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81 Cards in this Set

  • Front
  • Back
Name three things that signals allow organisms to do
-Allow organisms to respond to their environment

-help maintain homeostasis

-provide the ability to control function(s)
What Is Needed For Signaling?
Mechanism for Generation

A Signaling Molecule

A Receptor

Cellular messaging mechanism

Biochemical pathway
Describe Endocrine signaling, give an example
Long distance

Insulin
Describe Paracrine signaling, give an example
Medium to short distance

Histamine
What is autocrine signaling?
give an example:
When a cell signals itself

prostoglandin signaling
What are estrogens and androgens, steroids examples of?
Nuclear hormone signaling
Name some hormones involved in nuclear hormone signaling
estrogens

androgens

steroids
Name four types of receptors seen in signaling and describe their speed
-Ligand-gated ion channel
very fast

-GPCRs
fast

-Kinase-linked receptors
slow

-Nuclear receptors
really slow
Do cells ever actually touch each other in order to signal?
yes. Plasma membrane attached signaling. In the immune system.
What is an autocoid?
A locally-acting hormone
Name at least 5 properties of a receptor
Proteins with 1 or More (up to 5) Subunits

Transmembrane Proteins

Extracelluar Ligand Binding Domain

Intracelluar “Activation“ Domain

May be an Ion Channel

May or May Not Need to Interact With Other Membrane Proteins (Example - GPCRs vs guanylyl cyclase)

Very High Specificity For Ligands (Kd’s from 0.1-10 nM)

Ligand Binding Generates Different Types of Intracelluar Regulatory Events or “Second Message”
Name 4 main types of receptor
Ionotropic

Metabotropic

Kinase Based

Nuclear
How might one detect a hormone receptor?
With a binding assay
What is the basic idea of ligand binding/response theory?
Response is Proportional to Receptor Occupancy

L / (L + Kd)
What is a consequence of kinase cascades?
Signal amplification
What is significant about the fact that kinase cascades amplify signals?
Response of the Cell Often Occurs at Lower Ligand Values than the Kd of Receptors
What are the three main channel families?
Ligand-gated ion

Potassium

Sodium or calcium
What do voltage-gated cation channels generate?
Action potentials
What sort of channels are responsible for action potentials?
Voltage gated cation channels
what changes in proteins can drive signaling?

Give an example
conformational and phosphorylation

calmodulin
How many transmembrane domains do gPCRs have?
7
What STRUCTURAL change does calmodulin provide an excellent example of?
Conformation changes involved in activation
What example of cytokine sigaling was given in class? (Garrison)
Cytokine→Jak→Stat
Give a generic cascade following a growth factor signal
Growth Factor binds

Receptor dimerisation

Autophosphorylation of tyrosine residues

Docking of SH2 domain protein

SH2 acts as a dock for activation of Ras (GTP→GDP)

Ras is activated via GEF

MAPKKK, MAPKK, MAPK

Various transcription factors

Gene transcription
Where (what domains—extracellular or membranous) do g-protein coupled receptor ligands bind?
Both inside and outside the transmembrane helixes
Describe a general GPCR/Adenylyl cyclase cascade
Ligand binds to receptor, causes intracellular conformational change

Receptor acts as a GEF

Heterotrimeric g-protein dissociates into alpha and beta-gamma subunits catalyzed by exchange of ADP for ATP

Alpha (generally) binds to adenylyl cyclase at Switch II domain

Cyclase activates synthesis of cAMP from ATP

cAMP activates PKA

PKA phosphorylates targets (not necessarily diffused, however. AKAPs act as scaffolds.
What are the three functions of AKAPs?
Target PKA to site

Assemble signaling complexes that include substrate and phosphotase

Reorganize complexes
What are scaffolds for PKC called?
RACKs
What does phosphorylation change and not change?
Changes the activity (Km)

Does not change the maximum activity (Vmax)
Is a small amount of phosphorylation useful?
Generally not. Stoichiometric phosphorylation is usually necessary to potentiate change
Describe the action of pyruvate kinase
SOMEBODY PLEASE FILL THIS IN
Describe a general GPCR PLC cascade
General steps involving receptor binding, GProtein conformational change

Alpha activates PLC

PLC breaks down membrane-bound PIP2 into membrane bound DAG and cytosolic IP3

DAG activates PKC, PKC phosphorylates substrates

IP3 translocates to the nucleus and frees calcium

Calcium can do its work in the cell, such as gate channels, etc., activate calmodulin
Give a general description of calmodulin
It is an intracellular Ca+ receptor
What is the general pathway for receptor recycling?
GRKs phosphorylate

Arrestins bind, stop signaling

Receptor is internalized in a clathrin-bound vesicle

It is either recycled and modified by phosphatases
or
ubiquitinated and sent to a lysozome for destruction
What is the main difference between Class A and Class B receptor recycling?
Class A, rapid, nuclear translocation of pERK,

Class B, no nuclear translocation—however, cytosolic signaling via recruitment of other proteins
How is glycogen metabolism regulated by PKA and cAMP
Reduce glycogen synthesis, increase glycogen breakdown, and increase lipolysis
Name 4 ways Jak Stat can be inhibited
Phospotases on Jaks

Competition for Tyr binding

Ubiquitination of Jaks

Modification of stats by PIAS
What is the general Jak/Stat pathway?
Ligand binds to receptor

Receptors cross link

Jaks cross-phosphorylate

Jaks phosphorylate receptor tyrosines

Stats associate to phosphorylated receptor

Stats dimerize via SH2 domain

Stat Dimer translocates to nucleus
Give the basic TGF-Beta pathway
Type 2 receptor binds to ligand

Type 2 and 1 receptors dimerize and type 1 is phosphorylated by 2

Inhibitory Smads are kicked off Type 1

Smad 2 or 3 are phosphorylated by Type 1 receptor, unfold, dimerize with Smad 4 (mediated by SARA)

Complex translocates to nucleus
How would the amplitude of a signal be regulated?
Competition between pos and negative regulation
What influences the duration of a signal?
Induction of a negative signal
What motif does SH3 bind?
Proline-rich
Give 2 examples of cross-talk
Stats can be activated by Receptor Tyrosine Kinases.

TGF-B can activate MAP kinase via TAB-1

Ras/Map kinase signaling can inhibit Smad nuclear accumulation
Opposing and cooperative effects of TGF-B and Ras signaling: Give tissue examples
Epitheleial cells: antagonistic
Fibroblasts: cooperative
Give an example of nuclear cross-talk
GFAP expression is activated by BMP and LIF

Smads and STATs are required for full activity

The p300 coactivator integrates these two signals

Or just, p300 coactivator integrates Smads and STATs
What motif does a Pleckstrin homologous site bind?
PIP2, 3
Name 4 mechanisms employed to transmit signals
Kinases (phosphatases)

Creation of protein docking sites

Removal (proteolysis) of regulatory proteins

Regulation of localization
Are SH2 domains specific?
Yes
How does hedgehog work in the case of cubitus interuptus?
Patched naturally inhibits smoothened receptor

Hedgehog deinhibits smoothened by activating patched

Proteolyzed form of Ci is a repressor

Hedgehog signaling inhibits proteolysis of Ci

Full-length protein activates transcription
How can hedgehog signal to distant cells?

How was this tested?
Precursor SHH is cleaved into a membrane-tethered form and a “free” form

Free form is able to diffuse in a gradient

Tested by use of conditioned media from SHH transfected cells
How does Protein kinase B (Akt) inhibit apoptosis? (In brief)
PI3 kinase activates PKB which inhibits the activation of the procaspase 9 – caspase 9 – procaspase 3 – caspace 3 apoptotic cascade
Name two ways to identify receptors
Affinity chromatography

Affinity labeling
How do PDGF, FGF and Eph RTKs bind receptors and what is the commonality?
PDGF: Ligand dimerizes

FGF: FGF is bound to proteoglycan

Eph: Eph receptors dock via ligand for bidirectional signaling

Commonality: dimerization of receptors
What motif does SH2 bind?
Phosphotyrosine
What is Smad 4 required for?
DNA binding, transcriptional activation
What is Smad 4 not required for?
Receptor interaction, nuclear translocation
What does Smad 6 do?
Competes with Rsmads
What does Smad 7 do?
Blocks Rsmad phosphorylation
What does inhibin do?
Competes with ligand for TGF-B binding
Shup:What is the purpose of the AB region of the nuclear receptor?
Phosphorylation sites. Bind cytoplasmic molecules
Shup:What is the purpose of the C region of the nuclear receptor?
DNA binding
Shup:What is the purpose of the D region of the nuclear receptor?
This is the hinge region
Shup:What is the purpose of the E region of the nuclear receptor?
Ligand binding
Shup:What two regions minimally confer the entire function of a nuclear receptor molecule?
C and E. DNA binding and Ligand binding
Shup:What two areas are responsible for Nuclear localization?
D and E (hinge and ligand binding)
Shup:What is the part of a receptor that varies the most?
The N-terminus
Shup:Where is the most homology in nuclear receptors?
The DNA binding domain
Shup:What are two important regions found in the Zn fingers?
The D-box (dimerization) and P-box (DNA binding)
Shup:Which box is diagnostic for the type of receptor?
The P-box
Shup: What are the two main types of hormone response elements that are involved in DNA binding?
Palindromic and direct repeat
Shup: What is RXR?
It is a critical protein that forms dimers with direct repeat receptors.
Shup: What three receptors form heterodimers via direct repeats and RXR?
RAR, Vitamin D, Thyroid receptor
Shup: How do steroid receptors bind?
Homodimers with palindromic sequences
Shup: What sort of mutants often lead to no transcription?
Ligand or DNA binding. Dominant negative phenotype. Sequester wildtype.
Shup: Explain CHIP
Use formaldehyde to fix DNA + complexes

Shear DNA

Unfix

Immunoprecipitate with antibodies
Shup: What are 2 ways that corepressors work?
Prevent recruitment of coactivators

Have HDAC activity

Physically block activators
Shup: What is one way receptor antagonists do what they do?
Recruit co-repressors. Tamoxifen is a good example.
Shup: What is the AF-2 region critical for?
Transcriptional activation
Shup: Explain the importance of proteosome activity
In the absence of proteosome, there is no receptor cycling, transcription stops
Describe the RTK to Ras pathway for EGF
RTK binds ligand--2 ligands, 2 receptors

receptors dimerize

receptors phosphorylate each other at tyrosine residues

association with Grb-2 (has SH2 and SH3 domains)

Grb-2 binds Sos, Sos acts as GEF for Ras

Ras covers inacivation domain of Raf

-->Mek, Erk, transcription factors
Describe the AKT (PKB) pathway
PI3 Kinase

PIP2 to PIP3

PDK1, AKT BIND TO PIP3

PDK1 activates PKB (AKT)

With ligand, inhibits apoptosis via caspase regulation

without ligand, apoptosis