RTKs & GPCRs

Question 1

RTK structure

Answer 1

receptor tyrosine kinase

• extracellular ligand binding domain
• single pass transmembrane domain
• intracellular kinase domaine

Question 2

RTK role

Answer 2

• couple ligand binding (growth factors) to downstream signaling + gene transcription
• all receptors (except for insulin) control rate of cell proliferation and grown
• insulin receptor controls glucose homeostasis

Question 3

RTK ligands

Answer 3

• act as dimers: PGDF, fibroblast GF, VEGF (vascular endothelial), NGF (nerve), m-CSF (colony), IGF-1, insulin
• acts as a monomer: EGF

Question 4

RTK signaling (key events)

Answer 4

• receptor dimerization through ligand binding, increases receptor’s affinity for one another, brings close together
• receptor dimerization allows intracellular tyrosine kinase domains to cross phosphorylate on tyrosine residues
• signaling proteins have domains (4) that recognize P-Try (phosphotyrosine)

Question 5

SH2 domains

Answer 5

recognize P-Tyr + 2AAs on C-terminus

-contains 3 binding pockets

Question 6

PTB domains

Answer 6

recognize P-Tyr + 2-3AAs at N-term

Question 7

SH3 domains

Answer 7

recognize and bind to proline (AA) rich sequences

Question 8

PH domains

Answer 8

recognize phospholipids

i.e. phosphotidylinositol bi + tri phosphates, PIP3, PI3

Question 9

proteins with SH2 domains

Answer 9

GRB2
p85 (PI3K, 2x)
PLC gamma (2x)
STAT

Question 10

proteins with SH3 domains

Answer 10

GRB2 (MAPK, 2x)

Question 11

MAPK pathway (first step)

Answer 11

mitogen activated protein kinase

-upon ligand binding, the receptor dimerizes, cross phosphorylation of tyrosine residues (have Ras anchored 2x nearby)

Question 12

GEF

Answer 12

GDP/GTP exchange factor

• protein cofactor
• may inactivate or activate a protein, etc.

Question 13

Ras - structure

Answer 13

only PM-bound Ras involved in signaling. covalent attachment of hydrophobic anchors

1. enzyme attaches hydrophobic farnesyl residue at a C-term cysteine residue on Ras, attaching to PM
2. (in many Ras isoforms) second hydrobic anchor, a fatty acid residue covalently binds to a different C-term cys residue

Question 14

Ras - structure

Answer 14

only PM-bound Ras involved in signaling. covalent attachment of hydrophobic anchors

1. enzyme attaches hydrophobic farnesyl residue at a C-term cysteine residue on Ras, attaching to PM
2. (in many Ras isoforms) second hydrobic anchor, a fatty acid residue covalently binds to a different C-term cys residue

Question 15

MAPK - GRB2

Answer 15

protein that binds to phosphotyrosine
SH3-SH2-SH3
-SH2 domain binds to P-Tyr

Question 16

MAPK - SOS

Answer 16

SOS protein recruited to PM

• can bind to SH3 domain on GRB2
• is a Ras-GEF
• since Ras is nearby, can exchange GDP –> GTP

Question 17

MAPK - Ras

Answer 17

-Ras-GTP, activated by SOS can bind Raf

Question 18

MAPK - Ras

Answer 18

-Ras-GTP, activated by SOS can bind nearby Raf

Question 19

MAPK - Raf

Answer 19

• is activated by binding of Raf to Ras-GTP paired with other activity in PM
• serine/threonine protein kinase
• once activated, can phosphorylate, and activate MEK

Question 20

MAPK - MEK

Answer 20

• phosphorylated/activated by active Raf
• protein kinase
• MEK-Pcan phosphorylate/activate ERK

Question 21

MAPK - ERK

Answer 21

extracellular regulated kinase
ERK-P dimerizes
-travels to nucleus to phosphorylate/activate transcription factors related to cell proliferation
-cytoplasmic targets

Question 22

inactivation of MAPK pathway (4)

Answer 22

1. spontaneous hydrolysis of GTP to GDP, inactivates Ras
2. Ras-GAP hydrolyzes Ras-GTP
3. protein phosphatases (tyr and tyr/ser) dephosphorylate + deactivate every component of the signaling pathway
4. internalization of RTK via clathrin-mediated endocytosis

Question 23

overview of MAPK pathway

Answer 23

RTK –> YP –> GRB2 (SH2) –> SOS (recog SH3, Ras-GEF) –> Ras-GTP –> Raf (ser/thr k) –> MEK-P (pk) –> ERK-P + ERK-P –> cytoplasmic/nuclear targets

Question 24

PIP3

Answer 24

phosphotidyl inositol-3,4,5-triphosphate

-PI is pat of every PM, each hydroxyl can by phosphorylated by a specific lipid kinase

Question 25

PI3K - structure

Answer 25

- family of lipid kinases, phosphorylate PI at C3 OH - PIP3k Type A: two subunits 1. p85: regulatory, has 2SH2 domains 2. p110: catalytic, binds Ras-GTP inactive: p85 binds to p110, blocks kinase activity

Question 26

PI3K signaling pathway

Answer 26

1. RTK activation 2. p85's SH2 domains bind 2 P-Tyr on RTK p85 no longer bound to p110 3. p110 can bind to Ras-GTP (GRB2, SOS), now fully active 4. PI3K can phosphorylate PI-4,5-P2 at C3 hydroxyl 5. increased levels of PIP3 in PM

Question 27

Akt (PKB) - structure

Answer 27

contains PH and kinase domains - with low [PIP3] in PM, the PH domain is bound to the kinase domain, inhibiting its enzyme activity. protein not bound to PM - with high [PIP3] in PM, recruited to PM

Question 28

Akt pathway

Answer 28

PDK-1 (PI dependent k -1)and Akt are serine/threonine kinases - when [PIP3] in PM increases, they're recruited to the PM by their PH domains. both bind to PIP3, close proximity - full Akt activation requires two phosphorylations 1. PDK-1 phosphorylates Akt on threonine residue at the activation loop of its kinase domain 2. mTORC2: phosphorylates Akt on a hydrophobic serine residue

Question 29

Roles of Akt (3)

Answer 29

1. anti-apoptotic 2. glucose metabolism/ energy homeostasis 3. target of mTORC1

Question 30

TOR

Answer 30

(target of rapamycin, natural biological activator) - serine/threonine kinase - TORC1 + TORC2, bound to different regulatory proteins - mTORC1: regulated by GF, increases cell growth through inc protein synthesis. inhibited by rapamycin. - binds and is activated by Rheb-GTP - mTORC2: phosphorylates/activates Akt. resistant to rapamycin.

Question 31

Rheb

Answer 31

(Ras homologue enriched in brain, but ubiquitous) - GTPase - no Rheb-GEF - Rheb-GAP is TSC1/2

Question 32

TSC1/2

Answer 32

-active TSC1/2 complex is a GAP for Rheb, inactivating it (Rheb-GTP to -GDP) -active Akt (ser/thr k) + ERK-P phosphorylate + inhibit TSC2, thereby inhibiting its Rheb-GAP function -with a build up of Rheb-GTP, have increased levels of activated mTORC1 leading to increasing protein synthesis and cell proliferation [Akt phos/inactivates the inhibitor (TSC1/2), thus activated mTORC1]

Question 33

tuberus sclerosis complex

Answer 33

- hyperactive mTORC1 (mutation in TSC1/2, no GAP activity) - increased protein synthesis, increased cell size - but MAPK not affected by mutation, so no matching increased in cell proliferation - leads to tumors/giant cells

Question 34

termination of PI3K/Akt pathways (2)

Answer 34

tumor suppressing enzymes (protein and lipid phosphatases) act to decrease PIP3 in PM - p10: desphosphorylates C3 in PIP3 - Ship2: desphosphorylates C5 in PIP3 -mutation in PI3K or Akt pathway can lead to tumors

Question 35

PLCy (gamma) pathway

Answer 35

(PI4K and PI5K always present, have PIP2 in PM) PLCgamma has two SH2 domains + PH domain -SH2 domains, can bind to active RTK, which then phosphorylates + activates PLCgamma -PLCgamma cleaves PIP2 in PM to IP3 and DAG -DAG remains associated with PM w/ 2 FA chains -IP3 travels to ER, opens Ca2+ channels, inc Ca2+ in cytoplasm -w/ Ca2+ DAG

Question 36

proteins with PH domains

Answer 36

PI3K Akt (PKB) PDK-1 PLC gamma

Question 37

RTKs + cancer

Answer 37

-ErbB Receptors (erythroblastosis oncogene B) breast cancer, 25% gene amp/overexp of Erb2 (Her2) -N term trunctation can lead to dimerization and activation of receptor in absence of ligand -can design mAbs for EC portion of receptor -can inhibit kinase domain

Question 38

JAK/STAT

Answer 38

- receptor binding causes dimerization, but may not have intrinsic tyrosine kinase activity - recruits soluble JAK (dimerized). binds, cross phos on tyr residues of itself + receptor - phos-tyr residues of receptor recognized by SH2 domains on STAT proteins (signal transducers and activators of transcription) - STAT binds + is phosphorylated by JAK - STAT can dimerize, dissociate and travel to nucleus