229.SignalTrans2.102616 - Signal Transduc.on Mechanisms...

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Unformatted text preview: Signal Transduc.on Mechanisms: Messengers and Receptors 14 10/26/16 Local and long distance cellular signaling Ÿ Ligands are molecules produced by cells that act on the same and other cells. Ÿ Ligands exert their effects by binding to and ac>va>ng cognate receptors. Types of chemical signals. • Paracrine signaling: ligands travel short distances to reach target cells. • Autocrine signaling: ligands produced by a cell bind to and alter the func>on of same cell type. • Juxtacrine signaling: When membrane bound ligands bind to receptors on their immediate neighbors. Ligand and receptor bearing cells are in physical contact. • Endocrine signaling: Ligands travel long distances via circula.on from the site of produc>on to their target cells/>ssues. Signal transduc.on cascades. • Ligands bind to receptors and ac>vate them. • Ac>vated receptors transduce signals by altering downstream molecular pathways. • These pathways may produce cellular responses at the cytosolic level. • These pathways convey informa>on to the nucleas producing changes in gene expression. Characteris.cs of signal transduc.on cascades. • Signal integra.on: Mul>ple signals and cascades converge, diverge or operate in parallel to produce synergis>c or antagonisi>c effects and cellular responses. • Signal amplifica.on: Stepwise transmission of signals amplifies them several fold. One molecule can ac>vate several downstream effectors par>cularly if they are enzymes. • Ligand-­‐receptor interac.ons: Receptor interac>ons resemble enzyme-­‐substrate interac>ons in specificity and affinity and kine>cs. Characteris.cs of signal transduc.on cascades -­‐ 2. • Ligand receptor affini.es are denoted by the dissocia.on constant Kd which represents the ligand concentra>on needed to occupy half the (cognate) receptors. The lower the Kd the higher the ligand-­‐receptor affinity. • Receptor downregula.on: Required for signal termina>on can occur by endocytosis or desensi>za>on. Signal transduc.on mechanisms: G protein linked receptors. • G (guanine nucleo>de binding) proteins are monomeric or trimeric; composed of α, β, γ subunits. • Trimeric G proteins associate with 7 membrane spanning receptors (GPCR). • G protein is bound to GDP in inac>ve state. • Binding of ligand to receptor ac>vates it and G protein which exchanges GDP for GTP. Signal transduc.on by G proteins. • Ligand binding to receptor produces a conforma>onal change in receptor. • Ac>vated receptor binds Gα subunit and causes it to dissociate from the β and γ subunits and exchange GDP for GTP. Signal transduc.on by G proteins-­‐contd. • Ac>vated G protein either ac>vate (Gs) or inhibits (Gi) downstream effectors. protein. • It slowly hydrolyzes GTP by its intrinsic GTPase ac>vity. This ac>vity is enhanced by other (GAP and RGS) proteins. Signal transduc.on by G proteins-­‐termina.on. • As GTP is hydrolyzed the signal decays and is terminated when it is converted to GDP. • The GDP bound Gα subunit re-­‐associates with β and γ subunits. • Then the cycle re-­‐ini>ates again if the receptor con>nues to be occupied by ligand and stays ac>vated. Signal transduc.on downstream of G proteins. • Ac>vated GS protein associates with the enzyme adenylyl cyclase which converts ATP to cyclic AMP (cAMP) a second messenger. Gi proteins inhibit adenylyl cyclase. Ac.vity of G proteins and adenylyl cyclase. • Adenylyl cyclase remains ac>ve as long as Gα subunit is bound to GTP. • Upon GTP hydrolysis to GDP, Gα dissociates from AC which gets deac>vated. • cAMP is also hydrolyzed to AMP (inac>ve) by phosphodiesterase. • What would result from cons>tu>ve ac>vity of this pathway? cAMP and the ac.va.on of protein kinase A. • In the absence of cAMP, PKA exists as a hetero-­‐tetramer of 2 regulatory and 2 cataly>c subunits. This associa>on inhibits the cataly>c subunits. • When the R subunits bind cAMP they dissociate from the cataly>c subunits which become ac>ve. • The ac>vated cataly>c subunits phosphorylate their targets. Gq proteins produce other second messengers. • Another class of G proteins Gq ac>vate phospholipase C (PLC). • PLC converts PIP2 to the second messengers DAG and IP3. What is the difference between them ? IP3 mobilizes Ca2+ from internal stores. Cytosolic Ca2+ and DAG ac.vate protein kinase C. • Cytosolic Ca2+ and DAG ac>vate another kinase, protein kinase C (PKC). • PKC phosphorylates (Ser; Thr residues) and ac>vates its downstream targets. Protein kinase associated receptors. • They are single pass transmembrane proteins that have discrete receptor and effector domains. • These receptors either possess intracellular tyrosine kinase domains (receptor tyrosine kinase) or • Are associated with membrane bound tyr kinases without any ligand bindingcapacity (non-­‐receptor tyrosine kinases). Ac.va.on of receptor tyrosine kinases • Ligand (growth factor) binding causes receptor dimeriza>on and clustering. • Tyrosine kinase domain of one molecule auto-­‐ phosphorylates Y residues on another. • Phosphoryla>on and conforma>onal changes in the IC domain allow other proteins to bind to the receptor. Ac.vated RTKs bind SH2 domain proteins. • IC domains of ac>vated RTKs bind adaptor proteins that have src homology 2 (SH2) domains. • SH2 proteins like GRB2 recruit and ac>vate SOS which are guanine nucleo>de exchange factors (GEFs). • Ac>vated GEFs catalyze GDP-­‐GTP exchange on monomeric G proteins (Ras) to and ac>vate them. Ac.vated Ras ini.ates the MAPK signaling pathway. • Ac>vated Ras ini>ates a series of Ser-­‐ Thr kinase reac>ons, the mitogen ac.vated protein kinase (MAPK) pathway. • These pathways eventually ac>vate gene transcrip>on. Muta.ons and malfunc.ons in signaling processes. • Dominant nega>ve receptors inhibit the func>on of wild type receptors. • What kind of muta>ons produce a dominant nega>ve phenotype? Cons.tu.vely ac.va.ng muta.ons cause cancer. • Cons>tu>ve ac>va>on of some signaling pathways causes cancer. Why ? • Examples-­‐dele>on muta>ons in the EC domain of the EGFR receptor. • Cons>tu>ve ac>vity of Ras due lack of GTP hydroly>c ac>vity. Steroid hormones and nuclear hormone receptors. • Steroid hormones (estrogen, testosterone etc.) bind to cytosolic receptors. • Why do these receptors exist in the cytoplasm ? • Ligand binding changes the conforma>on of the receptors which translocate to the nucleas and ac>vate transcrip>on. • What are the pros and cons of these versus membrane bound receptors ? ...
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