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Signal Transduction Cell-cell signalling at a distance: Regulation of haploid and diploid specific genes in yeast mating a •Each haploid cell secretes a mating type pheromone eg. Alpha factor •Each haploid cell expresses a mating pheromone receptor eg. Alpha factor receptor •When a haploid cell ‘receives’ a signal from the opposite mating type, a signal transduction cascade is initiated •The consequences of this include arrest in the cell cycle in G1 (so that both cells are 1N), formation of ‘shmoo’ morphology, and expression of proteins involved in cell fusion Modes of cell-cell signaling 1. Direct cell-cell or cell-matrix (integrins and cadherins) 2. Indirect: Secreted molecules. A. Endocrine signaling. The signaling molecules are hormones secreted by endocrine cells and carried through the circulation system to act on target cells at distant body sites. B. Paracrine signaling. The signaling molecules released by one cell act on neighboring target cells (neurotransmitters). C. Autocrine signaling. Cells respond to signaling molecules that they themselves produce (response of the immune system to foreign antigens, and cancer cells). Cell Membrane Receptor Relays (‘transducers’) and second messengers Signal molecule Cell Response Steroid hormones This class of molecules diffuse across the plasma membrane and bind to Receptors in the cytoplasm or nucleus. They are all synthesized from cholesterol. They include sex steroids (estrogen, progesterone, testosterone) corticosteroids (glucocorticoids and mineralcorticoids) Thyroid hormone, vitamin D3, and retinoic acid have different structure and function but share the same mechanism of action with the steroids. Steroid Receptor Superfamily. They are transcription factors that function either as activators or repressors of transcription. Steroid Hormone Biosynthesis P450 superfamily Cholesterol Pregnenolone Testosterone Estradiol threonine (Thr) serine (Ser) H H + H3N+ C COO H 3N C COO CH OH CH2 CH3 OH Many enzymes are regulated by covalent attachment of phosphate, in ester linkage, to the side-chain hydroxyl group of a particular amino acid residue (serine, threonine, or tyrosine). Protein Kinase O Protein OH + ATP Protein O P O + ADP O Pi H2O Protein Phosphatase A protein kinase transfers the terminal phosphate of ATP to a hydroxyl group on a protein. A protein phosphatase catalyzes removal of the Pi by hydrolysis. Phosphorylation may directly alter activity of an enzyme, e.g., by promoting a conformational change. Alternatively, altered activity may result from binding another protein that specifically recognizes a phosphorylated domain. E.g., 14-3-3 proteins bind to domains that include phosphorylated Ser or Thr in the sequence RXXX[pS/pT]XP, where X can be different amino acids. Binding to 14-3-3 is a mechanism by which some proteins (e.g., transcription factors) may be retained in the cytosol, & prevented from entering the nucleus. Protein Kinase O Protein OH + ATP Protein O P O + ADP O Pi H2O Protein Phosphatase Protein kinases and phosphatases are themselves regulated by complex signal cascades. For example: Some protein kinases are activated by Ca++- calmodulin. Protein Kinase A is activated by cyclic-AMP (cAMP). Adenylate Cyclase (Adenylyl cAMP NH2 Cyclase) catalyzes: ATP cAMP + PPi N N Binding of certain hormones (e.g., epinephrine) to the outer N N surface of a cell activates H2 O Adenylate Cyclase to form 5' C 4' H H 1' cAMP within the cell. O H 3' 2' H Cyclic AMP is thus considered P O OH O to be a second messenger. O- Phosphodiesterase enzymes NH2 cAMP catalyze: cAMP + H2O AMP N N The phosphodiesterase that N N cleaves cAMP is activated by phosphorylation catalyzed by H2 O 5' C 4' Protein Kinase A. H H 1' O H 3' 2' H Thus cAMP stimulates its own P O OH degradation, leading to rapid O O- turnoff of a cAMP signal. Protein Kinase A (cAMP-Dependent Protein Kinase) transfers Pi from ATP to OH of a Ser or Thr in a particular 5-amino acid sequence. Protein Kinase A in the resting state is a complex of: • 2 catalytic subunits (C) • 2 regulatory subunits (R). R2C2 R2C2 Each regulatory subunit (R) of Protein Kinase A contains a pseudosubstrate sequence, like the substrate domain of a target protein but with Ala substituting for the Ser/Thr. The pseudosubstrate domain of (R), which lacks a hydroxyl that can be phosphorylated, binds to the active site of (C), blocking its activity. R2C2 + 4 cAMP R2cAMP4 + 2 C When each (R) binds 2 cAMP, a conformational change causes (R) to release (C). The catalytic subunits can then catalyze phosphorylation of Ser or Thr on target proteins. PKIs, Protein Kinase Inhibitors, modulate activity of the catalytic subunits (C). MAPK/ERK pathway kinase cascade from extracellular mitogen to transcription control in nucleus Epidermal growth factor & receptor growth factor receptor bound protein 2 guanine nucleotide exchange factor SOS G Protein Signal Cascade Most signal molecules targeted to a cell bind at the cell surface to receptors embedded in the plasma membrane. Only signal molecules able to cross the plasma membrane (e.g., steroid hormones) interact with intracellular receptors. A large family of cell surface receptors have a common structural motif, 7 transmembrane -helices. Rhodopsin was the 1st member of this family to have its 7-helix structure confirmed by X-ray crystallography. Rhodopsin PDB 1F88 Rhodopsin is unique in that it senses light. Most 7-helix receptors have domains facing the extracellular side of the plasma membrane that recognize & bind particular signal molecules (ligands). Rhodopsin PDB 1F88 The signal is passed from a 7-helix receptor to an intracellular G-protein. Seven-helix receptors are thus called GPCR, or G-Protein-Coupled Receptors. Approx. 800 different GPCRs are encoded in the human genome. G-protein-Coupled Receptors may dimerize or form oligomeric complexes within the membrane. Ligand binding may promote oligomerization, which may in turn affect activity of the receptor. Various GPCR-interacting proteins (GIPs) modulate receptor function. Effects of GIPs may include: altered ligand affinity receptor dimerization or oligomerization control of receptor localization, including transfer to or removal from the plasma membrane promoting close association with other signal proteins G-proteins are heterotrimeric, with 3 subunits , b, g. A G-protein that activates cyclic-AMP formation within a cell is called a stimulatory G-protein, designated Gs with alpha subunit Gs. Gs is activated, e.g., by receptors for the hormones epinephrine and glucagon. The b-adrenergic receptor is the GPCR for epinephrine. hormone signal outside GPCR plasma The subunit of membrane a G-protein (G) gg cytosol binds GTP, & AC GDP bbGTP can hydrolyze it to GDP + Pi. GTP GDP ATP cAMP + PPi & g subunits have covalently attached lipid anchors that bind a G-protein to the plasma membrane cytosolic surface. Adenylate Cyclase (AC) is a transmembrane protein, with cytosolic domains forming the catalytic site. hormone signal outside GPCR plasma The complex membrane of b & g gg cytosol subunits Gb,g AC GDP bbGTP inhibits G. GTP GDP ATP cAMP + PPi The sequence of events by which a hormone activates cAMP signaling: 1. Initially G has bound GDP, and ,b, & g subunits are complexed together. hormone signal outside GPCR plasma membrane gg cytosol AC GDP bbGTP GTP GDP ATP cAMP + PPi 2. Hormone binding to a 7-helix receptor (GPCR) causes a conformational change in the receptor that is transmitted to the G protein. The nucleotide-binding site on G becomes more accessible to the cytosol, where [GTP] > [GDP]. G releases GDP & binds GTP (GDP-GTP exchange). hormone signal outside GPCR plasma membrane gg cytosol AC GDP bbGTP GTP GDP ATP cAMP + PPi 3. Substitution of GTP for GDP causes another conformational change in G. G-GTP dissociates from the inhibitory bg complex & can now bind to and activate Adenylate Cyclase. hormone signal outside GPCR plasma membrane gg cytosol AC GDP bbGTP GTP GDP ATP cAMP + PPi 4. Adenylate Cyclase, activated by the stimulatory G-GTP, catalyzes synthesis of cAMP. 5. Protein Kinase A (cAMP Dependent Protein Kinase) catalyzes phosphorylation of various cellular proteins, altering their activity. Turn off of the signal: 1. G hydrolyzes GTP to GDP + Pi. (GTPase). The presence of GDP on G causes it to rebind to the inhibitory bg complex. Adenylate Cyclase is no longer activated. 2. Phosphodiesterase catalyzes hydrolysis of cAMP AMP. Turn off of the signal (cont.): 3. Receptor desensitization occurs. This process varies with the hormone. Some receptors are phosphorylated via specific receptor kinases. The phosphorylated receptor may then bind to a protein b-arrestin, that promotes removal of the receptor from the membrane by clathrin-mediated endocytosis. 4. Protein Phosphatase catalyzes removal by hydrolysis of phosphates that were attached to proteins via Protein Kinase A. Signal amplification is an important feature of signal cascades: One hormone molecule can lead to formation of many cAMP molecules. Each catalytic subunit of Protein Kinase A catalyzes phosphorylation of many proteins during the life-time of the cAMP. The stimulatory Gs, when it binds GTP, activates Adenylate cyclase. An inhibitory Gi, when it binds GTP, inhibits Adenylate cyclase. Different effectors & their receptors induce Gi to exchange GDP for GTP than those that activate Gs. In some cells, the complex of Gb,g that is released when G binds GTP is itself an effector that binds to and activates other proteins. Structure of G proteins: PDB 1GIA The nucleotide binding site in G consists of loops that extend out from the edge of a 6-stranded b-sheet. Three switch domains have been identified, that change GTPgS position when GTP substitutes for GDP on G. Inhibitory G These domains include residues adjacent to the terminal phosphate of GTP and/or the Mg++ associated with the two terminal phosphates. O GTP hydrolysis N NH H H O O O O N N NH2 O P O P O P O CH2 O O O O H H H H OH OH GTP hydrolysis occurs by nucleophilic attack of a water molecule on the terminal phosphate of GTP. Switch domain II of G includes a conserved glutamine residue that helps to position the attacking water molecule adjacent to GTP at the active site. PDB 1GP2 PDB 1GP2 Gb - side view of b-propeller Gb – face view of b-propeller The b subunit of the heterotrimeric G Protein has a b-propeller structure, formed from multiple repeats of a sequence called the WD-repeat. The b-propeller provides a stable structural support for residues that bind G. The family of heterotrimeric G proteins includes also: transducin, involved in sensing of light in the retina. G-proteins involved in odorant sensing in olfactory neurons. There is a larger family of small GTP-binding switch proteins, related to G. Small GTP-binding proteins include (roles indicated): initiation & elongation factors (protein synthesis). Ras (growth factor signal cascades). Rab (vesicle targeting and fusion). ARF (forming vesicle coatomer coats). Ran (transport of proteins into & out of the nucleus). Rho (regulation of actin cytoskeleton) All GTP-binding proteins differ in conformation depending on whether GDP or GTP is present at their nucleotide binding site. Generally, GTP binding induces the active state. Phosphatidylinositol Signal Cascades O O H2 C O C R2 R1 C O CH O H2 C O P O O H 1 6 OH OH H OH 2 H 5 OH phosphatidyl- H H 3 4 inositol H OH Some hormones activate a signal cascade based on the membrane lipid phosphatidylinositol. IP3 may instead be phosphorylated via specific kinases, to IP4, IP5 or IP6. Some of these have signal roles. E.g., the IP4 inositol-1,3,4,5-tetraphosphate in some cells stimulates Ca++ entry, perhaps by activating plasma membrane Ca++ channels. Protein Kinase B (also called Akt) becomes activated when it is recruited from the cytosol to the plasma membrane surface by binding to products of PI-3 Kinase, e.g., PI-3,4,5-P3. Other kinases at the cytosolic surface of the plasma membrane then catalyze phosphorylation of Protein Kinase B, activating it. Activated Protein Kinase B catalyzes phosphorylation of Ser or Thr residues of many proteins, with diverse effects on metabolism, cell growth, and apoptosis. Downstream metabolic effects of Protein Kinase B include stimulation of glycogen synthesis, stimulation of glycolysis, and inhibition of gluconeogenesis. Signal protein complexes: Signal cascades are often mediated by large "solid state" assemblies that may include receptors, effectors, and regulatory proteins, linked together in part by interactions with specialized scaffold proteins. Scaffold proteins often interact also with membrane constituents, elements of the cytoskeleton, and adaptors mediating recruitment into clathrin-coated vesicles. They improve efficiency of signal transfer, facilitate interactions among different signal pathways, and control localization of signal proteins within a cell. AKAPs (A-Kinase Anchoring Proteins) are scaffold proteins with multiple domains that bind to regulatory subunits of Protein Kinase A phosphorylated derivatives of phosphatidylinositol various other signal proteins, such as: • G-protein-coupled receptors (GPCRs) • Other kinases such as Protein Kinase C • Protein phosphatases • Phosphodiesterases AKAPs localize hormone-initiated signal cascades within a cell, and coordinate activation of protein kinases as well as rapid turn-off of such signals. WAVE (Wiskott-Adlrich related) proteins are scaffolding proteins which interact with actin and recruit other components, including PKA.
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