RTKs & GPCRs Flashcards
RTK structure
receptor tyrosine kinase
- extracellular ligand binding domain
- single pass transmembrane domain
- intracellular kinase domaine
RTK role
- 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
RTK ligands
- act as dimers: PGDF, fibroblast GF, VEGF (vascular endothelial), NGF (nerve), m-CSF (colony), IGF-1, insulin
- acts as a monomer: EGF
RTK signaling (key events)
- 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)
SH2 domains
recognize P-Tyr + 2AAs on C-terminus
-contains 3 binding pockets
PTB domains
recognize P-Tyr + 2-3AAs at N-term
SH3 domains
recognize and bind to proline (AA) rich sequences
PH domains
recognize phospholipids
i.e. phosphotidylinositol bi + tri phosphates, PIP3, PI3
proteins with SH2 domains
GRB2
p85 (PI3K, 2x)
PLC gamma (2x)
STAT
proteins with SH3 domains
GRB2 (MAPK, 2x)
MAPK pathway (first step)
mitogen activated protein kinase
-upon ligand binding, the receptor dimerizes, cross phosphorylation of tyrosine residues (have Ras anchored 2x nearby)
GEF
GDP/GTP exchange factor
- protein cofactor
- may inactivate or activate a protein, etc.
Ras - structure
only PM-bound Ras involved in signaling. covalent attachment of hydrophobic anchors
- enzyme attaches hydrophobic farnesyl residue at a C-term cysteine residue on Ras, attaching to PM
- (in many Ras isoforms) second hydrobic anchor, a fatty acid residue covalently binds to a different C-term cys residue
Ras - structure
only PM-bound Ras involved in signaling. covalent attachment of hydrophobic anchors
- enzyme attaches hydrophobic farnesyl residue at a C-term cysteine residue on Ras, attaching to PM
- (in many Ras isoforms) second hydrobic anchor, a fatty acid residue covalently binds to a different C-term cys residue
MAPK - GRB2
protein that binds to phosphotyrosine
SH3-SH2-SH3
-SH2 domain binds to P-Tyr
MAPK - SOS
SOS protein recruited to PM
- can bind to SH3 domain on GRB2
- is a Ras-GEF
- since Ras is nearby, can exchange GDP –> GTP
MAPK - Ras
-Ras-GTP, activated by SOS can bind Raf
MAPK - Ras
-Ras-GTP, activated by SOS can bind nearby Raf
MAPK - Raf
- 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
MAPK - MEK
- phosphorylated/activated by active Raf
- protein kinase
- MEK-Pcan phosphorylate/activate ERK
MAPK - ERK
extracellular regulated kinase
ERK-P dimerizes
-travels to nucleus to phosphorylate/activate transcription factors related to cell proliferation
-cytoplasmic targets
inactivation of MAPK pathway (4)
- spontaneous hydrolysis of GTP to GDP, inactivates Ras
- Ras-GAP hydrolyzes Ras-GTP
- protein phosphatases (tyr and tyr/ser) dephosphorylate + deactivate every component of the signaling pathway
- internalization of RTK via clathrin-mediated endocytosis
overview of MAPK pathway
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
PIP3
phosphotidyl inositol-3,4,5-triphosphate
-PI is pat of every PM, each hydroxyl can by phosphorylated by a specific lipid kinase
PI3K - structure
- 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
PI3K signaling pathway
- RTK activation
- p85’s SH2 domains bind 2 P-Tyr on RTK
p85 no longer bound to p110 - p110 can bind to Ras-GTP (GRB2, SOS), now fully active
- PI3K can phosphorylate PI-4,5-P2 at C3 hydroxyl
- increased levels of PIP3 in PM
Akt (PKB) - structure
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
Akt pathway
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
Roles of Akt (3)
- anti-apoptotic
- glucose metabolism/ energy homeostasis
- target of mTORC1
TOR
(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.
Rheb
(Ras homologue enriched in brain, but ubiquitous)
- GTPase
- no Rheb-GEF
- Rheb-GAP is TSC1/2
TSC1/2
-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]
tuberus sclerosis complex
- 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
termination of PI3K/Akt pathways (2)
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
PLCy (gamma) pathway
(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
proteins with PH domains
PI3K
Akt (PKB)
PDK-1
PLC gamma
RTKs + cancer
-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
JAK/STAT
- 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
