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59 Cards in this Set
- Front
- Back
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What are some examples of non-membrane associated receptors?
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Steroid Receptor
Retinoic Acid Receptor Nitric Acid Receptor Other Transcription Factors |
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cholera Toxin
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ADP-ribosylation Gs1-alpha -->
Inhibits GTP disassociation from Gs1-alpha --> causing it to continually activate Adenylate Cyclase this affects signaling in the intestinal track |
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Pertussis Toxin
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ADP ribosylation of Gi-alpha inhibits GTP binding-->
can't turn off AC can't turn off PLC --> increased IP3 & DAG --> increases Ca++ --> increased PKC activity -> constriction of sm. muscle |
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Gs2-alpha
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involved in olfactory sensory signal transduction
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Gi-alpha
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3 subtypes
Inhibits AC PLC K+ channel |
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G0-alpha
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not affected by toxins
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Gx-alpha
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neuronal cell expression
Activates PLC K+ channel closing |
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Heterotrimeric G proteins are
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transducers
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how is the beta-gamma G protein subunit anchored?
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Isoprenoid
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talk to me about GPCR
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Integral membrane protein
7 membrane spanning domains Conserved Cys has thioester linkage w/Palmitic that forms loop which interacts w/hetrotimeric G proein |
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How is G-alpha attached to membrane?
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myristic amide linkage to amino Glycine
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how could you measure Adenylate Cyclase activity?
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measuring amount of cAMP sytnesized from ATP
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what is special about GTP-gamma-S
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it's a non-hydrolyzable GTP
Activates Heterotrimeric & Small GTP binding proteins (smg) |
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what can be used to activate Heterotrimeric & smg Gs/Gi ?
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GTP-gamma-S
Aluminum Fluoride (NaFl + AlCl3) |
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What can be used to inactivate Heterotrimeric & smg Gs/Gi ?
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GTP-beta-S
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Cholera toxin does what in the intestines?
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causes Gs to stay active
--> increasing cAMP --> keeping PKA active --> disrupts Cl- transport into cell --> water & Na loss --> even greater water loss |
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what hydrolyzes cAMP?
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phosphodiesterase
cAMP --> AMP |
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what inhibits phosphodiesterase?
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Caffeine
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PKA
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2 regulatory & 2 catalytic subunits
regulatory stay together Targets Ser & Thr --> Arg-Arg-X-Ser |
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other than cAMP, what else can regulate PKA?
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Peptide inhibitors that contain pseudo Phosphorylation sites
Type 1: R-R-X-not Ser/Thr/Tyr Type 2: R-R-X-S |
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These kinases unfold from Regulatory Subunits
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PKG
Myosin Light Chain Kinase (MLCK) |
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activates MLCK
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Calmdulin (which needs Ca to work)
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Walk through what happens when epinephrine binds receptor
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1. Epi binds Beta-adrenergic receptors activate G
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Walk through the Epi pathway
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1. Epi binds Beta-adrenergic receptor activates Gs-Alpha
2. Gs-alpha-GTP activates AC --> Gs-alpha-GDP 3.AC increases cAMP levels 4. cAMP activates PKA 5. PKA increases cytosolic leves of Glucose PKA activates Glycoten Phosphorylase PKA inactivates Glycogen Synthase 2 ways a. directly phosphorylating Glycogen Synthase b. Activating Phosphatse Inhibitor (Phosphatase removes P from Glycogen Synthase --> activating it) PKA activates CREB (who needs CBP/P300) to increase transcription of enzymes that convert pyruvate back to glucose Beta Anergenic Receptor (feed Forward) PKA P-lates Beta-anergenic receptor --> desensitizing it (quick Neg feedback & homologous desens) |
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How does Protein Kinase C (PKC) get activated?
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Ca++ & DAG activates PKC
PLC cleaves out IP3 & DAG IP3 opens Ca++ channels |
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How is PKG activated?
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cGMP causes regulatory subunit to unfold
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what are 2 types of desensitization?
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HOMO
Hetero |
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Homologous Desensitization
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desensitization due to the presence of the activating ligand for that receptor
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Heterologous Desensitization
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desens due to an activating ligand of a different receptor
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How is the beta Anergenic Receptor regulated?
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Desensitized
--> PKA instant -->beta ARK Up regulated by PKA --> CREB increases transcription of beta-AR min-hours |
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what kind of kinase is PKC?
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Ser Thr
multiple genes/splices tissue specific expression |
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How is PLC activated?
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Different ligand activates beta AR stimulates different G protein
Gq binds GTP & activates PLC |
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How would you know if PLC is in a pathway?
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monitor levels of IP3 &/or DAG in response to GTP-gamma-S
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Calmodulin & Troponin both have
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Ca++ binding EF hand loops
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2 main categories of Ca binding domains
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Extracellular
Cytosolic |
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Extracellular Ca binding proteins
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Ca binding increases thermal stability & resistance to proteaolysis
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Cytosolic Ca binding proteins
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reversibly binds Ca
modulates activity |
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Helix-Loop-Helix
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loop 12 AA-w/conserved Gly & phobic AA
7 Oxygens bind Ca-w/5 being COO- |
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Calmodulin regulates
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MLCK
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MLCK P-lates
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myosin causing muscle to contract
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Troponin
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binds Ca & muscles flex
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Annexin inhibits
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PKC by direct interaction
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How do you characterize signal transduction pathway?
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1. Physiological response to ligand/stimulus
2. ID ligand & receptor & purify/ID genetically 3. Use inhibitors/activators to ID players 4. Determine identity of interacting proteins 5. talk to researchers working on different but similar projects |
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Platelet Derived Growth Factor Receptor activates
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RTK
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How is PKA networked with MAP kinase?
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PKA downregulates Raf
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Signal for Mito Proteins
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N-terminal sequence,
basic amphipathic alpha helix |
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Signal for Nuclear Proteins
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short stretch of basic sequences (eg., PKKKRKV
or bipartite signals ( two regions of basic amino acids separated by a spacer of more than four AA |
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Signal for Peroxisome Proteins
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C-term w/ these 3= S K/H L
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Signal for Lysosome Proteins
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N term Mannose-P
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Signal for Cholorplast Proeisn
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tends to be basic, not amphipathic, usually has a lot of ser/thr (hydroxlated residues
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How to test possible role of signal
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attach reporter to protein w/signal sequence
S35 Met |
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RER protein
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signal sequence at N-term binds SRP
SRP binds GTP & stops translation Peptide translocation Complex interacts w/ribosome --> loss of SRP w/GDP translation continues & peptide goes inside RER 20 phobic AA |
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How do membrane proteins get inserted?
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SRP & SRP receptor
ribosome syntesizes protein into membrane an signal sequences tell where to cleave leaving phobic TM integral |
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get protein into Mitochondria
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what was that signal sequence?
peptide binds outer receptor (like Tom20) transfer to GIP Transport through protein pore binds Inner membrane Translocase proteinase removes signal sequence binds chaperon in matrix unfolded & translocated |
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In vitro Import assays
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isolated organelle
vitro synthesized protein (35S met degrade outside organelle with protease (tells if it has outer parts&/or not in at all can treat organelle with Na-carbonate and centrifuge - only integral membranes in pellet |
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Clathrin + AP2 + ARF
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Plasma Membrane --> endosome
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Clathrin + AP1 + ARF
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Golgi-> Endosome
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COPI
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Retrograde: Golgi --> ER
ARF |
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COPII
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ER -> Golgi
Sar1-GTP |
