Signal Transduction Pathways Part 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
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
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
G-proteins switches itself off G-protein hydrolyzes GTP to GDP G-protein-GDP is inactive No activation of enzyme
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
Signaling can be turned off camp produced by adenylate cyclase and broken down by phospodiesterase
camp activates protein kinase A
Protein kinase A phosphorylates and regulates effecter proteins
Activation of GPCR leads to increase in gene transcription
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)
Cell responses mediated by camp
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)
Hydrolysis of PIP2
Some GPCRs increase Ca +2 and PKC activity
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
Cell responses mediate by IP 3 and diacylglcerol
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
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)
Phospho-amino acids
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
Types of RTKs
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
Activation of RTKinases
Activation of RTKinases
Platelet-derived growth factor (PDGF) Relay proteins transmit signal Only one receptor shown
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
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
Activation of Ras by RTK ligand or signaling molecule Cell A Cell B
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
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.
MAPKinase pathways
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
Phosphoinositide docking sites
PIP 3 forms docking site Activation of PI 3-kinase enhances cell survival and cell division.
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
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)
Pathways can intersect Overview of pathways
Modulation of pathways Phosphatases dephosphorylateproteins. GTPase activator proteins turn off ras G- proteins. Movement of transducing proteins within cell. Import to nucleus
Signals are transient Signal is transmitted, then turned off. Time course for activation and inactivation