Bioc 325 Lecture 1-2023 PDF

 Course Coordinator: Dr. Ayad Jaffa; DTS 4th floor, email: [email protected]  Teaching Assistant:  Signal transduction (3rd edition), 2015. Authors: IJsbrand M. Kramer. ISBN: 9780123948038  eBook ISBN: 9780123948199  Signal Transduction: Principles, Pathways, and Pro...

 Course Coordinator: Dr. Ayad Jaffa; DTS 4th floor, email: [email protected]  Teaching Assistant:  Signal transduction (3rd edition), 2015. Authors: IJsbrand M. Kramer. ISBN: 9780123948038  eBook ISBN: 9780123948199  Signal Transduction: Principles, Pathways, and Processes 1st Edition  by Lewis Cantley (Editor), Tony Hunter (Editor), Richard Sever (Editor), Jeremy Thorner (Editor)  BIOC 325 Course: Fall Schedule 2022 (Tentative) Dates Topic August 30 Topic 1: Signal Transduction: Definition and Pharmacological Introduction September Topic 2: Roles of Structural Domains and Scaffold Proteins in Signal 4 Transduction September Topic 3: 7TM G-protein Coupled Receptors: 6  General Features September  Effector Systems and Second Messengers (Part 1): Adenylate Cyclase 11 September  Effector Systems and Second Messengers (Part 2): PLCβ 13 September  Calcium in Signaling 18  Proteins associated with GPCR (Part 1): GRKs, RGS September  Proteins associated with GPCR (Part 2): β-Arrestin, Dynamin, Clathrin 20  GPCR Desensitization, Internalization, and Recycling September  Special GPCRs: Protease-Activated Receptors (PARs) 25 September  GPCR Heterodimerization 27  Classical methods to assess signaling and coupling of GPCRs October 2  Topic 4: Receptor Tyrosine Kinase (RTKs) October 4  Important Kinases Downstream of RTKs Adaptor Proteins October 9  Topic 5: Mitogen-Activated Protein Kinases (MAPKs) and Transactivation of EGFRs by GPCRs October 11  Topic 6: Nuclear Receptors October 16 Article presentations and discussions October 18 Article presentations and discussions October 23 Article presentations and discussions EXAM 1 September 28 Exam 2 October 26  Student Presentation 15%  Midterm Exam 35%  Final Exam 45%  Class Participation 5% Introduction into Signal Transduction  Define signal transduction  Give an overview of the steps of signal propagation, and types and properties of the receptors.  Define receptor agonists and antagonists Receptors are structural proteins on the surface or inside of a cell that selectively receive and bind a specific substance (ligand) and elicit a specific response.  Membrane bound receptors ◦ G Protein coupled receptors Adrenergic receptors, Angiotensin II, Bradykinin ◦ Enzyme receptors Tyrosine kinase ◦ Ligand gated ion channel receptors Nicotinic, GABA, glutamate  Intracellular and nuclear receptors ◦ IP3 receptor (ER) ◦ Steroid hormones receptor  Receptors have 2 major properties A. Recognition B. Transduction Decker et.al. 2005,Nature Reviews Immunology 5, 675-687| doi:10.1038/nri1684  A. Recognition: The receptor protein must exist in a conformational state that allows for recognition and binding of a compound and must satisfy the following criteria: ◦ Saturability – receptors exist in limited numbers. ◦ Reversibility – binding must occur non-covalently due to weak intermolecular forces (H-bonding, van der Waal forces). ◦ Stereo-selectivity – receptors should recognize only one of the naturally occurring optical isomers [(+ or -), (d or l), or (S or R)]. ◦ Agonist specificity – structurally related drugs should bind well, while physically dissimilar compounds should bind poorly. ◦ Tissue specificity – binding should occur in tissues known to be sensitive to the endogenous ligand. Binding should occur at physiologically relevant concentrations.  B. Transduction: The second property of a receptor is that the binding of an agonist must be transduced (propagated) into functional response (biological or physiological). Different receptor types are linked to effector systems: ◦ either directly ◦ or through simple or more-complex intermediate signal amplification systems. “The relaying of molecular signals (for example, as contained in a hormone) or physical signals (for example, sensory stimuli) from a cell's exterior to its intracellular response mechanisms.” McGraw-Hill Dictionary of Scientific and Technical Terms, 6th edition, published by The McGraw-Hill Companies, Inc.  1. Initiation of signal 2. Transmission of signal 3. Nuclear or cytoplasmic events 4. Biological effects  Initiation: interaction of ligand with its receptor.  Transmission: the action by which the receptor transmits the signal into the cell.  Nuclear or cytoplasmic events  Biological effects  Signal transduction is involved in multiple biological processes: ◦ Proliferation ◦ Migration ◦ Apoptosis  Any defect in signaling pathways can lead to different diseases ◦ Cardiovascular diseases ◦ Alzheimer disease ◦ Cancer  Signal transduction refers to any process by which a cell converts one kind of signal or stimulus into another.  Involves ordered sequences of biochemical reactions inside the cell, which are activated by second messengers, resulting in a signal transduction pathway.  Such processes are usually rapid; they last for: ◦ milliseconds in the case of ion flux, ◦ minutes for the activation of protein- and lipid-mediated kinase cascades, ◦ or hours and even days for gene expression.  Signal transduction always involves the following phenomena: ◦ Signal reception ◦ Signal integration ◦ Signal amplification ◦ Signal reaches its target (Functional response) http://www.biochem.mpg.de/en/rd/oesterhelt/web_page_list/ShortDesc_ST_cascade/ The signal transduction cascade (or chain) is responsible for  Integration ◦ Integration indicates that several receptors activate/deactivate one and the same catalyst which thereby acts as a signal integrator. ◦ In eukaryotic signal transduction networks, cross-talk between different systems adds another level of integration.  Amplification ◦ Amplification typically consists of activation of a catalyst, such as a protein kinase, which amplifies the input of a single unit (photon or molecule) into the phosphorylation of many target molecules.  Adaptation ◦ Adaptation is defined as return of the signaling system to the pre-stimulus level while the stimulus persists. This enables cells to perceive changes in stimulus size rather than absolute stimulus levels (feedback). ◦ Example: Adaptation of the eye to bright sunlight or dim moonlight.  Substances that bind to Receptors to serve a biological activity.  It is a signal triggering molecule, binding to a site on a target protein to transfer information.  Types: ◦ peptides (short protein) ◦ small molecules  neurotransmitter Ligand  hormone  pharmaceutical drug  Toxin  Lipids Receptor ◦ Photon/light particles  Ligand-Receptor interaction follows simple mass-action relationships, i.e. only one ligand occupies each receptor and binding is reversible  When a Ligand (L) combines with a receptor (R), it does so at a rate which is dependent on the concentration of the ligand and the concentration of the receptor. k1 [L] + [R] [L-R] L = Ligand k2 R = receptor LR = ligand-receptor complex [L].[R].k1 = [L-R].k2 k1 = rate for association k2 = rate for dissociation KD = Dissociation Constant KA = Affinity Constant 1 [L].[R] k2 kD= = = kA [L-R] k1 KD: Dissociation Constant: represents the concentration of the ligand required to occupy 50% of receptor binding sites  Law of Mass Action Which of the following 2 drugs has better affinity? kD Activation of membrane receptors and target cell responses is proportional to the degree of receptor occupancy.  Radioimmunoassay (RIA)  Dose-response experiments  Use of Receptor antagonists: ◦ Competitive ◦ Non-competitive  Provides direct measure of the number (or density) of receptors in the LR complex.  Provides information on receptor density and ligand affinity and selectivity.  Ligand is radio-labeled (125I, 35S. or 3H). Bmax  Selection of proper radio-ligand:  Agonist vs. antagonist  Higher affinity for antagonists  Saturation binding curve-occurs at steady state conditions (equilibrium is theoretical only). Bmax: maximal number of binding sites  The amount of ligand bound at any time is solely determined by: ◦ the number of receptors ◦ the concentration of ligand added ◦ the affinity of the ligand for its receptor   Measures the functional response of a ligand (drug), which is an indirect assessment of receptor binding.  Can be in vitro, in vivo, or ex vivo. EC50: is the concentration at which the drug exerts 50% of its total activity  Depending on the effects that the ligands cause, they can be divided into ◦ Agonists (bind to active site of receptor leading to an effect) ◦ Antagonists (prevent agonist-mediated responses by preventing a drug from binding and eliciting its normal response) ◦ Partial agonists (have weak binding or effect alone; but have antagonistic effect in the presence of an agonist)   Not all agonists acting at the same receptor produce the same maximal response. C is partial agonist Three drugs with presumably different receptor affinities and different maximal effects.  Antagonists inhibit agonist-mediated responses by preventing a drug from binding and eliciting its normal response.  Two types of antagonists ◦ Competitive ◦ Non-competitive   Binds reversibly to the same site as the endogenous ligand or agonist.  Can be overcome by more [ligand]  Their presence produces a right- ward shift in both the binding and dose-response curves.  No change in Emax or Bmax, but different EC50  Similar dose-response curve shapes indicates the presence of a competitive agonist (competing for the same binding sites). A = agonist alone Note: There are also irreversible antagonists B = antagonist (one concentration) that bind covalently to the ligand binding A+B = agonist + antagonist site of the receptor. Ex: Aspirin  Does not prevent formation of the L-R complex, but impairs the conformational change which triggers a response.  Binds to an allosteric binding site: (site different than the agonist binding site)  Cannot be overcome by adding more agonist  Emax and Bmax are reduced, but EC50 remains the same for the unaffected receptors. A = agonist alone  Dose-response curves will have B = antagonist (one concentration) different shapes indicating A+B = agonist + antagonist different binding sites.  Neurotransmitters Hormones Neuropeptides (ex: Growth Hormone) Ions (ex: Na+; Ca2+)  After the binding of ligand to receptor (first messenger), specific molecules are activated to propagate this signal.  Second messengers are intracellular signaling molecules released/activated in the cell to trigger biological processes such as proliferation, differentiation, migration, survival, and apoptosis.  Second messengers are therefore one of the initiating components of intracellular signal transduction cascades.  Examples ◦ cAMP/GMP, Inositol Triphosphate (IP3), Diacylglycerol (DAG), and Calcium (Ca2+). Roles of Structural Domains and Scaffold Proteins in Signal Transduction 40