7: Cell Signalling And Transduction

Question 1

Second messengers

Answer 1

Small non-protein molecules that relay a signal to the interior of the cell

• cAMP
• IP3
• DAG
• Ca2+

Question 2

Basic signalling pathways

Answer 2

• GPCRs
  
  • activate G proteins which then kick off a cascade, involving second messengers (cAMP, IP3)
• Receptor kinases
  
  • kinases
  • when they bind a ligand their kinase activity is activated and they kick off a phosphorylation cascade

Question 3

GPCRs

Answer 3

• huge family of proteins
• 7 transmembrane domains (a helices)
• named because they activate heterotrimeric G proteins
• contain a variable region for ligands to bind on cytosolic side of membrane
• GTPases
  
  • attached to GTP = ON
  • Attached to GDP = OFF

Question 4

GTPases

Answer 4

• GTP = ACTIVE
• GDP = INACTIVE
• category 1
  
  • monomeric and small
  • ran, ARF, Sar 1
• category 2
  
  • trimeric (3 subunits)
  • a and g secure the G protein to the plasma membrane
  • peripheral membrane proteins
  • have small hydrophobic domain (can move around membrane)

Question 5

GPCR mechanism

Answer 5

1. Resting state
   
   • receptor not bound to ligand
   • Ga is bound to GDP and associated with GBY
2. Ligand binds receptor
   
   • Ga releases GDP and acquires GTP (active)
3. Ga and GBY subunits separate
   
   • Ga leaves
4. G protein subunits activate or inhibit target proteins
   
   • imitates signal transduction events
   • target protein = effector protein
5. Ga subunit hydrolyzes its bound GTP to GDP
   
   • now inactive
   • target protein also inactivated
6. Subunits recombine to form an inactive G protein

Question 6

Terminating GPCR response

Answer 6

1. GRK (G protein receptor kinase) phosphorylates the GPCR
2. Arresting binds the phosphorylated GPCR (now inactive)
3. Arrestin recruits AP2
4. AP2 recruits a clathrin coat
5. GPCR is internalized by endocytosis
6. GPCR in the endosome may have several fates

Question 7

Adenylyl Cyclase

Answer 7

• makes cAMP from ATP
  
  • removes to phosphates and creates rung structure
• after ligand binds GPCR, G protein is activated, Ga subunit activates adenylyl cylase
  
  • adenylyl cyclase makes cAMP
  • GTP on Ga is hydrolyzed
  • Ga dissociated from adenylyl cyclase so no more cAMP

Question 8

cAMP

Answer 8

• second messenger with many roles
• phosphodiesterase (PDE) will shut down signal
  
  • convert cAMP to AMP
• main target is PKA (protein kinase A)

Question 9

PKA

Answer 9

• has 2 catalytic subunits (kinase activity) and 2 regulatory subunits (structural)
• regulatory subunits normally bind to catalytic subunits (inactive form)
• cAMP binds to the regulatory subunits of PKA and catalytic subunits are released
• catalytic subunits now available to phosphorylate/activate

Question 10

Fight or flight response

Answer 10

• mediated by cAMP/PKA
• PKA turns on transcription factors by going into nucleus and phosphorylation gets CREB
  
  • also liberate glucose for use in muscles
  • glycogen turned into glucose

Question 11

PLC

Answer 11

• phospholipase C
• makes lipid derived second messengers
  
  • IP3 and DAG
• phosphotidylinositol with phosphates on inositol ring = PIP2
• PLC will cleave PIP2 into IP3 + DAG
• if a protein has a PH domain it will bind to PIP2
  
  • PLC has a PH domain
  • PLC will bind to PIP2 and cut it into IP3+DAG
  • cuts between phosphate and glycerol
• IP3 made up of the phosphate+inositol
• DAG is the diagylglycerol tail

Question 12

PLC mechanism

Answer 12

1. Ligand binds GPCR
   
   • activates the G protein
2. Ga stimulates PLC
   
   • PLC cuts PIP2
3. DAG remains in the membrane
4. IP3 released into cytosol

Question 13

IP3 and DAG

Answer 13

• DAG stays in membrane and activates PKC

- IP3 goes into cytoplasm and binds IP3 receptors on ER to release Ca2+

Question 14

Calcium

Answer 14

• second messenger
• released by SER by IP3
• concentration of Calcium in SER is 10,000x higher than in cytoplasm
• once cell filled with calcium, ATP driven pumped will pump it back into SER
• need DAG and Ca2+ for PLC activation

Question 15

Calmodulin

Answer 15

• calcium binding protein
• contains 4 binding sites
• has very low affinity for calcium, so it is only activated when calcium concentration is VERY HIGH

1. Calmodulin binds 4 calcium ions
2. Calmodulin changes conformation, resulting in active complex
3. The 2 globular hands of the complex wrap around a binding site on a target protein

Question 16

Calcium release following fertilization of animal egg

Answer 16

1. Blocks polysperm
   
   • exocytosis of vesicles to change coat proteins so that no other sperm can fuse
2. Egg activation
   
   • resumes metabollic pathways after floor of Ca2+
   • ripple effect of calcium

Question 17

Receptor kinases

Answer 17

• when they bind a ligand their own kinase activity is stimulated and starts a phosphorylation cascade
• pathway often involved in growth
  
  • ligand is often a growth factor
  • involved in mitosis

Question 18

Tyrosine kinases

Answer 18

-activated by phosphorylation on a tyrosine residue

Question 19

Serine/threonine kinases

Answer 19

-activated by phosphorylation on a serine/threonine residue

Question 20

RTKs

Answer 20

• receptor tyrosine kinase
• has a binding site for ligand on outside
• single transmembrane domain (a helix)
• tyrosine amino acid residues on cytoplasmic portion

Question 21

RTK mechanism

Answer 21

• once the ligands bind, 2 receptors cluster together
• AUTOPHOSPHORYLATION: each receptor puts phosphates on its neighbouring receptor
  
  • receptor kinase now active
• proteins with SH2 domain can now bind to phosphorylated tyrosines (to become phosphorylated)
  
  • eg. GRB2

Question 22

Ras

Answer 22

• small, monomeric G protein
• anchored at inner surface of plasma membrane by lipid group
• Ras-GTP = ON
• Ras-GDP = OFF
• when ras is on, it will lead to cell proliferation
• SOS is the GEF (guanine exchange factor) for Ras (to activate it)

Question 23

Ras mechanism

Answer 23

• receptor kinase activates GRB2 (which has SH2 domain)
• GRB2 activates SOS
• SOS puts GTP on Ras
• Ras is activated
• Ras triggers extensive phosphorylation cascade (Ras-MAP pathway)

Question 24

Ras-MAP pathway

Answer 24

• Ras phosphorylates Raf (MAPKKK)
• RAF phosphorylated MEK (MAPKK)
• MEK phosphorylates MAPK
• MAPK goes into nucleus and turns on transcription factors (eg. Ets and Jun)
  
  • turning on genes involved in cell proliferation

-a GAP will then turn off Ras

• all members of this pathway can cause cancer when mutated
  
  • mutation in gene results in no responding to on/off signalling
  • 30% of all cancers have something wrong in this pathway

Question 25

Proto-oncogenes

Answer 25

-genes that have the potential to become cancer causing

Question 26

Cross-talk in signalling

Answer 26

- pathways not linear - interconnected web - components of one pathway participate in other pathways