Signalling - Lehninger Chapter 12

Biochemistry 471/671 - M. Mossing

11/18/07, 9:52 PM

Biosignalling

• 12.1 Molecular Mechanisms
• 12.2 Gated Ion Channels
• 12.3 Receptor Enzymes
• 12.4 G-Protein Coupled Receptors
• 12.5 Scaffolding and Membrane Rafts
• 12.6 Micro-organisms and plants
• 12.7 Sensory Transduction
• 12.8 Steroid Hormone Receptors
• 12.9 Cell Cycle
• 12.10 Cancer and Apoptosis

12.1 Molecular Mechanisms

• Specificity - Receptors bind specific ligands and Chemical analogs,
• toxins, inhibitors are Antagonists
• Synthetic activators are Agonists
• Affinity can be measured in crude preparation
• Cooperativity can make the Receptor response curve steeper
• Amplification by a cascade of enzyme activation events (often phosphorylation)
• Desensitization - negative feed back
• often limits the duration of the Signal response
• Allows recalibration of the sensitivity of the response to continued stimulation
• Integration of multiple signals through common intermediates, co-localization

12.1a Scatchard analysis; Linear transformation of binding data

• Old fashioned, statistically flawed
• Receptor + Ligand ⇔ RL
• Kd = [R][L]/[RL]
• Separation of Bound (RL) and Free (L) Ligands
• Plotting Bound ([RL]) vs [L]) total gives a hyperbolic plot with RLmax = Rtotal
• (only if no nonspecific binding)
• Plotting Bound/Free vs. Bound ([RL]/[L] vs [RL]) is a Scatchard Plot
• Kd = (R_T - [RL]) [L]/[RL]
• [RL]/[L] = (R_T - [RL])/Kd
• Rearranging to the form y = mx + b
• Gives: [RL]/[L] = -1/Kd ([RL]) + (R_T/Kd)

12.2 Gated Ion Channels - by ligands or Voltage

• Allow ion-selective, passive transport in response to ligand binding or membrane potential
• The electrochemical driving force is made up of 2 terms
• A chemical (concentration gradient) term
• and an electrical potential term
• ΔG = RT ln (C_in/C_out) + zFΔψ
• Transient channel openings result in ion fluxes
• Ion flux changes membrane potential more than concentration (except for Ca²⁺_in)
• Flux continues until channel closes or ΔG = 0
• RT ln (C_in/C_out) = -zFΔψ
• E -- the equilibrium potential is the Δψ required to balance the concentration gradient

12.2a Nerve cell function

• At rest K⁺ channel is open; K⁺_in > K⁺_out Δψ = -60 mV
• Acetylcholine receptor channels open upon binding ligand
• Na⁺ and Ca²⁺ can pass to cell interior membrane depolarizes
• Prolonged ligand binding result in ligand-bound closed state
• Acetyl choline is hydrolyzed by acetylcholinesterase in synapse
• Voltage gated Na⁺ channels open (Na⁺ out => in) upon depolarization (amplify the signal)
• Voltage gated K⁺ channels open (K⁺ in => out) membrane repolarizes locally
• Wave of depolarization and repolarization (action potential) transmitted along the axon to the next synapse
• Voltage gated Ca²⁺ channels allow Ca²⁺ influx
• Ca²⁺ triggers SNAP/SNARE membrane fusion event that releases acetylcholine from exocytotic vesicles into the synapse

12.3 Receptor Enzymes

• Receptor Tyrosine Kinases (RTKs)
• activate intracellular kinase activity upon binding extracellular ligand
• Insulin Receptors (IR) bind 2 insulin peptide with 2 α chains
  • β chains (auto-)phosphorylate each other
  • P- β subunits now active tyrosine kinases
  • Active RTK initiates a signal transduction cascade
• Receptors coupled to JAK-STAT cascades
• Janus Kinase - Signal Transducers and Activators of Transcription
• Receptor Guanyl Cyclases

12.3a Insulin Signal Transduction Cascades

• Promote Cell growth, Glucose uptake and storage
• Insulin Receptor phosphorylates Insulin Receptor Substrate-1 (IRS-1)
• Adaptor proteins Grb2 and Sos bind to P-Tyr-IRS-1 via SH2 domain
• Sos activates Ras GTPase
• Ras.GTP activates Raf-1 kinase (MAPKKK)
• Raf-1 kinase activates MEK kinase (MAPKK)
• MEK kinase activates ERK kinase (MAPKinase)
• ERK kinase activates Elk1 transcripional activator
• P-Tyr-IRS-1 binds Phosphoinositol 3 kinase (PI3K)
• PI3K converts PIP₂ + ATP => PIP₃
• PIP₃ activates protein kinase b (PKB)
• PKB phosphorylates Glycogen Synthase Kinase (GSK) to INACTIVATE
  • GSK can no longer phosphorylate Glycogen Synthase
  • Unphospohrylated GS makes more glycogen
• PKB also promotes vesicle fusion to place more GLUT4 transporters in the plasma membrane

12.4 G-Protein Coupled Receptors

• Receptors have 7 transmembrane helices
• Interact with heterotrimeric G-Proteins
• Gα binds GDP at rest
• Gβ and Gγ complete the heterotrimer
• Activation of receptor leads to:
• GDP release and GTP binding to Gα
• Dissociation of β&gamma
• Slow hydrolysis of GTP to GDP returns the system to the resting state

12.4a The βAdrenergic Receptor (βAR)

• Binds epinephrine (Adrenaline) Kd (5μM)
• Agonist Isoproterenol Kd (0.4μM)
• Antagonist Propanolol Kd (0.005μM) blocks activation
• Gs.GTP activates adenylyl cyclase (AC)
• AC make cAMP from ATP
• cAMP binds to Protein Kinase A (PKA) regulatory subunits
• PKA kinase subunits initiate kinase cascade
• Desensitization
• β&gamma binds to βAdrenergic Receptor Kinase (βARK)
• βARK phosphorylates Serines on βAdrenergic Receptor
• βAR-P binds βarrestin
  • preventing rebinding of Gα_S
  • Stimulating endocytosis
  • βarrestin also serves as a scaffold for MAPK cascade

12.4b Second Messengers

• cAMP produced by AC; AC is regulated by many receptor pathways
• Somatostatin Receptor activates Gα_i inhibits AC
• Distinct effects can be obtained by localization to scaffolds
• Phosphatidylinositols => DAG + Inositol phosphates
• Gα_q.GTP activates Phospholipase C
• PLC cleaves PIP3 to DAG + IP3
• IP3 activates Ca²⁺ channels on the ER
• Increased [Ca²⁺] + DAG activate Protein Kinase C
• PKC initiates yet another kinase signalling cascade
• Ca²⁺ also binds to Calmodulin
• Calmodulin binds to many targets including CamKinases

12.5 Scaffolding and Membrane Rafts

• Up to 3500 signalling genes (> 500 kinases) in the human genome
• Pre-assembled complexes can coordinate signalling pathways
  • Assembly through specific protein binding modules/domains
  • SH2 and PTB domains bind phospho-Tyrosine
  • SH3 domains bind Proline rich target peptides
  • Plextrin Homology (PH) domains bind PIP3
• Scaffolding and adaptor proteins are multivalent
• Membrane Rafts and Caveolae can further enhance or insulate signalling pathways

12.7 Sensory Transduction

• Vison - Rhodopsin is a 7tmR, transducin is a htG-protein
• Signal transduction through cGMP and ligand gated channel
• Olfactory Receptors 7tmRs
• Gα_olf -> AC -> cAMP - or - PLC -> IP3 -open Ca²⁺ channel -> [Ca²⁺]_in -> open Cl^- channel -> depolarization
• Taste Receptors also 7tmRs
• Gustducin -> AC -> [cAMP] -> PKA -> closing K⁺ channel
• Common Features of 7tmR-htmG protein systems
• Gα.GTP activate an enzyme, enzyme makes a small molecule signal
• Gα.GTP -> Gα.GDP can be modulated by GAP or RGS proteins

12.7a Vison -

• A photon converts bound Rhodopsin ligand 11cis -> all trans retinal
• Gα releases GDP, β&gamma and Rhodopsin and activates PDE
• PDE converts cGMP to GMP
• Low [cGMP] causes Na⁺, Ca²⁺ channels to close
• membrane hyperpolarizes neural signal initiated
• Recovery / Adaptation
• [Ca²⁺] drops as Ca²⁺-Na⁺ antiporter
• low [Ca²⁺] stimulates Guanylyl Cyclase
• After prolonged stimulation low [Ca²⁺] and Recoverin stimulate Rhodopsin Kinase
• P-Rhodopsin binds Arrestin, Retinal ligand dissociates

12.8 Steroid Hormone Receptors (includes retinoids like vitamin D)

• Do not reside in membranes
• Hormones themselves are "lipids" - traverse the membrane by passive diffusion
• SR + Hormone -> SR.H + DNA -> SR.H.DNA complex
• Direct activation of gene expression

12.9 Cell Cycle

• Cells grow and divide according to an irreversible 4-stage program
• G1 - S (DNA synthesis) - G2 - M (Mitosis and cell division)
• DNA replication and Cell division are controlled by cyclin dependend kinases
• Cyclins (regulatory subunits) are synthesized and degraded at specific points in the cell cycle
• Progress through the cell cycle is highly regulated by
• Extracellular signalling pathways (growth factors)
• Internal surveillance, cycle checkpoints for DNA damage, synthesis, etc

12.10 Cancer and Apoptosis

• Cancer is inevitable because of 2 laws
• Survival of the fittest
• Murphy's law -- Anything that can go wrong - will.
• Cell growth in a multicellular organism is mostly halted in adults
• Many redundant signalling pathways can activate or suppress growth in different cells
• Mutations in signalling pathways can lead to uncontrolled growth
• Cells that grow out-compete, and interfere with normal cells

# 12.10a Cancer Genes - Oncogenes - gain of function - activate cell growth constitutively (dominant) - Proto-oncogenes normally activate growth, cell cycle only when stimulated - Tumor Supressor genes normally controll cell growth - Mutations lead to loss of function (recessive) - Both genes need to be inactivated to observe the cancer phenotype

12.10b Apoptosis

• In addition to growth suppression cells have an active suicide program
• Triggered by immune system responses
• Cell cycle checkpoint repeated failures
• Protease cascade of caspases