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Definition of enzyme-linked cell surface receptors, and examples of
- enzymatic activity is in the same protein (not linked to another protein)
- receptor tyrosine kinase
- receptor serine kinase
- receptor guanlyl kinase
Definition of growth factor
small secreted proteins capable of stimulating proliferation, differentiation or motility.
How are EGF-like growth factors secreted?
- As a proform
- go to secretory pathway as a transmembrane domain, then get cleaved at the membrane to be excreted at the extracellular side
How did they figure out that EGF has a receptor?
- EGF only stimulates growth/proliferation of certain cells.
- experiment: radiolabelled EGF leads to saturable binding on cell surface
some characteristics of EGFR (ERBB1)
- C-terminus has homology with SRC protein (soluble tyrosine kinase)
- Single TMD
- ectodomain (sticks out extracellularly-> where ligand binds)
- exposure to EGF stimulates incorporation of 32P-ATP into cell membranes
Is the dimerization of EGFR ligand mediated, or receptor mediated?
The crystal structure of a dimerized EGFR show that the ligand does not bind at the interface, thus it is receptor mediated.
What are the 4 domains of EGFR and what do they do?
- Domain 1+3 bind the ligand
- Domain 2 is the dimerization arm
- Domain 4 interacts with dimerization arm to inhibit dimerization in absence of ligand.
How is ERBB2 different from ERBB1?
ERBB2 is constitutively active, and does not require growth factor.
Domains 1 and 3 are linked together, thus dimerization arm is always available (will dimerize with any other ERBB other than itself[no ERBB2 dimers])
SH2 domains and SH3 domains
Located on GRB2 (recruited to membrane, and binds SOS)
SH2- 100 a.a., bind to phosphorylated tyrosines and the 3 amino acids following the T residue.
SH3- 60a.a., binds proline rich target sequences. 2-B-sheets placed at right angles, with binding surface of highly hydrophobic residues; variable loops that flank the pocket determine the binding specificity.
Explain the MAP kinase cascade
- Active RAS recruits, binds, and activates MAPKKK by phosphorylation
- GTP hydrolysis leads to dissociation of RAS from MAPKKK
- MAPKKK activates MAPKK via phosphorylation
- MAPKK activates MAPK via phosphorylation
- MAPK dimerizes, and enters nucleus to alter transcription
What are the stages of the cell cycle? What is the R-point?
- G1- external signals influence the cell's decision to enter cell cycle-late G1 is R point
- S- synthesis of DNA (replication
- G2- premitotic phase- all the other organelles undergo replication
- M-mitosis phase- actual division of cell.
What are the hallmarks of cancer cells?
- Self-sufficiency in growth signals
- insensitivity to anti-growth signals
- evade apoptosis
- limitless reproductive potential
- sustained angiogenesis
- tissue invasion and metastasis
- genetic instability
any gene that encodes a protein able to transform cells in culture or induce cancer in animals
What are 3 mechanisms of proto-oncogenes turning to oncogenes?
- Gene gets translated infront of a new promoter sequence
- genetic rearrangement leads to gene amplification (a whole section of chromosome is amplified)
- specific coding sequence within the protein that somehow promotes growth of cells (hyperactivity of gene)
What are some differences between ERBB1, and v-ERBB
no ectodomain, thus unresponsive to EGF; constitutively active.
What are 3 different levels of RTK defects?
- 1) level of receptor (ligand independent firing/receptor overexpression)
- 2) level of ligand regulation (autocrine loop- cell makes the ligand itself, and then responds to it. ex. viral oncogene v-sis makes sis protein, and then responds to it)
3)level of signalling (>90% of pancreatic cancers have RAS mutations, ex. no GTPase activity)
Why are mutations in RAS troublesome? Where are mutations usually found?
Oncoprotein of RAS binds GTP, but has no catalytic activity, thus constitutive activity of its function.
- most mutations found in residue 12 or 61 (both critical for GTPase activity)
- glycine 12, glutamine 61; co-ordinate gamma phosphate for hydrolysis
- Binds to domain 3 of EGFR/ERBB1, thus EGFR can't bind ligand-> No dimerization
binds to domain 4 of ERBB2, receptor is able to dimerize, but can't undergo conformational change to induce intracellular signal
Good for breastcancer, as ERBB2 is usually over-expressed in breast cancers
- binds to domain 2 of EGFR/ERBB1
- blocks dimerization
What are some short comings of the 3 mAB discussed for ERBB1/ERBB2
target ectodomains, thus will not work if mutation results in loss of ectodomain.
Due to large size, might not be able to effectively interact with cells at center of mass
Costly to produce/administer IV
only works in 10% of non-small cell lung cancers, but if it works-> spectacular results
Binds to intracellular phosphorylation site in the kinase domain, not allowing the transphosphorylation of the receptor
certain mutations, heighten the receptors's affinity to iressa, thus could prescreen for these mutations
Structure of insulin A-chain? Structure of insulin B-chain? How are they connected as insulin?
A-chain: 2 imperfect a-helices running anti-paralell, joined by a disfulfide bond and a turn
A-chain cystines disulfide bond to B-chain cysteines.
How is insulin release regulated?
Insulin released when blood glucose levels increase
- glucose enters B-cells, undergoes glycolysis to create ATP
- Increases ATP:ADP rati, leading to closing of ATP-sensitive potassium channel
- Closing of K+ channel depolarizes membrane, leading to opening of calcium channels
- Increase in intracellular calcium causes release of insulin from granules
- blood insulin acts on liver, muscle, adipose tissue
- Constitutively covalent dimer (a2b2; a outside of membrane, b inside of membrane)
- -insulin binds to a-subunit on the receptor and induces the autophosphorylation of B-subunits on tyrosine residues
- autophosphorylation leads to activity
How does Insulin signalling work in cells?
- Phosphorylated B-subunit recruits IRS1 through its PTB domain
- receptor phosphorylates IRS1 on its tyrosines
- PI3K is recruited through its SH2 domain to IRS1
- PI3K phosphorylates PIP2 to PIP3, increasing PIP3:PIP2 ratio
- PDK1 and AKT are recruited to membrane through their pH domains
- PDK1 phosphorylates AKT to activate it.
Then AKT translocates GLUT4, and inhibits GSK3 activity
What does the plexin domain do? (PH domain)
- binds phosphorylated PIs (PIP2 and PIP3)
- allows proteins recruitment when PIP2/PIP3 form
What are the two actions of AKT?
1)active AKT translocates glut4 from cytoplasm to cell membrane, allowing cellular uptake of glucose.
- 2) AKT phosphorylates GSK3 (always active) to decrease its activity
- GSK3 normally phosphorylates everything including GS(glycogen synthase) which decrease GS's activity. Thus in presence of insulin, GS activity increases as AKT phosphorylates GSK3 to shut it off.
Why might receptors not respond to insulin in diabetes?
- Many different pathways
- Could be inhibitory phosphorylation of IRS proteins (on serine, instead of tyrosine residues), preventing further signalling.
- Could be tyrosine dephosphorylation by over-expression of phosphatase
How is insulin similar to insulin-like growth factor?
- similar in primary structure, especially A+B strands
- synthesized in liver, stimulates chondrogenesis (bone growth+tissue growth)
- IGF1 serum levels correlate with growth
- (at birth, they correlate with birth weight; during growth, they correlate with amount of growth)
How does excess of IGF1 change the disease state in different developmental times?
Excess in IGF1 prior to closure of epiphyses-gigantism
Excess in IGF1 all the time -> acromegaly
- small secreted molecules (~160a.a.) that control aspects of growth, and differentation of specific types of cells
- constructed of four a-helices folded into a characteristic arrangement
four examples of cytokines
ex. prolactin: during pregnancy, induces epithelial cells in mammary glands to differentiate into acinar cells that produce milk proteins
- interleukins: essential for proliferation and functioning of T and B cells
- interferons: anti-viral response
- erythropoietin: rbc formation
- cytokine which triggers production of erythrocytes by inducing proliferation and differentiation of erythroid progenitor cells
- synthesized by kidney cells that monitor concentration of oxygen in the blood.
How are cytokine receptors different than ERBB receptors?
Cytokine receptors are constitutive dimers, with no intrinsic kinase activity. (associate with JAK for kinase activity)
only bind one ligand per receptor
JAK/STAT pathway of cytokine receptor signalling
- cytokine binds cytokine receptors causing conformational change
- changes conformation of bound JAK proteins (2 per receptor), allowing it to activate.
- Activated JAK phosphorylates the tyrosine residues on cytokine receptor tail, initiating signalling.
- STAT gets recruited to receptor via SH2 domain
- JAK phosphorylates STAT protein, causing STAT to dimerize
- Dimerization of STAT reveals its nuclear localization signal
- STAT will alter transcription directly (usually interacts with promoter)
How is cytokine signalling terminated?
- SHP1 has an SH2 domain (knockout mice die due to excess RBC/WBC)
- SHP1s SH2 domain binds to one of the last phosphorylated tyorsine's in the receptor tail.
- SHP1 then also associates with JAK to dephosphorylate it, leading to inactive JAK
- SHP1 then depolarizes other tyrosine residues
How was Eoro mantyranta's EPO receptor different than WT?
his receptor was truncated at the c-terminus, thus SHP1 could not recruit to the membrane, and JAK was constitutively active, leading to increased RBC production (25-50% higher RBC levels, but normal EPO levels)
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