogen!) Hence, the importance of receptors! Steroid hormone cortisol Cortisol acts on metabolism of proteins, carbohydrates and lipids Made in Adrenal Gland Diffuses across PM In blood causes increased blood pressure and glucose concentrations. Gives a quick burst of energy. the Stress Hormone secreted during fight or flight responses. The nuclear receptor superfamily NO is smooth muscle relaxation All hormone receptors bind as either heterodimers or homodimers-to simplify things it is shown only as a monomer here. -NO diffuses locally, half life is 2-30 seconds -Nerve cell releases acetylcholine which binds endothelial cell. -Relaxation of smooth muscle cells around blood vessels causes the vessels to expand increasing blood flow cgmp mediates local signaling by NO: Viagra and nitroglycerin Cell surface receptor-mediated signaling Acetylcholine is released by the autonomic nerves of the penis. Local blood vessel dilation, Penile erection. G-Protein Coupled Receptors Nitroglycerin relieves Angina cgmp can regulate kinases, ion channels, and phosphodiesterases Viagra maintains erection by -keeping cgmp levels high -Keeping smooth muscle relaxed Enzyme-linked receptors Viagra inhibits phosphodiesterase that normally lowers cyclic GMP levels Angina is pain resulting from poor blood flow to heart -nitroglycerine used for 100 years -is converted to NO 4

5 Three classes of cell-surface receptor A Neutrophil Chasing a chemoattractant The neutrophil is guided by GPCR signaling G protein-coupled receptors and their effectors Many different mammalian cell-surface receptors are coupled to a trimeric signal-transducing G protein. Function in: Light detection odorant detection detection of certain hormones and neurotransmitters G-protein-linked receptor Demonstration of functional domains in G protein-coupled receptors: chimeric proteins Ligand 7 Pass Transmembrane proteins Light green region binds ligand G-protein Orange region binds G-protein Expressed in Xenopus oocytes See B. Kobilka et al., 1988, Science 240:1310 5

6 Clinical relevance 10 minute Break Half of all known drugs work through G-G protein coupled receptors The genome project has revealed vast numbers of new GPCRs (new drug targets?) The G-ProteinG G-proteins are composed of three subunits: α, β, γ G-proteins disassemble into two signaling proteins when activated Tethered to membrane by hydrophobic lipid anchor In response to ligand the G-protein is released from the GPCR into two signaling proteins: α subunit and βγ complex 20 known G-proteins G in mammals (different targets) The α subunit switches itself off by hydrolyzing its bound GTP A G o protein opens a K+ channel in the heart muscle After a few seconds the GTP on the α subunit is hydrolyzed by the intrinsic GTPase activity. This inactivates the subunit, which dissociates from the target and re-associates with the βγ complex. Both the activated α subunit and the βγ complex can regulate target proteins. G oβγ The Heart s contraction is controlled by two sets of nerve fibers: one speeds and one slows contraction. To slow, nerves release acetylcholine. Acetylcholine binds a GPCR on heart releasing the G-proteins. Here the G o βγ complex is the active signaling component-binding to the K+ channel and thus opening it: less frequent contractions

7 G-proteins activate enzymes that catalyze the synthesis of messenger molecules G-protein coupled receptors signal by several mechanisms An example enzyme is Adenylate cyclase The intracellular message molecules are called Second Messengers The signal is thus greatly amplified. We will examine four GPCR second messengers: camp Inositol triphosphate (IP3) Diacylglycerol (DAG) Ca+ cgmp is also a second messenger camp increase activates gene transcription Synthesis and degradation of cyclic AMP G s camp activity mediated by camp-dependent protein kinase (A kinase or PKA). This signaling pathway controls many processes in cells including hormone synthesis. ATP is always present in cells. Adenylate cyclase forms camp from ATP by a cyclization reaction that removes two phosphates and joins the remaining phosphate to the sugar. camp phosphodiesterase is continually active and breaks the bond to form AMP. cgmp is an analogous second messenger Earl Sutherland, discovered camp Effect of Cholera toxin on Gsα PKA Structure and regulation Cholera toxin is a hexameric protein produced by bacteria: Vibrio cholerae which causes Cholera. camp levels 100 fold higher: In epithelial cells this permits massive flow of water from blood into intestines Important tool in early studies of Gs proteins. NAD+=nicotinamide adenine dinucleotide 7

8 CREB links camp signals to transcription The two branches of the inositol phospholipid pathway PKC capk=pka Modification of a common phospholipid precursor generates several second messengers: synthesis of DAG and IP 3 Phospholipase C causes an increase in intracellular calcium 25 of the known GPCRs activate PLC 2 messengers: -DAG=PKC -IP3=Calcium release Cytosolic Ca 2+ levels are tightly controlled Calcium signaling in Rat brain glial cells Ca 2+ is actively pumped to the cell exterior and into the ER and mitochondrion. 8

9 Ca2+ calmodulin structure The Activation of Cam Kinase II Dumbell structure when inactive. Alpha helix jack-knifes when Ca2+ bound to surround target protein (CaM Kinase II). CaM Kinase II phosphorylates a wide range of cellular proteins PLC-mediated responses Two major pathways by which G-proteinG protein-linked cell- surface receptors generate small intracellular mediators Gs Gq Adenylate cyclase PLC PKA (capk) calmodulin Figure from Molecular Biology of the Cell, 3 rd edn. Turning off GPCRs: arrestin Multiple G proteins transduce signals to different effector proteins Figure from Molecular Biology of the Cell, 4th edn. Figure from Molecular Biology of the Cell, 4th edn. 9

10 camp-mediated responses 10