Principles of Signal Transduction/ G-Protein Coupled Receptors/ Receptor protein-tyrosine kinase- Lecture 5-8

Question 1

What are the ligands used in G-protein coupled receptor pathways?

Answer 1

neurotransmitters (epinephrine, serotonin, dopamine); histidine; sensory stimuli (light/ odorants), many prescription drugs

Question 2

What are the receptors used in G-protein coupled receptor pathways?

Answer 2

proteins with 7 transmembrane domains that interact with ligands (binding extracellularly) to initiate conformational change in the receptor and allow for intracellular domain of the receptor to interact with heterotrimeric G proteins

Question 3

What are the subunits of the heterotrimeric G proteins in the G-protein coupled receptor pathway?

Answer 3

alpha, beta, and gamma (beta and gamma are normally found together but alpha is bound to GTP when alone and GDP when in complex with the other two)

Question 4

What is the activation pathway of G-protein coupled receptors?

Answer 4

upon ligand binding, receptor adopts a conformation allowing for interaction with the alpha subunit of a Heterotrimeric G-protein (with alpha subunit bound to GDP and the other two subunits)
alpha subunit releases GDP and binds GTP
alpha subunit dissociates from beta-gamma complex, now both active and able to initiate downstream effects

Question 5

What is the process of inactivation for a G-protein in the G-protein coupled receptor pathway?

Answer 5

alpha subunit hydrolyzes GTP to GDP (with the help of a membrane bound RGS protein because its a crappy GTPase on its own) after activating its target protein
alpha reassociates with beta-gamma

Question 6

Describe the downstream effects of the Gs pathway.

Answer 6

Gs (the alpha subunit at least) activates adenylyl cyclase which converts ATP to cAMP
four cAMP bind to the the two regulatory subunits of PKA (inactive it is two regulatory subunits and two catalytic subunits) and activates it (now regulatory subunit-cAMP complex and two separate active catalytic subunits)
which can phosphorylate targets

Question 7

What are examples of targets of active catalytic subunits of PKA?

Answer 7

glycogen phosphorylase (breaks down glycogen)
glycogen synthase (inactivated by PKA phosphorylation ensuring glycogen is not synthesized at the same time as breakdown)

Question 8

What is the downstream effect of the Gi pathway?

Answer 8

inhibition of adenylyl cyclase

Question 9

What is the downstream effect of the Gq pathway?

Answer 9

activates phospholipase Cbeta
phospholipase Cbeta breaks down the membrane phospholipid PIP2 into IP3 and DAG which each have different downstream targets

Question 10

What is the downstream effect of IP3?

Answer 10

activates release of calcium inside the cell, activating PKC

among other things

Question 11

What is the downstream effect of DAG?

Answer 11

directly activates PKC

Question 12

What is the downstream effect of the Gt pathway?

Answer 12

activated by rhodopsin receptor, this activates cGMP phosphodiesterase which breaks down cGMP

Question 13

What is the mechanism of cholera toxin?

Answer 13

adds an ADP-ribose molecule to the alpha subunit of Gs, resulting in its inability to catalyze the hydrolysis of GTP to GDP (it remains active permanently) keeping adenylyl cyclase activity prolonged, resulting in rises in cAMP which activates PKA
prolonged PKA sctivation (in the intestinal epithelium) causes efflux of Cl- and water into the gut –> watery diarrhea

Question 14

What is the mechanism of pertussis toxin?

Answer 14

addition of ADP-ribose molecule to alpha subunit of Gi which prevents biding to GPCRs and thus prevents action

Question 15

What are the ligands used in the Receptor Tyrosine Kinase pathway?

Answer 15

growth factors (platelet-derived growth factor, epidermal growth factor, fibroblast growth factor, insulin, etc)

Question 16

What are the general steps of signal transduction for the Receptor Tyrosine Kinase pathway?

Answer 16

binding of ligand to single pass transmembrane protein (RTK) causes receptor dimerization
dimerization causes auto/trans phosphoryolation at the active site and receptors become active
phosphorylation leads to receptor;s catalytic function and creates site for target protein recruitment

Question 17

What is the pathway for signal transduction of a MAP Kinase cascade in the Receptor Tyrosine Kinase pathway?

Answer 17

RTK is phosphorylated after ligand binding
Grb2 binds via SH2 domain (phospho-tyrosine binding)
Grb2 is constituatively bound to SOS via Grb2’s SH3 domains binding proline-rich regions in SOS
SOS activates Ras
Ras activates Raf
Raf phosphorylates MEK (a MAPKK)
MEK phosphorylates ERK (a MAPK)

Question 18

What is Ras?

Answer 18

a small G-protein which can bind GTP (inactive) and GDP (active) and has intrinsic GTPase activity

Question 19

SOS is a _____ for Ras.

Answer 19

GEF (guanine nucleotide exchange factor)

Question 20

What is the pathway for signal transduction of the Phosphatidylinosital-3 kinase (PI-3K) pathway of the Receptor Tyrosine Kinase pathway?

Answer 20

PI-3 (complex of regulatory subunit and catalytic subunit) binds to RTK via the SH2 domain of the regulatory subunit (p85), allowing the catalytic subunit (p110) to access PIP and PIP2 in the membrane and phosphorylate them on the 3 position of the inositol ring
phosphoinositides phosphorylated on the 3 position activate (directly and indirectly) Akt (alternatively PKB)
this phosphorylates substrates that promote cell survival and inhibit apoptosis
PTEN removes phosphate from 3 position of phosphoinositides (tumor supressor)

Question 21

What mutations in the RTK pathway most often result in cancers?

Answer 21

RTKs, Ras, or BRaf lead to activation in the absence of growth factors and constitutive activation of the PI-3K pathway

Question 22

What are the ligands for the Cytokine Receptor pathway?

Answer 22

prolactin, growth hormone, interferons, erythropoietin, etc.

Question 23

What are the steps of signal transduction for the Cytokine Receptor pathway?

Answer 23

cytokine receptors (each bound to a JAK1 and JAK2 on the intracellular portion) are dimerized by ligand, activating the JAK subunits that act as cross-phosphorylate and activate each other
these phosphorylate teh intracellular position of the cytokine receptor so it can bind SH2-containing proteins (SATs) which are transcription factors that dimerize once active and travel into the nucleus to bind DNA and activate expression of specific genes

Question 24

What are the ligands for the TGFbeta/BMP pathway?

Answer 24

teh TGFbeta/BMP family (heterodimers of type I and type II receptors)

Question 25

What are the receptors for the TGFbeta/BMP pathway?

Answer 25

serine/threonin kinases

Question 26

What are the steps of signal transduction for the TGFbeta/BMP pathway?

Answer 26

TGFbeta ligands induce type I and type II receptors to dimerize
Type II receptors (constituitively active) phosphorylate type I receptors which activates its kinase activity and phosphorylate rSMADs (their major substrates)
these dimerize with coSMADs
enter the nucleus and activate expression of specific genes

Question 27

What ligands are used in the Notch pathway?

Answer 27

single-pass, membrane bound ligands called Delta or Jagged

Question 28

What are the steps of signal transduction for the Notch pathway?

Answer 28

Notch (a single-pass, membrane bound structure) binds ligand on a passing cell, it’s intracellular domain (Nicd) is proteolytically cleaved by S2 cleavage (ADAM10 or TACE) and S3 cleavage (gamma secretase)
enters the nucleus and interacts with DNA-binding factor CSL, resulting in expression of target genes (without Nicd CSL inhibits expression of Notch pathway target genes)

Question 29

What are the ligands for the Wnt pathway?

Answer 29

family of secreted proteins that have a covalently attached fatty acid

Question 30

What is the signaling complex for the Wnt pathway?

Answer 30

Frizzled and LRP5/6

Question 31

What are the steps for signal transduction for the Wnt pathway in the absence of ligand?

Answer 31

complex of proteins (APC) in the cytoplasm lead to proteolytic destruction of beta-catenin via phosphorylation
In the nucleus, TCF/LEF (a DNA binding factor) inhibits expression of Wnt pathway target genes

Question 32

What are the steps for signal transduction for the Wnt pathway in the presence of ligand?

Answer 32

Wnt binds Frizzled-LRP5/6 receptor complex
Axin binds to LRP5/6, disrupting the APC complex while Dv1 (disheveled) binds Frizzled and leads to inhibition of CK1 and GSK3beta
beta-catinin no longer exists in the phosphorylated state and is stabilized so that it migrates to the nucleus and interacts with TCF/LEF and transcriptional co-activators to activate expression of the Wnt pathway target genes

Question 33

What other roles does beta-catinin play?

Answer 33

binds intracellular region of cell-cell adhesion molecules (cadherins) to help tether them to the actin cytoskeleton providing stability to cell-cell junctions

Question 34

What role does Wnt mutations play in colorectal cancer?

Answer 34

mutations in APC leads to underexpression of APC, leading to beta-catinin to continually be stable and the pathway to be locked in the on position
APC, then, is a tumor supressor gene

Question 35

What ligands activate the Hedgehog pathway?

Answer 35

small family of secreted proteins with unusual biosynthesis

Question 36

What receptor is primarily used in the Hedgehog signaling pathway?

Answer 36

Patched (Ptch1)- a 12 transmembrane domain protein

Question 37

What are the steps of signal transduction in the Hedgehog pathway in the absence of ligand?

Answer 37

Ptch1 inhibits signaling activity of Smoothened (Smo) catalytically
Absence of Smo signaling Gli (pathway responsive transcription factor) is phosphorylated by PKA, CK!, adn GSK3beta
this proteolytically cleaves into a repressor form (GliR) which enters teh nucleus and activates expression of the HH pathway target gene

Question 38

What are the steps of transduction in the Hedgehog pathway in the presence of ligand?

Answer 38

ligand binds Ptch1, which relieves Ptch1-mediated inhibition of Smo
Smo signals (in a poorly understood manner) to prevent phosphorylation and proteolytic cleavage of Gli
full length, activated Gli (GliA) enters the nucleus and activates expression of HH pathway target genes

Question 39

What do mutations in Ptch1 result in?

Answer 39

Gorlin syndrome

it is therefor a tumor suppressor gene

Question 40

What do mutations in Sonic HH result in?

Answer 40

holoprosencephaly (can cause cyclopia)