Signal Transduction Pathways. Part 2
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1 Signal Transduction Pathways Part 2
2 GPCRs G-protein coupled receptors > 700 GPCRs in humans Mediate responses to senses taste, smell, sight ~ 1000 GPCRs mediate sense of smell in mouse Half of all known drugs work on GPCRs
3 G-proteins relay signal from GPCR G-protein couples the GPCR to an enzyme or ion channel, that transduce the signal Activation of GPCR by ligand causes change in G- protein G-protein releases GDP and binds GTP GPCR acts as a guanine nucleotide (GTP) exchange factor or GEF
4 G-proteins relay signal from GPCR G-protein made of three protein subunits: a, b, and g a hydrolyzes GTP to GDP and turns off G-protein coupled enzyme Activates enzyme
5 G-proteins switches itself off G-protein hydrolyzes GTP to GDP G-protein-GDP is inactive No activation of enzyme
6 Some G-proteins regulate camp production Adenylyl cyclase (AC) converts ATP to camp Some G-proteins activate AC Some G-proteins inhibit AC Ø Activation of GPCRs can regulate levels of camp
7 Signaling can be turned off camp produced by adenylate cyclase and broken down by phospodiesterase
8 camp activates protein kinase A
9 Protein kinase A phosphorylates and regulates effecter proteins
10 Activation of GPCR leads to increase in gene transcription
11 Regulation of glycogen by PKA Glycogen synthase (INACTIVE) Protein kinase A Glycogen synthase (ACTIVE) glycogen glucose Glycogen phosphorylase (ACTIVE) Protein kinase A Glycogen phosphorylase (INACTIVE)
12 Cell responses mediated by camp
13 Some G-proteins stimulate phospholipid breakdown G-protein activates the enzyme phospholipase C-b (PLCb) PLCb hydrolyzes a specific membrane phospholipid: Phosphatidylinositol 4,5-bisphosphate (PIP 2 ) Products of this reaction are Inositol 1,4,5-trisphosphate (IP 3 ) à causes release of Ca +2 from ER Diacylglycerol (DAG)à activates protein kinase C (PKC)
14 Hydrolysis of PIP2
15 Some GPCRs increase Ca +2 and PKC activity
16 Protein kinase C Binds Ca 2+ and diacylglycerol Binding of these ligands activates PKC and brings the kinase to the membrane PKC phosphorylates effector proteins some of the same as PKA, and some different effectors to PKA
17 Cell responses mediate by IP 3 and diacylglcerol
18 Signaling from GPCRs G-protein à adenlyl kinase à camp G-protein à PLCb à inositol 1,4,5-trisphosphate + diacylglycerol à Ca +2 G-protein coupled to ion channel à allow influx of K + ions à
19 Enzyme coupled receptors Receptor tyrosine kinases receptor protein is a kinase Receptor associated with a tyrosine kinase two different proteins Receptor tyrosine phosphatase - receptor protein is a phosphatase Others: Ser/Thr kinases, His-kinase, Guanylate cyclase (cgmp)
20 Phospho-amino acids
21 Receptor tyrosine kinases Signal proteins that work through RTKs Growth factors promote cell division, survival, differentiation Insulin - metabolic regulator Ephrin - Eph receptor cell adhesion, direct movement of cells during development
22 Types of RTKs
23 Signaling molecule activates RTK Signaling molecule binds to receptor Binding causes change in structure of receptor formation of receptor dimer Change in structure activates tyrosine kinase One kinase phosphorylates other kinase in dimer Phosporylated tyrosine residues are docking sites for relay proteins pass on signal
24 Activation of RTKinases
25 Activation of RTKinases
26 Platelet-derived growth factor (PDGF) Relay proteins transmit signal Only one receptor shown
27 Ras G-protein One subunit monomeric, not three as with other G- proteins mentioned earlier Key protein in many signaling pathways Functions as a switch Mutations that activate Ras cause cancer
28 Ras G-protein Active when GTP is bound Inactive when GDP is bound Activated by receptor tyrosine kinases à Ras-GEF à Ras GEF = guanine nucleotide exchange factor Stimulates exchange of GDP for GTP Inactivated by Ras-GAP (GTPase activating protein) Stimulates Ras to hydrolyse GTP to GDP
29 Activation of Ras by RTK ligand or signaling molecule Cell A Cell B
30 MAPkinase Serine/Threonine kinase involved in many pathways. Originally identified in insulin stimulated cells. Activated by GPCRs and RTKs Phosphorylates downstream targets to transmit signal. We will look at two MAPkinases ERK1 and ERK2
31 Ras activates MAPkinase Ras activates a kinase cascade. Active Ras binds to, and activates Raf kinase. Signal is amplified by kinases phosphorlyating multiple copies of target.
32 MAPKinase pathways
33 PI 3-kinase Phosphoinositide 3-kinase Phosphorylates polar head group of phospholipid phosphatidylinositol. Phosphorylates 3-position on inositol (PIP 3 ) Activated by GPCRs and RTKs Phosphorylated phospholipid is a docking site for transducing proteins
34 Phosphoinositide docking sites
35 PIP 3 forms docking site Activation of PI 3-kinase enhances cell survival and cell division.
36 PTEN phosphatase Removes phosphate from PIP 3 Turns off pathway to cell division and survival PTEN gene is mutated or lost in many cancer patients. Mutation in PTEN leads to uncontrolled growth (cancer) and increased cell death
37 Role of phosphatases Reverse phosphorylation events. Turn off pathways dependent upon phosphorylation. Can transmit signal as well as turn off signal. Some protein activated by dephosphorylation Src tyrosine kinase activated by dephosphorylation Active src promotes movement of cells and cell division (cancer metastasis, or migration of cancer cells)
38 Pathways can intersect Overview of pathways
39 Modulation of pathways Phosphatases dephosphorylateproteins. GTPase activator proteins turn off ras G- proteins. Movement of transducing proteins within cell. Import to nucleus
40 Signals are transient Signal is transmitted, then turned off. Time course for activation and inactivation
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