Lecture 3

1. CELL SIGNALING AND MOTILITY (BIOL 3373) Lecture 3

2. WHAT IS The CELL SIGNALING? • How cells receive and respond to signals from their surroundings • On one hand, cell signaling regulates gene expression and controls the cell fate (proliferation, motility, differentiation and programmed cell death, or apoptosis). • On the other hand, cell signaling allows for the organization of cells into tissues, which, in turn, generate organs. In addition, cell signaling is essential for the maintenance of cells, tissues and organs.

3. CELL SIGNALING • Communication among cells is referred as intercellular signaling. • Cells communicate with each other through signaling molecules. • Signaling molecules could be: • proteins, small peptides, amino acids, • nucleotides, • steroids, retinoids, fatty acid derivatives, • nitric oxide, carbon monoxide

4. Forms of signaling molecules : Steroid hormones Steroid hormones are a group of hormones that belong to the class of chemical compounds known as steroids. All steroid hormones are derived from cholesterol. They are transported through the bloodstream to the cells of various target organs where they carry out the regulation of a wide range of physiological functions. These hormones often are classified according to the organs that synthesize them. The adrenal cortex produces the adrenocortical hormones, which consist of the glucocorticoids and the mineralocorticoids. Glucocorticoids such as cortisol control many metabolic processes, including the formation of glucose and the deposition of glycogen in the liver. Mineralocorticoids such as aldosterone help maintain the balance between water and salts in the body, predominantly exerting their effects within the kidney. The androgens are the male sex hormones, responsible for the development and maintenance of reproductive function in the male. The principal androgen, testosterone, is produced by the testes. Estrogens are female sex hormones. They are secreted mainly by the ovaries. Estradiol is the most potent of the estrogens. the estrogens promote the development of the primary and secondary female sex characteristics. Progestins, the most important of which is progesterone, are the other type of female sex hormone and are named for their role in maintaining pregnancy (pro-gestation).

5. Forms of signaling molecules- Gas - NO Nitric oxide (NO) is a gas molecule produced and released by endothelial cells (signaling cells)and rapidly diffuses across the membranes. NO binds to an enzyme inside the smooth muscle cells (target cell), inducing cell relaxation

6. Forms of signaling molecules Neurotransmitters Neurotransmitters are chemicals produced and released by neurons. Neurotransmitters travel across the synapse and allow communication between neurons.

7. Forms of signaling molecules Neurotransmitters

8. Forms of signaling molecules • Peptide Hormones and Growth Factors

9. CELL SIGNALING • Cells that produce and release the signaling molecules are signaling cells. • Cells that receive the signal are target cells • Targets cells posses specific receptorsthat recognize signaling molecules. receptor signaling cell signaling molecules target cell

10. CELL SIGNALING Depending on the distance that the signaling molecule has to travel, we can talk about four types of signaling: Contact- dependent signaling requires cells to be in direct membrane-membrane contact. The signaling molecule remains bound to the surface of the signaling cells. It influences only cells (target cells) contact it via a specific protein receptor. Contact-dependent signaling is especially important during development and immune response from Alberts et al., Molecular Biology of the Cell. 6th edition.

11. CELL SIGNALING In paracrine signaling the signaling molecules are local mediators and affects only target cells in the proximity of the signaling cell. An example is the conduction of an electric signal from one nerve cell to another or to a muscle cell. In this case the signaling molecule is a neurotransmitter. from Alberts et al., Molecular Biology of the Cell. 6th edition.

12. CELL SIGNALING In autocrine signaling cells respond to molecules they produce themselves. So signaling cells are also target cells. Examples include cancer cells as they can produce signals that stimulate their own survival and proliferation. autocrine signaling signaling cell = target cell from Alberts et al., Molecular Biology of the Cell. 6th edition.

13. CELL SIGNALING Large multicellular organisms use long- range signaling that coordinate the behavior of the cells in remote part of the body. Synaptic signaling is performed by neurons that transmit signals electrically along their axons and release neurotransmitters at synapses, which can be located far away from the neuron cell body. from Alberts et al., Molecular Biology of the Cell. 6th edition.

14. CELL SIGNALING In endocrine signaling signaling molecules (hormones) are produce by endocrine cells and sent through the blood stream to distant cells

15. The Three Stages of Cell Signaling • Earl W. Sutherland discovered how the hormone epinephrine acts on cells • Sutherland suggested that cells receiving signals went through three processes: • Reception • Transduction • Response

16. EXTRACELLULAR FLUID CYTOPLASM Plasma membrane Reception Transduction Response 1 2 3 Receptor Activation of cellular response Relay molecules in a signal transduction pathway Signal molecule Overview of cell signaling Cell signaling consists of 3 stages

17. Three Stages of Cell Signaling: 1 Reception CYTOPLASM EXTRACELLULAR FLUID Plasma membrane Reception 1 1 The receptor and signaling molecules fit together (lock and key model) Receptor Signaling molecule Signaling molecule binds to the receptor protein at the cell surface

18. 1- Reception: A signal molecule binds to a receptor protein, causing it to change shape • The binding between a signal molecule (ligand) and receptor is highly specific • A conformational change in a receptor is often the initial transduction of the signal • Most signal receptors are plasma membrane proteins

19. Receptors in the Plasma Membrane

20. Intracellular Receptors • Some receptor proteins are intracellular, found in the cytosol or nucleus of target cells • Small or hydrophobic chemical messengers can readily cross the membrane and activate receptors • Examples of hydrophobic messengers are the steroid and thyroid hormones of animals • An activated hormone-receptor complex can act as a transcription factor, turning on specific genes

21. Hydrophobic signaling molecules can cross the plasma membrane and bind to nuclear receptors

22. Hormone (testosterone) EXTRACELLULAR FLUID The steroid hormone testosterone passes through the plasma membrane. Plasma membrane Testosterone binds to a receptor protein in the cytoplasm, activating it. Receptor protein Hormone- receptor complex The hormone- receptor complex enters the nucleus and binds to specific genes. DNA The bound protein stimulates the transcription of the gene into mRNA. mRNA NUCLEUS New protein The mRNA is translated into a specific protein. CYTOPLASM

23. The nuclear receptor superfamily Nuclear receptors are inactive without signaling molecules (ligands) When the nuclear receptor interacts with the specific ligand, it becomes activate and induces the transcription of target genes

24. Three Stages of Cell Signaling: 2 Transduction CYTOPLASM EXTRACELLULAR FLUID Plasma membrane Reception Transduction 1 1 2 Receptor 2nd Messenger! Relay molecules in a signal transduction pathway Signaling molecule The signal from the receptor is converted into a intracellular message that produces a cellular response.

25. Includes a NETWORK of molecular and cellular events conveying a SIGNAL from the outside to the inside of the cell. SIGNAL TRANSDUCTION: The study of the molecular circuits responsible for the generation of a cellular response after the delivery of a signal.

26. SIGNAL TRANSDUCTION Signaling molecules (ligands) bind to receptors on target cells. After the binding with the ligand, the receptor activates one or more intracellular signaling within the target cells, that involves several proteins ( transducer Protein). The intracellular signaling modulates the activity of target proteins (also known as effector proteins) thereby the behavior of the cells. from Alberts et al., Molecular Biology of the Cell. 6th edition.

27. Three Stages of Cell Signaling: 3 Response CYTOPLASM EXTRACELLULAR FLUID Plasma membrane Reception Transduction Response 1 2 3 Receptor Activation of cellular response Relay molecules in a signal transduction pathway Signaling molecule Cellular responses can be different and complex (i.e. change in gene expression, cell motility, cell growth, cell differentiation, cell death), depending on cell types.

28. Cytoplasmic and Nuclear Responses • The response may occur in the cytoplasm or in the nucleus • Many signaling pathways regulate the synthesis of enzymes or other proteins, usually by turning genes on or off in the nucleus • The final activated molecule in the signaling pathway may function as a transcription factor

29. Cytoplasmic and Nuclear Responses • Many other signaling pathways regulate the synthesis of enzymes or other proteins, usually by turning genes on or off in the nucleus • The final activated molecule may function as a transcription factor

30. Fine-Tuning of the Response • There are four aspects of fine-tuning to consider • signal Amplification (and thus the response) • Specificity of the response, • Efficiency of response, • Initiation of the signal, • Termination of the signal,

31. Signal Amplification figure 1 • At each step of many signal transduction pathways, the number of activated participants in the pathway increases. This is referred as signal amplification, • For example, one epinephrine-activated GPCR activates 100s of Gas-GTP complexes, which in turn activate 100s of adenylyl cyclase molecules, that each produce hundreds of cAMP molecules, and so on. The overall amplification associated with epinephrine signaling is estimated to be ~108-fold (figure 1)

32. Specificity of the response • Different kinds of cells have different collections of proteins • These differences in proteins give each kind of cell specificity in detecting and responding to signals • The response of a cell to a signal depends on the cell’s particular collection of proteins • Pathway branching and “cross-talk” further help the cell coordinate incoming signals

33. Specificity of the response Signal molecule Receptor Relay molecules Response 2 Response 3 Response 1 Cell A. Pathway leads to a single response Cell B. Pathway branches, leading to two responses Activation or inhibition Response 5 Response 4 Cell D. Different receptor leads to a different response Cell C. Cross-talk occurs between two pathways

34. The same signal can induce different responses in distinct cell types

35. Efficiency of response depends on: • The expression of specific receptors; • The bioavailability of transducer molecules: • Expression levels/half-life • Localization within the cell • Modality of activation/inactivation • The bioavailability of effectormolecules: • cytoskeletal elements (morphological changes) • transcription factors (changes in gene expression) • proteolytic enzymes (cell death)

36. INITIATION of Signal: RECEPTOR ACTIVATION/ InACTIVATION • For EACH signaling pathway there is a common feature that defines a sequence of events: • The receptor is in an INACTIVE STATE in the absence if the signal • When the signal arrives, it BINDS to the receptor, which MODIFIES its conformation • This change in the receptor ACTIVATES other molecules (i.e. a downstream signaling cascade)

37. Termination of the Signal: • Inactivation mechanisms are an essential aspect of cell signaling • When signal molecules leave the receptor, the receptor reverts to its inactive state.

38. THE SIGNALING PATHWAY VIA G-PROTEIN

39. G-protein coupled receptors G- protein coupled receptors consists of 3 components: trans-membrane receptors (GPCRs) known also as serpentine receptors. 2. G proteins: Guanine nucleotide-binding proteins, trimeric G protein. 3. Effectors: Effectors can be different : adenylyl cyclase or Phosphodiesterase (PDE) or phospholipase C-β (PLC- β) or ion Channel. Receptor adenylyl cyclase ion Channel PDE PLC- β G protein Effector

40. These effectors in turn regulate the intracellular concentrations of secondary messengers, such as cAMP, cGMP, diacylglycerol (DG), IP3, sodium (Na+), potassium (K+) or calcium cations (Ca2+), which ultimately lead to a physiological response, usually via the downstream regulation of gene transcription. adenylyl cyclase ion Channel PDE PLC- β cGMP Na+, cAMP Ca2+ K+ DG IP3 Second messengers

41. G-protein coupled receptors Several types of receptors are associated with G proteins, such as hormones, neurotransmitters, growth factors, glycoproteins, cytokines, odorants and photons

42. N C -Terminal chain -Terminal chain NH2 Extracellular loops HO2C Transmembrane Membrane VII VI V IV III II I helix G-Protein binding region Variable intracellular loop Intracellular loops G-protein-coupled receptors Single protein composed of 7 transmembrane domains with an extracellular amino terminus and an intracellular carboxyl terminus. Intracellular C terminus changes conformation in response to a stimulus and this results in changes in the ability to recruit G proteins.

43. G-protein G proteins are composed by 3 subunits, α,β and γ. The α subunit contains the GDP binding site as well as the GTP hydrolysis domain.

44. ADENYLYL CYCLASE : EFFECTOR OF GPCR First messenger (signal molecule such as epinephrine) Adenylyl cyclase G protein G-protein-linked receptor GTP ATP Second messenger cAMP Adenylyl cyclase is a multipass transmembrane protein that converts ATP to form cyclic AMP (cAMP). Protein kinase A Cellular responses

45. Cyclic AMP (cAMP) Phosphodiesterase Adenylyl cyclase Pyrophosphate H2O ATP Cyclic AMP AMP P P i • The Adenylyl Cyclase catalyzes the reaction that leads to the production of cAMP. • cAMP is synthesized from ATP through a cyclization reaction that removes two phosphate group as pyrophosphate. • cAMP is an unstable molecule because it is soon hydrolyzed to AMP by specific Phosphodiesterases.

46. Ligand Cell membrane G Protein Receptor ß ß g g a a Binding site for G-protein opens Activation of G-protein The binding of the ligand to the receptor changes the receptor conformation. The new receptor conformation allows the binding of the G protein. When the G protein assembles to the receptor it alters its own conformation, therefore the GDP binding site within the α subunit is distorted and GDP is released. 1 2 3 ß g a GDP GTP

47. Activation of G-protein GTP binds Fragmentation and release • 4. The binding of GTP to the α subunit promotes the closure of the nucleotide binding site within the α subunit. • 5. Therefore the α subunit changes its conformation and • 6. separates from both the β-γ dimer and the receptor. • This process is active while the ligand is bound to the receptor • One ligand can activate several G protein: Signal amplification 4 6 5 ß g ß ß g g a a a GTP α subunit changes conformation

48. Activation of G-protein GTP as-subunit GDP Adenylyl cyclase Binding site for a subunit GTP hydrolysed to GDP catalysed by asubunit Binding P ATP cyclic AMP ATP cyclic AMP Active site (open) Active site (closed) Active site (closed) Signal transduction (con) a Subunit recombines with b-g dimer to reform inactive G protein. • 7. The α-GTP subunit interacts with the Adenylyl cyclase • 8. Adenylyl cyclase becomes active and converts ATP in cAMP. • 9. The cycle is completed when GTP is hydrolyzed to GDP within the α subunits. This causes the re-association of α subunit with β-γ dimer and the binding of G-protein to the receptor, which terminates the cycle. 7 8 9

49. Adenylate cyclase cyclic AMP ATP Activation PKA P P Enzyme (inactive) Activation of protein Kinase A (PKA) • Thetarget proteins of cAMP vary depending on cell types, however the best characterized cAMP target is the PROTEIN KINASE A (PKA). • PKA phosphorylates serine or threonine residues on target proteins thereby regulating their activity. • Among the target proteins of PKA the phosphodiesterases are responsible to lower cAMP concentration thus keeping PKA activation short and localized. Enzyme (active) Target Protein

50. Target Proteins of protein Kinase A (PKA) Examples of a signaling pathway mediated by cAMP-PKA include the activation of transcription regulator, the CRE binding protein (CREB). PKA phosphorylates CREB on a single serine. Active phosphorylated CREB (p-CREB) recruits the coactivator CREB-binding protein (CBP) (not known) to the regulatory sequence (CRE) present in many genes (i.e. somatostatin gene) that are activated by cAMP. CREB signaling plays an important role in the learning and memory process in the brain.