Signal Transduction: G-Protein Coupled Receptors

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1 Signal Transduction: G-Protein Coupled Receptors Federle, M. (2017). Lectures 4-5: Signal Transduction parts 1&2: nuclear receptors and GPCRs. Lecture presented at PHAR 423 Lecture in UIC College of Pharmacy, Chicago. MOLECULAR SIGNALING In general, signaling involves: o Binding of a ligand to a receptor o Relaying information from the receptor by receptor modification of small molecules in a scaffold cascade Scaffold protein = provides a dock for smaller signaling molecules to form a cascade for the incoming signal o Amplification of this information throughout the cell with the use of various signaling molecules o Integration of this information in some way Activation/repression of gene transcription, protein modifications (phosphorylation, etc.) G PROTEINS Two main molecular switches to activate proteins: o Protein phosphorylation = addition of a phosphate group to the protein o GTP-binding = addition of a phosphate group to dephosphorylated guanine base to form GTP Addition of GTP to a protein GTPase = G protein = GTP binding protein o These terms are interchangeable. They all refer to signaling proteins that bind GTP molecules Two forms of GTPases o Large heterotrimeric G proteins o Small monomeric GTPases G protein regulators o GAPs = GTPase activating proteins These actually turn OFF G proteins. I know SILLY The reason they are called activating proteins is because they activate the ability to hydrolyze the GTP to GDP, essentially inactivating the G protein (GTP is the active form) o GEFs = Guanine nucleotide exchange factors

2 These turn ON G proteins Form GTP from GDP SIGNALING COMPLEXES Signaling complexes provide speed, efficiency, and specificity The benefit of having a protein scaffold with enzymes already assembled is that there is a higher specificity and speed in response to the signal binding to the receptor because enzymes are already in the right place Sometimes the complex does not form until the receptor is actually phosphorylated and activated Phosphoinositide dock = phosphorylation of phospholipids on the membrane creates a docking location for signaling complexes o Now enzymes can come to the membrane and all interact with one another o Docking domains PH = binds the phosphoinositide molecule on the membrane Like the start of the docking protein PTB = phosphotyrosine binding These residues usually found on the activated receptor SH2 = binds phosphotyrosine resides SH3 = binds proline-rich regions Feedback loops in the signaling cascade help to regulate the response o Positive feedback enzyme activity remains even after the signal is gone o Negative feedback can be a short delay or long delay Short delay the signal causes a response and the negative feedback quickly dampens this response Long delay the signal causes a response, which then fluctuates between being dampened and heightened Ways that a receptor can dampen response to a signal Adaptation and Desensitization o Receptor sequestration receptor is taken back into the cell o Receptor down-regulation less of the receptor is being made o Receptor inactivation usually inactivated on the cytosolic side o Inactivation of signaling protein signal can t be passed into the cell o Production of inhibitory protein this protein stops the signal from being transduced

3 G-PROTEIN COUPLED RECEPTORS 40% of modern drugs target this receptor These signaling cascades can be very rapid because of amplification of the signal Domains o Extracellular ligand binding domain o Transmembrane 7 alpha helices domain o Intracellular regulatory domain When ligand binds to the extracellular domain, this causes a twist in the transmembrane helices which then activates the receptor When the receptor is activated, the intracellular domain is able to activate G proteins Trimeric G protein o Has 3 domains: alpha, beta, gamma o Alpha contains GDP o Alpha and gamma are always associated with the cell membrane o When ligand binds to receptor, the transmembrane conformational change allows the alpha subunit to dissociate from the beta and gamma subunits This allows phosphorylation of GDP to GTP Subunits are now activated o Essentially, the receptor itself acts as a GEF because it activates the G protein o These subunits can now interact with target proteins and effector proteins Activated alpha subunit: RGS = effector protein that acts exclusively with alpha subunit o Is a GAP because it will hydrolyze the GTP and deactivate the alpha subunit When GTP is hydrolyzed to GDP, alpha subunit will re-associate with gamma/beta subunits and the trimeric protein is now deactivated again Activated beta/gamma subunit: Interacts with target proteins o Various G-protein families Gs = alpha subunit activates adenylyl cyclase and Ca 2+ channels Gi : Alpha subunit inhibits adenylyl cyclase Beta/gamma subunit activated K + channels decrease cell excitability o K + channel acts as effector protein Gq = activates PLCβ

4 SECOND MESSENGERS Adenylyl cyclase = membrane protein that is activated by Gs o Releases camp = secondary messenger PKA = camp-dependent protein kinase o A protein that phosphorylates it s target proteins Specifically the serine and threonine amino acids 2 catalytic subunits function is to activate kinase activity 2 regulatory subunits function is to bind camp and regulate the kinase activity o PKA downstream effects: Transcription regulation CRE binding protein (CREB) will be phosphorylated by PKA and then is translocated to camp response element (CRE) on DNA CREB binding protein (CBP) will be recruited to CREB and activate transcription of the genes on the CRE Can regulate ion channels and GEFs gives positive and negative feedback PLCβ = phospholipid C-β. Activated by Gq o Activated PLCβ goes on to covert PIP2 to IP3 and diacylglycerol o PIP2 = phosphatidylinositol 4,5-bisphosphate a membrane bound effector protein o IP3 = inositol 1,4,5-triphosphate A water soluble second messenger that releases Ca 2+ from the endoplasmic reticulum and induces Ca 2+ ligand gated channels o Diacylglycerol = a lipid anchored second messenger that activates protein kinase C (PKC) by bringing it up to the cell membrane PKC = A Ca 2+ dependent protein kinase. Phosphorylates serine and threonine amino acids

5 KNOW THESE STRUCTURES (IP3, PIP2, diacylglycerol) Ca 2+ o Concentrations of Ca 2+ Cytosol = 0.1 um Endoplasmic reticulum = 100 um Extracellular space = 1,000 um o Concentrations maintained by ion transporters and energy driven pumps on the plasma membrane and ER membrane o Calmodulin = allosteric regulator of other proteins Activated when Ca 2+ binds to it 4 binding sites; undergoes cooperative binding with Ca 2+ = the more Ca 2+ that binds to it, the more likely it is to bind another Ca 2+ molecule o CaM Kinase = protein that is activated by calmodulin Auto-phosphorylates and can remain active after calmodulin leaves