Module 5-Membranes and signalling (Lec 13-14) VERSION V (Posted a day after posting version IV).pdf

BIOC*4580 – Membrane Biochemistry Winter 2024 Membranes and Signalling Lehninger Chapter 12 Signals initiate at the membrane Signaling molecules are generally released by one of three general mechanisms: 1. Exocytosis the signal molecule is stored in secretory vesicles, and released upon a signal, (...

BIOC*4580 – Membrane Biochemistry Winter 2024 Membranes and Signalling Lehninger Chapter 12 Signals initiate at the membrane Signaling molecules are generally released by one of three general mechanisms: 1. Exocytosis the signal molecule is stored in secretory vesicles, and released upon a signal, (e.g. insulin) this is the main route in eukaryotes 2. Cleavage the signal is part of a protein that is released from its parent membrane by proteolysis, or by cleavage of a GPI anchor (e.g. epidermal growth factor) 3. ABC transporter signal molecule exits the cell via an ABC transporter (e.g. prostaglandins) G-Protein Coupled Receptor (GPCR) signaling In multicellular animals, GPCRs are the largest group of plasma membrane receptors. Consists of 3 components: 1) Plasma membrane receptor with 7 transmembrane helices e.g. epinephrine receptor 2) heterotrimeric guanosine nucleotide-binding protein (G protein) 3) Effecter enzyme in the plasma membrane that generates a 2nd messenger Second Messengers include: - cAMP - cGMP - inositol 1,4,5-trisphosphate (PIP3) 4 GPCR Topology 7 TM Helices, often additional extra-membrane helices in IL/EL loops N-terminus is extracellular, C-terminus is intracellular 3 intracellular loops (IL1->3), 3 extracellular loops (EL1->3) Disulfide bonds typically connect EL1-EL2, and EL2-EL3 EL2 (the gate) controls access to ligand binding site Helix 8 lies along the membrane surface, and is amphipathic and lipidylated Note TM3 is in center, other helices wrap around it Kimber, after Buehlar Key GPCR architectural features The outer leaflet portion contains the ligand binding site This region is diverse between families Shifts here are small upon binding (typically 1 Å or less) The inner leaflet portion is more conserved, and forms a signaling module Here large conformational changes convey the signal to the cytoplasmic G-protein Trends Pharmacol Sci. 2012 Jan; 33(1): 17-27 GPCR movement upon ligand binding Grey is the unliganded protein, cyan shows conformation upon ligand binding TM6 in β2 AR moves 14 Å – bending at a conserved Pro kink TM5 also typically moves in concert (connected by IL3) This opens up a large cleft for the Gprotein to bind The β-Adrenergic Receptor is a GPCR Adrenergic receptors = protein receptors in the plasma membrane that bind epinephrine (adrenalin) – four general types: α1, α2, β1, β2 β-adrenergic receptors = applies to both β1 and β2 subtypes GPCR PDB 2YCW More than a third of all drugs target GPCRs (e.g., “beta blockers” used to treat hypertension, cardiac arrythmias, glaucoma, anxiety, migraines all target the GPCR β-adrenergic receptor) G-protein activation cycle Gα with GDP bound - inactive complex Binding a GPCR-ligand complex opens up the Gα subunit, allowing GTP to bind instead of GDP. Gα dissociates from the Gβ /Gγ complex. The Gα -GTP activate adenylyl cyclase which catalyzes the synthesis of cAMP from ATP. cAMP binds and activates cAMP– dependent protein kinase (PKA) PKA phosphorylate specific Ser or Thr residues of target proteins bringing about the cellular response to epinephrine Lehninger, 6th Ed./8th Ed Fig. 12-5 Activation of cAMP-Dependent Protein Kinase (PKA) R2C2 complex is catalytically inactive because the autoinhibitory domain of each R subunit occupies the substratebinding cleft of each C subunit The autoinhibitory domain is an intrinsically disordered region that can change its shape to occupy the binding sites of varied proteins. cyclic AMP is an allosteric activator of PKA – binding of cAMP yields two active C subunits Fluorescence Resonance Energy Transfer (FRET) Fluorescence resonance energy transfer (FRET) = measures the nonradiative transfer of energy between fluorescent probes attached to each protein – used to determine if two proteins interact and where in the cell they interact Measuring [cAMP] with FRET When [cAMP] is low, R and C subunits of PKA are associated and FRET is exhibited When [cAMP] rises, R and C subunits of PKA dissociate and FRET ceases Measuring PKA Activity with FRET When PKA is inactive, the Ser residue is not phosphorylated, 14-3-3 has no affinity for the Ser residue, and FRET is not observed When PKA is active in the cell, the Ser residue is phosphorylated, 14-3-3 binds the Ser residue, and FRET is observed Several Mechanisms Cause Termination of the βAdrenergic Response Methods of termination: – epinephrine concentration drops below the Kd for its receptor – Gsα has intrinsic GTPase activity that switches it to its inactive form by converting its bound GTP to GDP. – cAMP is hydrolyzed to 5′ AMP by cyclic nucleotide phosphodiesterase Cyclic AMP acts as a second messenger for many regulatory molecules (e.g., Dopamine, epinephrin, glucagon, serotonin, somatostatin etc.,) Stimulatory G protein (Gs) – Activates adenylyl cyclase inhibitory G protein (Gi) = inhibits adenylyl cyclase and lowers [cAMP] – activated by the binding of somatostatin to its receptor – structurally homologous to Gs The GTPase activity of most G proteins is weak. GTPase activator proteins (GAPs) = increase the GTPase activity of G proteins by about 105 –fold Guanosine nucleotide-exchange factors (GEFs) = catalyze the exchange of bound GDP with GTP Example: the β-adrenergic receptor The β-Adrenergic Receptor is Desensitized by Phosphorylation and by Association with Arrestin Desensitization decreases the response while the signal persists β-adrenergic receptor kinase (βARK) = phosphorylates several Ser residues near the receptor’s C-terminus when epinephrine is bound to the receptor β-arrestin (βarr or arrestin 2) = protein that binds to the receptor following receptor phosphorylation and blocks receptor sites that interact with the G protein Adaptor Proteins Confine Signaling to a Specific Region of the Cell Adaptor proteins = noncatalytic proteins that hold together other protein molecules that function in concert AKAPs (A kinase anchoring proteins) = have multiple, distinct protein-binding domains The mechanism of G protein activation and inactivation via the binding of GDP and GTP Example: Ras, the G protein prototype In the nucleotide-binding site, Ala146 main chain hydrogen bonds to the guanine oxygen, allowing GTP, but not ATP, to bind switch I and switch II = regions are exposed when GTP is bound and can interact with downstream proteins – Thr35 and Gly60 main chain hydrogen bond with the oxygens of the γphosphate of GTP P loop (phosphate-binding) = contains a Lys residue that binds the γ phosphate of GTP Ras, the G-Protein Prototype (a ~20 kDa minimal signaling unit) (Active conformation of Ras) GTP Hydrolysis Flips the Switches in Ras When GTP is hydrolyzed, the loss of hydrogen bonds allows the switch I and switch II regions to relax into a buried conformation Ras turns to its inactive conformation Defects in G proteins leads to a variety of diseases About 25% of human cancers display a mutation in the G protein Ras that eliminates its GTPase activity This causes Ras to be constantly active promoting cell division “Activating” and “Inactivating” Mutations in Gα “activating” mutations = lead to a continuously elevated [cAMP] – found in ~40% of adenomas “inactivating” mutations = cause individuals to be unresponsive to hormones that act through cAMP Cholera Toxin (secreted by Vibrio cholerae ) Blocks the GTPase Activity of Gs Cholera toxin = enzyme that catalyzes transfer of the ADP-ribose moiety of NAD+ to an Arg residue of Gsα Blocks the GTPase activity of Gsα leaving Gsα permanently active and leading to high intracellular [cAMP] High cAMP activates PKA which phosphorylates and activates CFTR Clchannel and a Na+-H+ exchanger in intestinal epithelium High NaCl efflux causes increased water loss as cells try to maintain osmotic balance See Page 424 Lehninger 8ed for detailed explanation of the cholera toxin mechanism of action The Mechanism of action of Cholera Toxin Fragment A1 Subunit A Enters the cell Catalyses transfer of ADP ribose from NAD+ to Gsα Fragment A2 Cholera Toxin (A Heterodimeric Protein) Subunit B Recognizes and bind to specific gangliosides on the surface of intestinal epithelial cells Provides a route for subunit A to enter the cell Block the GTPase activity of GSα GPCRs coupled to Gq acts through Phospholipase C e.g., Acetylcholine (muscarinic) Gq = associated trimeric G protein that activates the PIP2-specific phospholipase C (PLC) when an agonist binds its specific receptor PLC catalyses the cleavage of membrane phospholipid phosphatidylinositol 4,5bisphosphate (PIP2) to diacylglycerol (DAG) and inositol 1,4,5trisphosphate (IP3) DAG and IP3 = potent second messengers IP3 Opens the IP3-Gated Ca2+ Channel IP3-gated Ca2+ channel = receptor-gated channel in the endoplasmic reticulum that opens to release sequestered Ca2+ into the cytosol Elevated [Ca2+] and Diacylglycerol Activate Protein Kinase C (PKC) Protein kinase C (PKC; C for Ca+2) = binds and phosphorylates proteins that contain a PKC consensus sequence Proposed Mechanism of Action of the IP3Gated Ca2+ Channel (Ca+2 in the ER is maintained at a high level by the action of the SERCA pump.) Receptor Tyrosine Kinases (RTKs) ▪ Receptor tyrosine kinases (RTKs) = family of plasma membrane receptors with protein kinase activity – have an extracellular ligand binding domain and a cytoplasmic Tyr kinase domain ▪ Humans have ~ 60 genes encoding RTKs ▪ Active insulin receptor protein (INSR) = a dimer of αβ monomers ▪ α subunits contain the insulin-binding domain ▪ intracellular domains of the β subunits contain the protein kinase activity ▪ Stimulation of the Insulin Receptor Initiates a Cascade of Protein Phosphorylation Reactions Activation of Tyrosine Kinase by Autophosphorylation One molecule of insulin binds between the two subunits of INSR on the extracellular side. This causes the two intracellular Tyr kinase domains to move together, activating them. autophosphorylation = each β subunit phosphorylates three essential Tyr residues near the C-terminus of the other β subunit Opens the active site in each β subunit activating them Phosphorylate Tyr residues of target proteins Regulation of Gene Expression by Insulin insulin receptor substrate 1 (IRS1) = becomes the point of nucleation for a complex of proteins that carry the message from the receptor to end targets SH2 domain = binds phosphorylated Tyr residues in a protein partner The Membrane Phospholipid PIP3 Functions at a Branch in Insulin Signaling The enzyme phosphoinositide 3kinase (PI3K) can also bind to phosphorylated IRS-1 vis its SH2 domain. Activates PI3K which catalyses the phosphorylation of PIP2 to PIP3 PKB (also known as Akt) binds to PIP3 and is phosphorylated and activated by the protein kinase PDK1. Activation of PKB starts a cascade of events initiating the movement of GLUT4 to the plasma membrane and activation of glycogen synthase. Turning RTKs off Tyrosine phosphatases (membrane-bound and cytosolic) dephosphorylate pTyr in receptors and other targets Receptor-mediated endocytosis removes receptor and bound growth factor (ligand) from the cell surface Pancreatic β Cells Secrete Insulin in Response to Changes in Blood Glucose Glucose entry into pancreatic β cells via GLUT2 leads to increased glycolysis and increased production of ATP High [ATP] closes ATP-gated K+ channels Reduced efflux of K+ depolarizes the membrane Opens voltage-gated Ca2+ channels The increase in cytosolic [Ca2+] triggers the release of insulin by exocytosis Reduction of blood glucose by insulin reverses this process slowing or stopping the release of insulin ATP-Gated K+ Channels in β Cells Channels are octamers: – four identical Kir6.2 subunits – four identical SUR1 subunits Sulfonylurea drugs = oral medications used in the treatment of type 2 diabetes mellitus – bind to the SUR1 subunits, closing the channels and stimulating insulin release The Insulin Receptor Is A Prototype for Receptor Tyrosine Kinases All have cytoplasmic Tyr kinase domains Extracellular domains are variable reflecting the different specificities of each receptor to a different growth factor. VEGFR - Vascular Endothelial Growth Factor PDGFR - Platelet Derived Growth Factor EGFR - Epidermal Growth Factor TrkA - High Affinity Nerve Growth Factor FGFR - Fibroblast Growth Factor Stryer 15.25 Dimerization of extracellular domains upon growth hormone binding drives interaction of cytoplasmic domains Receptors are monomeric in the absence of their ligand (unlike the INSR) Binding HGH to one receptor creates a binding site for a second receptor driving dimerization of the receptor Phosphorylation of the kinase domain by its partner in the dimer activates the kinase itself The kinase domain then phosphorylates other protein targets leading to a cascade of reactions similar to insulin signaling leading to PIP2 signaling 1. Which factor is NOT involved in the specificity of signal transduction? a. interactions between receptor and signal molecules b. location of receptor molecules c. structure of receptor molecules d. structure of signal molecules e. transmembrane transport of signal molecules by receptor molecules 2. Which statement concerning receptor enzymes is correct? a. They are not usually membrane-associated proteins. b. They contain an enzyme activity that acts on a cytosolic substrate. c. They contain an enzyme activity that acts on the extracellular ligand. d. They have a ligand-binding site on the cytosolic side of the membrane. e. They have an active site on the extracellular side of the membrane. 3. Cholera toxins: a. block the GTPase activity of Gs. b. bind to gangliosides (cell surface oligosaccharides) c. associate with a G protein. d. All of the answers are correct. 4. _____ is NOT involved in signal transduction by the β-adrenergic receptor pathway. a. ATP b. Cyclic AMP c. Cyclic GMP d. GTP e. All of these are involved. 5. In signal transduction by the β-adrenergic receptor pathway (through the Gs G proteins), _____ catalyzes the synthesis of cyclic AMP. a. tyrosine kinase b. epinephrine c. Gsα d. adenylyl cyclase 6. Hormone-activated phospholipase C can convert phosphatidylinositol 4,5-bisphosphate to: a. diacylglycerol + IP3. b. diacylglycerol + inositol + inorganic phosphate. c. glycerol + inositol + inorganic phosphate. d. glycerol + phosphoserine. e. phosphatidyl glycerol + IP2. 7. Autophosphorylation of receptor tyrosine kinases depends on: a. dimerization of the receptor. b. phosphorylation of multiple Tyr residues. c. ligand binding. d. transmission of conformational changes through the membrane. e. All of the answers are correct. 8. Which statement concerning signal transduction by the insulin receptor is correct? a. Activation of the receptor protein kinase activity results in the activation of additional protein glycosylases. b. Binding of insulin to the receptor activates a protein kinase. c. Binding of insulin to the receptor results in a change in its primary structure. d. The receptor protein kinase activity is specific for tryptophan residues on the substrate proteins. e. The substrates of the receptor protein kinase activity are mainly proteins that regulate transcription. 9. Which feature of signal transduction comes from precise molecular complementarity between a signal molecule and its receptor? a. specificity b. cooperativity c. amplification d. desensitization e. integration 10. G protein–coupled activation of phospholipase C directly generates which second messenger? a. diacylglycerol b. inositol triphosphate c. calcium d. both diacylglycerol and inositol triphosphate e. both inositol-1,4,5-trisphosphate and calcium 11. Cholera toxin leads to the activation of the α subunit of G proteins through what kind of covalent modification? a. phosphorylation b. adenylation c. acetylation d. palmitoylation e. ADP-ribosylation 12. Salbutamol is an epinephrine agonist for the β-adrenergic receptor. What effects would be seen if a hepatocyte is treated with salbutamol? a. inactivation of adenylyl cyclase b. activation of the triacylglyceride synthesis c. activation of glycogen synthesis d. glucose export from cells e. both activation of the breakdown of glycogen and glucose export from cells 13. Which characteristic is NOT one attributed to a second messenger? a. intracellular signaling molecules b. molecules that can easily pass across membrane bilayers c. molecules that can act as allosteric effectors for signaling proteins d. molecules that can be rapidly converted between active and inactive forms e. molecules that are synthesized in response to receptor/signal interactions Good Luck!! Wish you all the best!!