BCOR 103 1st Edition Lecture 16 Outline of Last Lecture I. Cell Signaling and Cellular ResponsesII. Protein Kinase/PhosphataseIII. Signaling MoleculesOutline of Current Lecture I. ReceptorsII. Receptor Tyrosine KinasesIII. Transmitters/Second MessengersCurrent LectureCell Surface Receptors- Cell surface receptorso Molecule binding to receptor initiates signal transmissiono Most reside on cell surface of target cell (exception: steroid hormone receptors)o Transmit signal by regulating activity of intracellular proteinso Common receptor domains: Extracellular molecule-binding domain Membrane spanning domain Cytosolic catalytic domaino Receptor itself can respond to signalClasses of Cell Surface Receptors1. G protein-coupled receptors: most abundant type2. Receptor tyrosine kinases: only kind with enzymatic activity!3. Cytokine receptors: usually coupled to non-receptor tyrosine kinases4. Enzyme-linked receptors:a. Tyrosine phosphatasesb. Serine/threonine kinasesc. Guanlyly cyclaseG Protein-coupled receptors (GPCRs)These notes represent a detailed interpretation of the professor’s lecture. GradeBuddy is best used as a supplement to your own notes, not as a substitute.- Structure: serpentine receptorso Extracellular domain (ligand specificity)o 7 membrane spanning α-heliceso Cytosolic domain (functional domain)- Functions: >1,000 in mammalian cellso Coupled to G proteino Act as a molecular switch GDP-bound: inactive or “off” state GTP-bound: active or “on” stateo Bind variety of molecules Neurotransmitters, neuropeptides, peptide hormoneso Involved in many signaling pathways Smell, sight, taste, metabolism, cell survival, proliferation, ion channels*GPCRs bind to heterotrimeric G Proteins*A heterotrimeric G protein has three different polypeptides: α, β, and γ Regulation of Heterotrimeric GPCRs- Mammalian cells have 20 α-subunits, 5 β-subunits, and 12 γ-subunits- GTP and GDP regulate on/off state of GPCRs complexes to receive hormone/signal- The GTP/GDP regulation is an exchange reaction, NOT phosphorylation/dephosphorylation. Receptor Tyrosine Kinases (RTKs)- 59 RTKs found in the human genome!o Generally classified by extracellular domains, which determine specificity of ligand/receptor interactions and dimerization properties.Activation of RTKs- Activation requires auto-phosphorylation of cytosolic kinase domainChanges in RTKs after Activation- Auto-phosphorylation within kinase domain increases protein kinase activity- Auto-phosphorylation outside kinase domain creates binding sites for downstream signaling proteins (effectors)o Effector binds though: SH2 domains- Src Homology 2 PTB domains- phosphotyrosineResponses to RTK Activation- Activate or inhibit enzymatic activity through tyrosine phosphorylation- Stimulate enzymatic activity via conformational changes- Bridge interactions between receptor and enzymatic targets (adaptors)- Localize proteins to plasma membrane and/or promote complex formation at plasma membrane (docking proteins)Inactivating Receptor Tyrosine KinasesGeneral Mechanisms:1. Phosphatases (PTP): regulated by localization2. Ubiquitination (ubiquitin ligase Cbl)3. Negative feedback (PKC-induced inhibitory phosphorylation)4. Endocytosis5. Antagonistic ligands6. Hetero-oligomerization with kinase domain-deficient receptor mutantsEpidermal Growth Factor Receptor- Multiple, complex signaling pathways are downstream of all receptor tyrosine kinases.Cytokine Receptors- no kinase activity, separate polypeptide comes in- Cytokine receptors:o No biological activityo Bind to intracellular tyrosine kinases Ex: Src kinase family (immune cell signaling)o Stimulates growth, differentiation, and immune responsesSrc Kinases: Non-receptor Tyrosine Kinases- Identified in Rous sarcoma virus- Roles in differentiation, cell adhesion, motility, immune cell survival…- Phosphorylation of Y527 keeps Src in auto-inhibited inactive conformation- SH3 or SH12 binding or dephosphorylation of Y527 activates Src*Constitutive Src Kinase identified as First OncogeneReceptors Linked to other Enzymatic Activities- Tyrosine phosphataseso Remove phosphate groups from phosphotyrosineso Ligand-mediated dimerization inhibits phosphataseo Human cells have 21 protein-tyrosine phosphatases- Serine/threonine Kinaseso Phosphorylate serine and threonine residues of substrateo Exhibit open/closed auto-regulation of kinase activity- Cytoplasmic proteins with additional biochemical activityTransmitters and Second Messengers- Transmitters:o Intracellular molecules that are linked to receptorso Propagates and amplifies signal to intracellular targetso Common transmitters are: Second messengers: small molecules produced in response to initial signal Small G proteins: guanine-nucleotide binding proteins that modulate protein kinases cascades Protein kinases: phosphorylate enzymatic target or part of signaling cascade.- Ultimate targets:o Final target of signal transduction pathway Transcription factors Nuclear targets Cytoplasmic targetsSecond Messengers: cAMP Pathway- Second messengers: small molecules that initiate intracellular signalingo Examples: cAMP, Ca 2+, DAG, etc.- cAMP is synthesized from ATP by adenylyl cyclase and degraded to AMP by cAMP phosphodiesterase - cAMP acts downstream of hormone epinephrine to signal breakdown of glycogen to glucose in anticipation of muscle activity.Regulation of Protein Kinase A (PKA) by cAMP- epinephrine triggers cAMP synthesis via G-protein coupled receptors activation of adenylyl cyclase- cAMP binds to the two regulatory subunits of PKA- PKA regulatory subunits then phosphorylate serine residues on target proteinsPKA Directly Affects Enzymatic Activity of Proteins- PKA phosphorylation inhibits glycogen synthase- PKA phosphorylation activates phosphorylase kinaseAmplification of Signal via cAMP Second Messengers- Epinephrine response: release of glucose in anticipation of muscle usage- Activates 100 million intracellular molecules from one signaling