Cell Signaling and Signal Transduction PDF

Plasmodesmata Helical bundle Plasmo- it transcends your plasma are parallel helices that have ribbons membrane; des- desmosome tubule; and found in different sides of the bilipid layer m...

Plasmodesmata Helical bundle Plasmo- it transcends your plasma are parallel helices that have ribbons membrane; des- desmosome tubule; and found in different sides of the bilipid layer mata-opening. It is the opening that They form a tunnel structure that allows transcends your plasma membrane and specific materials to enter/exit the cell. always connected to your ER. β-barrel Are cytoplasmic channels that pass composed of beta pleated sheets, their through the cell walls of adjacent cells. tertiary structures are in different o Usually it is a canal that is made directions of two adjacent cells however it usually served as transporters or channel connects cells that are not proteins adjacent. It moves two different type of cells. Peripheral Proteins - If the protein is found only on one side of the bilipid layer Contain a dense central structure, the desmotubule, derived from the smooth ER Normal peripheral proteins of the two cells. The N-and C-terminals are exposed on one Like gap junctions between animal cells, side. plasmodesmata serve as a sites of Acts as secondary stallers, movement, or cell-to-cell communication, as substances placement of secondary messengers pass through the annulus surrounding the desmotubule. Membrane-anchored peripheral proteins The N- and C- terminal are anchored within the lipid bilayers. Lesson 5: Cell Signaling and Signal Types of proteins based on their location of N- Transduction and C- terminal protein 1. Type I transmembrane - C-terminal is inside the cell and N- outside Integral Proteins - If the proteins transcend in the 2. Type II transmembrane bilipid layer - The N-terminal is found within the cell and C-terminal is found outside the cell α-helix able to transcend these specific bilipid 3. Type III multipass transmembrane layer - It requires interaction of both C- and N- they interact within the same direction and terminals on one side the r-groups form a helical structure - The ligand will usually interact with both (called a-helix) sides. R-groups of it interact with the R-groups of the same amino acid to form a spiral form 4. Type IV transmembrane making it less soluble to water. 27  - Reverse of type II. Here C-terminal is the small one compared to N-terminal 5. Type V GPI anchored membrane/ membrane anchored proteins - It allows the connection of an interaction with the specific ligand SIGNAL TRANSDUCTION 1. G-Protein Coupled Receptors These effectors interact with the GPCR superfamily of proteins/7TM receptors that comprises 7 helices. ○ Plant and animal hormones Amino-terminus is present on the outside ○ Neurotransmitter of the cell, the seven helices that traverse ○ Opium derivatives the plasma membrane are connected by ○ Chemoattractants loops of varying length, and the ○ Odorants and tastants carboxyl-terminus is present on the inside ○ Photons of the cell. Three loops present on the outside of the Examples of Physiologic Processes Mediated by cell form ligand-binding pockets; three GPCRs and Heterotrimeric G Proteins loops present on the inside serve as binding sites for intracellular signaling proteins. II. GPCR G Protein Epinephrine - The stimulus or ligand would be the epinephrine/ adrenaline - The receptor would be the beta adrenergic receptor - The effector would be adenylyl cyclase - The physiologic response would be glycogen breakdown Serotonin Heterotrimeric; alpha(α), beta (β), and gamma (γ) - The receptor is serotonin receptor subunits where it is held at the plasma membrane - The effector would also be adenylyl by lipid chains that are covalently attached to the cyclase α and γ subunits. Light (stimulus) Signal Transduction by GPCR - rhodopsin(receptor) - visual excitation (physiological response) that allows you to see phosphodiesterase (effector) I. GPCR Receptors 1. A ligand binds to the receptor. 28  2. Receptor has increased affinity for the Gα these are messengers that interact with subunit, attracting it while subsequently the environment to the surface of the cells releasing attached GDP which is replaced (most likely your ligands) by GTP. 3. In GTP bound conformation, Gα Second messengers dissociates from the Gβγ domains and They transfer information from the surface activates the effector protein. The alpha of your plasma membrane/bilipid layer subunit attaches to your effector. towards your nucleus 4. Activation of the effector leads to the production of a second messenger, cyclic I. Second messengers AMP (cAMP), using ATP in the process. - cyclic adenosine monophosphate (cyclic 5. Gα subunits can turn themselves off by AMP, or simply cAMP) is a second hydrolysis of GTP to GDP and inorganic messenger that is capable of diffusing to phosphate(Pi) decreasing affinity to the other sites within the cell. effector protein. - The synthesis follows the binding of a first 6. Dissociation of Gα from the effector. messenger– a hormone or other ligand— 7. Desensitization, through phosphorylation, to a receptor at the outer surface of the of the active receptors by the G cell. protein-coupled receptor kinase (GRK) to - Often stimulates a variety of cellular prevent overstimulation. activities which enable cells to mount a 8. Binding of arrestin to the phosphorylated large scale, coordinated response GPCR to compete with the active site for following stimulation by a single activated Gα. extracellular ligand. - Other second messengers includeCa2+, Important Notes on GPCR phosphoinositides, inositol trisphosphate, - Gα subunits possess a weak GTPase diacylglycerol, cGMP, and nitric oxide activity, which allows them to slowly hydrolyze the bound GTP and inactivate II. Phosphatidyl inositol derived second themselves. messengers - Termination of the response is - Membrane phospholipids are converted accelerated by regulators of G protein into second messengers by a variety of signaling (RGSs) which increases the rate enzymes: of GTP hydrolysis by the G subunit. - phospholipases - The mechanism for transmitting signals - phospholipid kinases across the plasma membrane by G - phospholipid phosphatases proteins is of ancient evolutionary origin - Inositol-containing lipids can be and is highly conserved. phosphorylated by specific lipid kinases - One third of all prescription drugs act as that are activated in response to ligands that bind to GPCRs and a number extracellular messenger molecules, such of inherited disorders affect GPCRs. as acetylcholine. - Mutations in the GPCR usually affect the cascade of signals from the outside of the cells to the intracellular components therefore the cell fails to act or produce molecules accordingly. Primary messengers 29  - Inositol 1,4,5 trisphosphate (IP3) is a sugar phosphate– a small, water soluble molecule capable of rapid diffusion throughout the interior of the cell. - Calcium ions can also be considered as intracellular or second messengers because they bind to various target molecules, triggering specific responses. - Contraction of a smooth muscle cell and exocytosis of histamine containing secretory granules in a mast cell, both are triggered by elevated calcium levels. III. Second messengers through ligand-induced breakdown of PI Phosphorylation Secondary messengers-within the cytosol Step 1-2: Phosphorylation of phosphatidylinositol and nuclear membrane that transcends (PI) molecules by kinases leads to the formation signals of phosphoinositides, including How secondary messengers transfer phosphatidylinositol bisphosphate (PIP2) in the information through the process, plasma membrane. phosphorylation Phosphorylation- process where an Step 3: When a signal molecule activates a inorganic phosphate is transferred to a G-protein-coupled receptor (GPCR), the G-protein material dissociates into Gα and Gβγ subunits. The Gα subunit activates phospholipase C (PLC), an Protein-Tyrosine Phosphorylation effector enzyme. This process uses protein-tyrosine kinases, they are enzymes that Step 4: Activated PLC cleaves PIP2 into inositol phosphorylate specific tyrosine residues triphosphate (IP3) and diacylglycerol (DAG). The on protein substrates pathway splits: Tyrosine residues are found all over the amino acid sequences, these sequences DAG activates protein kinase C (PKC). will then concur change in conformation leading to change in function in terms of Step 7: IP3 diffuses through the cytosol and binds proteins to IP3 receptors on the smooth endoplasmic PTKs are divided in two groups depending reticulum, triggering calcium ion release into the on where it is located: cytosol. ○ Receptor protein-tyrosine kinases (RTKs), which are integral Final steps: Elevated cytosolic calcium activates membrane proteins that contain a PKC, leading to the phosphorylation of target single transmembrane helix and proteins. DAG can be further broken down into extracellular ligand binding domain arachidonic acid, which contributes to pain and ○ Non Receptor (or cytoplasmic) inflammation responses via eicosanoids. protein-tyrosine kinases, free or bound within an organelle or Important Notes functions as an enzyme found in - Protein kinase C isoforms have a number the cytosol of important roles in cellular growth and Example of ligand that bind to RTKs are differentiation, cellular metabolism, cell growth factors/hormones death, and immune responses. 30 Reception Dimerization of PTK - When RTK and IRS is bound together, the (Ligand-Mediated) receptor phosphorylates tyrosine residues It serves as an inactive monomer in the present on the docking protein bilipid layer. However, in the presence of a - The phosphorylated sites then act as ligand, their specific ligand will interact binding sites for additional signaling with both active sites of inactive PTK and molecules it will form a dimer and this dimer will - Docking proteins provide versatility to the serve as a site for phosphorylation. signaling process due to its ability to turn Phosphorylation will serve as an area or on subsequent signaling molecules interaction where other tyrosine - Recruit a primary docking protein where components and will have their specific other enzymes can connect domains such as SH2 and PTB ○ In the non-activated state, the Transcription Factors and RTKs receptors are present in the - They usually involve the STAT family, membrane as monomers which requires phosphorylation to be ○ Binding of a bivalent ligand activated. This phosphorylation should activates the receptors and come from the SH2 domain initiates dimerization and - STATs contain an SH2 domain together activation of the kinase activity with a tyrosine phosphorylation site that ○ Addition of phosphate groups to can act as a binding site for another SH2 the cytoplasmic domain of the Stat molecule other receptor subunit - Upon RTK interaction, tyrosine residue in ○ The newly formed phosphotyrosine these STAT proteins are phosphorylated residues of the receptor serve as initiating the interaction between the binding sites for target proteins phosphorylated tyrosine residue on one containing SH2 or PTB domains STAT protein and the SH2 domain on a second STAT protein, and vice versa Activation of Downstream Signaling Pathways - Once it is phosphorylated it would turn Receptor protein-tyrosine kinases (RTKs) into its active form which could be a STAT are autophosphorylated on one or more dimer that can be used for amplification or tyrosine residues for assembly of transcription machinery Activation results in formation of signaling - STAT dimers stimulate the transcription of complexes, in which SH2- or PTB specific genes involved in an immune containing signaling proteins bind to response. specific autophosphorylation sites Signaling Enzymes and RTKs Adaptor Proteins and RTKs - Enzymes can directly interact with the - Functions as linkers that enable two or inorganic phosphate found in RTKs. more signaling proteins to become joined Enzymes usually need inorganic together as part of a signaling complex phosphates that they derived from ATP - Contain an SH2 domain and one or more - Protein kinases, protein phosphatases, addition protein-protein interaction lipid kinases, phospholipases, and GTPase domain activating proteins - If enzymes have SH2 domains, these Docking Proteins and RTKs enzymes associate with activated RTK - Contain either a PTBdomain/SH2 domain and are turned on directly or indirectly and a number of tyrosine phosphorylation sites Important Notes on Protein-Tyrosine Phosphorylation 31  - Signal transduction by RTKs is usually states is aided by accessory proteins that terminated by internalization of the bind to the G protein. receptor The proteins include: - Receptor-binding protein Cbl possesses ○ GTPase-activating proteins (GAPs) an SH2 domain hence it will associate – stimulates hydrolysis with the active RTK receptor and later on ○ Guanine nucleotide-exchange catalyzes the attachment of a ubiquitin factors (GEFs) – stimulates molecule to the receptor dissociation of GDP - Ubiquitin is a small protein that is linked ○ Guanine nucleotide-dissociation covalently to other proteins, thereby inhibitors (GDIs) – inhibit the marking those proteins for internalization release of a bound GDP or degradation - Binding of the CBl complex to activated receptors is followed by receptor ubiquitination and internalization - GPCRs, internalized RTKs can have several alternate fates; they can be degraded in lysosomes, returned to the plasma membrane, or become part of endosomal signaling complexes and engage in continued intracellular signaling - Transfer of energy from messenger which is phosphorylation or inorganic phosphate. Conversion of ATP to ADP for energy. G PROTEIN CYCLE Ras Map-Kinase Pathway cont... Ras-MAP Kinase Pathway RAS PROTEIN Small GTPase that is anchored at the inner surface of the plasma membrane by a covalently attached lipid group that is embedded in the inner leaflet of the bilayer. Functionally similar to the heterotrimeric G proteins which acts as both a switch and a molecular timer, but the structure only 1a — GDI interaction inhibits the release of GDP contains one, small subunit. hence remaining inactive Exists in an active and an inactive GDP-bound form. 1b-2 — GEF interaction exchanges bound GDP for Ras-GTP activates downstream signaling a GTP proteins; turned off by hydrolysis of GTP. The cycling of monomeric G proteins, 3 — Binding and activation of target protein such as Ras, between active and inactive (typically a kinase or a phosphate) 32 4 — GAP binding accelerating GTP hydrolysis into 3 — Subsequent recruitment of the Grb2-Sos GDP proteins. 5 — Inactivation of G protein and dissociation with 4 — The GTP-GDP exchange of Ras which target protein activates Ras. 5 — Soluble Raf is recruited to the membrane and phosphorylated hence being activated. RAS-MAP KINASE CASCADE Ras-GTP can be thought of as signaling 6 — Raf phosphorylates and activates another hub because it can be interact directly with kinase named MEK. several downstream targets. Turned on in response to a wide variety of 7 — MEK phosphorylates and activates another extracellular signals and plays a key role in kinase named ERK. regulating vital activities such as cell proliferation and differentiation. 8 — MEK translocate to the nucleus where it Extracellular signals → Plasma membrane phosphorylates transcription factors. → Cytoplasm → Nucleus 9 — Phosphorylated TFs increases the affinity for Generalized Ras-Map Kinase Cascade DNA regulatory proteins leading to increased transcription. 10 — Expression of a MAPK phosphatase (MKP-1). 11 — Removal of phosphates by MPK-1 from the activated ERK. Adapting MAP Kinase to Different Signals The same basic pathway from RTKs through Ras to the activation of transcription factors, as illustrated is found in all eukaryotic investigated, from yeast through flies and nematodes to mammals. Specificity in MAP kinase pathways is also achieved by spatial localization of the component proteins through scaffolding proteins. 1-2 — Binding of growth factor to its receptor leads to the autophosphorylation of tyrosine residues of the receptor. 33  of G proteins, each of which can activate an adenyl cyclase effector, each of which can produce a large number of cAMP messengers in a short period of time. IMPORTANT NOTES ON RAS-MAP KINASE CYCLIC AMP ACTIVATION PATHWAY Adenylyl cyclase, an integral membrane Raf is a serine-threonine protein kinase protein that consists of two parts, each which activates protein kinase MEK and containing six transmembrane helices. later activates up to two other protein The enzyme’s active site is located on the kinase ERK1 and ERK2. inner surface of the membrane in a cleft Over 160 proteins that can be situated between two similar cytoplasmic phosphorylated by ERKs have been domains. identified, including transcription factors, The breakdown of the cAMP is protein kinases, cytoskeletal proteins, accomplished by a phosphodiesterase, apoptotic regulators, receptors, and other which converts the cyclic nucleotide to a signaling proteins. 5’ monophosphate. The pathway leads to the activation of genes involved in cell proliferation, including cyclin D1, which plays a key role in driving a cell from G1 into S phase. All MAPKs have a tripeptide near their catalytic site with the sequence Thr-X-Tyr where the MAPKK phosphorylates MAPK on both the threonine and tyrosine residue of this sequence, thereby activating the enzyme. CYCLIC AMP cAMP is synthesized by adenylyl cyclase, an integral membrane protein whose catalytic domain resides at the inner surface of the plasma membrane. The binding of a single hormone molecule at the cell surface can activate a number 34  A single cell may have dozens of different receptors sending signals to the cell CYCLIC AMP CASCADE interior simultaneously. cAMP exerts most of its effects by Once they have been transmitted into the activating PKA, the response of a given cell, signals from these receptors can be cell to the cAMP is typically determined by selectively routed along a number of the specific proteins phosphorylated by different signaling pathways that may this kinase. cause a cell to divide, change shape, Most of the PKA substrates carry out activate a particular metabolic pathway, or different functions which is facilitated by even undergo apoptosis. AKAPs which provide a structural framework of scaffold for coordinating Examples of Converge, Divergence, and protein-protein interactions by Crosstalk Among Signaling Pathways sequestering PKA to specific locations within the cell. 1. Convergence As a consequence, PKA accumulates in Although binding different ligands, all of close proximity to one or more substrates. them can lead to the formation of phosphotyrosine docking sites for the SH2 domain of the adaptor protein Grb2 in close proximity to the plasma membrane. The recruitment of the Grb2-Sos complex results in the activation of Ras & transmission of signals down the MAP kinase pathway. As a result of this convergence, signals from diverse receptors can lead to the transcription and translation of a similar set of growth promoting genes in each target cell. CROSS-TALK, CONVERGENCE, AND DIVERGENCE Signals from a variety of unrelated receptors, each binding to its own ligand, can converge to activate a common effector, such as Ras or Raf. Signals from the same ligand, such as EGF or insulin, can diverge to activate a variety of different effectors and pathways, leading to diverse cellular responses. Signals can be passed back and forth between different pathways, a phenomenon known as cross-talk. 35 2. Divergence Evidence of signal divergence has been evident virtually in all of the examples of signal transduction that have been described. Cyclic AMP and an insulin receptors illustrates how a single stimulus sends signals out along a variety of different pathways. 3. Cross-talk Cyclic AMP was depicted earlier as an initiator of a reaction cascade leading to glucose mobilization. Cyclic AMP activates PKA, the cAMPdependent kinase, which can phosphorylate and inhibit Raf, protein that heads the MAP kinase cascade. However, both PKA and the kinases of the MAP kinase cascade phosphorylate the transcription factor CREB on the same serine residue, activating the transcription factor and allowing it to bind to specific sites on the DNA. 36