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308 Cards in this Set
- Front
- Back
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ER (endogenous agonist)
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17B estradiol
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ER agonist
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ethinyl estradiol
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ER antagonist
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fulvestrant
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AR endogenous agonist
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testosterone
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AR agonist
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methyltestosterone
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AR antagonist
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spironolactone
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PR endogenous agonist
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progesterone
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PR agonist
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medroxyprogesterone
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PR antagonist
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mifepristone
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GR endogenous agonist
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cortisol
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GR agonist
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prednisone
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GR antagonist
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mifepristone
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TR endogenous agonist
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T3, T4
tri-iodothyronine levo-thyroxine |
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TR agonist
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levo-thyroxine
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TR antagonist
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none
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VDR endogenous agonist
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calcitriol
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VDR agonist
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calcitriol
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VDR antagonist
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none
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MR endogenous agonist
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aldosterone
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MR agonist
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fludrocortisone
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MR antagonist
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spironolactone
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RAR endogenous agonist
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all trans and 9 cis retinoic acid (vitamin A)
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RAR agonist
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accutane
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RAR antagonist
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none
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RXR endogenous agonist
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9 cis retinoic acid (vitamin A)
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RXR agonist
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accutane
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RXR antagonist
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none
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PPARalpha endogenous agonist
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fatty acids
ecosanoids prostanoids |
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PPAR alpha agonist
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clofibrate
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PPAR alpha antagonist
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none
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PPAR beta endogenous agonist
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fatty acids
ecosanoids prostanoids |
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PPAR beta agonist
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none
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PPAR beta antagonist
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none
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PPAR gamma agonist
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rosiglitazone
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PPAR gamma antagonist
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none
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LXR endogenous agonist
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oxysterol
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LXR agonist
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Drugs under testing that promote reverse cholesterol transport and reduce atherosclerosis in mice
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LXR antagonist
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none
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FXR endogenous agonist
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bile acids
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FXR agonist
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obeticholic acid
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FXR antagonist
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guggulsterone
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PXR endogenous agonist
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5b-pregnane-3,20-dione
PROMISCUOUS |
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PXR agonist
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hyperforin (SJW)
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PXR antagonist
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sulforaphane at very high concentrations (cruciferous veg)
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CAR endogenous agonist
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no high affinity
low affinity: bile acid and bilirubin inverse agonist: steroids (androstanes) |
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CAR agonist
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phenobarbital
inverse agonist: clotrimazole |
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CAR antagonist
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none
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ER function
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1. breast
2. uterus 3. CV 4. bone |
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AR function
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1. development and maintenance of male reproductive tract
2. anabolism of protein in skeletal muscle |
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PR function
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A:
1. uterine development 2. reproductive function: inhibition of endothelial cell proliferation B: mammary gland development |
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GR function
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mediates response to stress by increasing:
1. glucose production in the liver 2. fat redistribution 3. muscle breakdown 4. bone loss |
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TR function
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maintains BMR
necessary for normal development, especially of CNS |
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VDR function
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1. regulation of Ca and phosphorus absorption and homeostasis
2. bone development 3. control of cell growth and differentiation |
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MR function
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sodium reabsorption in the distal tubule
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RAR function
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1. embryonic development
2. maintenance of visual pigments 3. maintenance of skin |
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PPAR alpha function
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liver: upregulation of genes involved in
FA transport FA beta oxidation to increase liver uptake and catabolism of FAs |
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PPAR beta function
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studies suggest:
regulation of cholesterol trafficking in macrophages promotes formation of Type 1 muscle fibres (long distance running) |
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PPAR gamma function
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fat cell differentiation
induces expression of adipocyte specific genes |
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LXR function
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1. increases CYP7A1 (cholesterol --> bile acids)
2. controls cholesterol intestinal absorption 3. cholesterol catabolism 4. reverse cholesterol transport from peripheral tissues to liver for excretion in bile 5. induces SREBP1c and other lipogenic genes to increase lipogenesis 6. inhibits PPAR-alpha mediated FA oxidation by interfering with PPAR binding to target sites |
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FXR
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decreases bile acid secretion into the intestine
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PXR
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1. regulates phase I and phase 2 enzymes (esp 3a4, ugts)
2. regulates drug transporters, esp pgp |
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CAR
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1. regulates enzymes, esp. 3a4, 2d6, ugts
2. regulates drug transporters, esp pgp |
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which receptors are homodimers
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ER
PR AR GR MR |
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which is the heterodimerization partner
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RXR
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Draw out the chart left at the top of the matthews page
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ok
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Cytoplasm or nucleus
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GFAP: cytoplasm
PTEL: nucleus |
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3A4 regulation of NRs
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Inducers: GR, VDR, PXR, CAR
Inhibitor: LXR |
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ER - isoforms ?
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ERα ERβ
2 different genes |
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AR- isoforms ?
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none
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PR - isoforms ?
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PR-A
PR-B From a single gene, with different promoters, overlap in function |
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GR- isoforms ?
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none
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TR- isoforms ?
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TRα1
TRα2 TRβ1 TRβ2 2 genes 4 splice variants |
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VDR- isoforms ?
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none
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MR- isoforms ?
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none
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RAR- isoforms ?
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RARα RARβ
RARγ 3 genes |
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RXR- isoforms ?
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RXRα
RXRβ RXRγ 3 genes |
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PPAR - isoforms ?
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PPARα β γ
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LXR- isoforms ?
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LXRα
LXRβ |
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FXR- isoforms ?
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Only α in humans (also β in rodents)
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PXR- isoforms ?
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no
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CAR- isoforms ?
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no
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All p-box options
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Steroids: CGSCKV
Estrogen: CEGCKA other: CEGCKG |
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What are anti-androgens
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Inhibitors of androgen synthesis
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GS amino acids
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Glycine serine
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EG amino acids
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Glutamic acid glycine
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Androstenedione
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Common precursor for estrogen and testosterone
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Drug that activates PXR
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Rifampin
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3 dietary ligands for ER
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Soy
Red clover Red wine |
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Type of binding of type 1 aromatase inhibitor
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Noncompetitive
Irreversible Suicide inhibitor |
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Type of binding of type 2 aromatase inhibitor
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Competitive, reversible
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What is misoprostil
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PG analogue
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MOA anti-androgens
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Inhibit 5-α reductase enzyme that converts T into DHT (most potent AR agonist)
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Groupings of NRs
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Group 1 (NR1) = heterodimers
Group 2 (NR2) = RXR Group 3 (NR3) = homodimers |
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Saw palmetto
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5 α reductase inhibitor for benign prostatic hyperplasia
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Which receptor is promiscuous
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Which receptor is promiscuous
PXR |
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Soy
Red clover Red wine |
Dietary ligands for ER
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Where is aromatase highly expressed
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Placenta
granulosa cells of the ovarian follicle |
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MOA anti-estrogens
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Testosterone ----[aromatase]---> 17-β-estradiol
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What is an anti-estrogen (and what is it not)
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Prevents estrogen production
not receptor antagonist |
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4 mechanisms that alter NR activity (other than basic agonists, antagonists, inverse agonists)
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1. Receptor mutations
2. Targeting of coregulators and accessory proteins 3. Targeting endogenous NR ligand synthesis 4. Environmental factors (ex. diet |
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3 herbs that induce 3a4 by activating PXR
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Hyperforin
Ginkgo biloba Kava kava |
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How are aromatase inhibitors classified
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Type 1: steroidal inactivator
type 2: nonsteroidal inactivator also 3 generations 1st generation blocked all estrogens (highly potent) |
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Generations of SERMs
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1st gen: tamoxifen
2nd gen: raloxifene |
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Which agonist works by phosphorylating its receptor
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phenobarbital
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2 examples of anti-androgens
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Finasteride
dutasteride |
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Example of anti-estrogens (2)
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Letrozole
exemestane |
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Which generation and type is letrozole
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Generation 3 type 2
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Which generation and type is exemestane
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Generation 3 type 1
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Place of aromatase inhibitors in therapy
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adjuvant with tamoxifen
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Drugs metabolized by 3a4 (3)
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Atorvastatin
tamoxifen warfarin |
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NHP 5 α reductase inhibitor
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Saw palmetto
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SJW significance (other than hyperforin being agonist of PXR)
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3a4 inducer
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Kava kava significance
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3a4 inducer
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Heterodimers with RXR are named DR#
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DR1: PPAR
2: RAR 3: VDR 4: TR 5: RAR |
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Which is permissive of the RXR heterodimers - Aos or classicals
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AOs
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Indication of anti-androgens
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Male pattern baldness
benign prostatic hyperplasia |
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ChIP-Seq (7 steps)
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1. Mix NR + DNA
2. Shear (cut up) the DNA 3. Add specific antibody 4. Immunoprecipitate 5. Purify the complexes 6. Remove the protein (reverse crosslinks) 7. Sequence the DNA fragments |
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TAP-LC/MS - what does it stand for
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Tandem affinity purification liquid chromatography/mass spectrometry
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Methodology of TAP-LC/MS
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1. Start with cells overexpressing flag-tagged GR
2. Lyse the cells 3. Incubate with flag m2 beads 4. Wash the beads to remove non-specifically bound proteins 5. Elute the protein and trypsin digest 6. Analyze by LS/MS/MC 7. Generate protein interaction map |
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High/low doses of ___ cause cushing's syndrome
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Cortisol
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Adverse effects of PPARγ agonists like rosiglitazone (2)
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Cardiac events
Weight gain |
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What domain has this structure
Three layered sandwich of 11-12 alpha-helices Short region of beta sheet |
LBD
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Name a method used for determining novel protein interactions
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TAP-LC/MS
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Which is LXXXIXXXL/I
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CoRNR box
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Which is LXXLL
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NR box
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Sequence on corepressor that interacts with the region around AF2 to cause repression
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CoRNR Box
LXXXIXXXL/I |
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Sequence of CoRNR box
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LXXXIXXXL/I
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What is the conformational change upon ligand binding that causes corepressor to unbind
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Helix 12 reorients
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Sequence on coactivators that bind to AF2 region and cause activation
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NR box
LXXLL |
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RAR stands for
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Retinoic acid receptor
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CAR stands for
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Constitutive androstane receptor
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FXR stands for
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Farnesoid X receptor
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PPAR stands for
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Peroxisome proliferator activated receptors
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LXR stands for
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Liver X receptor
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1 examples of coactivator
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SRC1,2,3
steroid receptor coactivator |
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Examples of receptors that move to the nucleus when activated by ligand (4)
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GFAP
GR PR AR FXR |
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Examples of receptors that are constitutively DNA bound (4)
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PLET
TR PPAR LXR ER |
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2 examples of corepressors
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SMRT: silencing mediator of retinoic acid and thyroid hormone receptor
NCOR: nuclear receptor co-repressor |
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Which types of receptor bind ligands with high affinity
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Steroid (homodimers)
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Ligands of heterodimer receptors (5)
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Low affinity ligands
Fatty acids cholesterol Metabolite Vitamins |
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Reverse endocrinology (4 steps)
|
Opposite of what we have classically done:
Physiology --> hormone/ligand --> Receptor --> genomic information |
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Steps to adoption (5)
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1. Tissue distribution
2. identification of ligands (natural/synthetic) 3. Pharmacological studies 4. ID of target genes 5. Transgenic mice 1. Knockout 2. Overexpression |
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Knockout phenotype: RXR
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Embryonic and post-embryonic development
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Long term spatial memory
Stress Erythropoiesis energy metabolism |
GR knockout phenotype
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Knockout phenotype: PPARs, LXR
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Glucose and lipid homeostasis
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Knockout phenotype: GR (4)
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Long term spatial memory
Stress Erythropoiesis energy metabolism |
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LXRα tissue distribution
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Liver
intestine macrophage |
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Knockouts of __ are lethal
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NCOR
SMRT IGF-I EPO SHP1 |
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TRα1 expression
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Widely expressed
Particularly high in cardiac and skeletal muscle |
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TRα2
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Widely expressed
unable to bind hormone |
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TRβ1
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Brain
Liver Kidney |
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TRβ2
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Hypothalamus
pituitary |
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2 drugs that inhibit the production of thyroid hormone
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Propylthiouracil
methimazole |
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Who is at high risk of not enough D (4)
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Over 50
dark skin don't go outside much Wear covering clothing |
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Pathway of vit D
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Skin: 7-dehydrocholesterol --> vit D3
Liver: vit D3 (from skin and diet) --> 25-OH-D3 Kidney: 25-OH-D --> 1,25-OH-D3 |
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2 AKAs for vit D
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1,25OHD=calcitriol
vitD3=cholecalciferol |
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Which type of vit D do you get from diet
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3
|
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Which type from supplements
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2
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What are dietary sources
|
Fish
Meat |
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The story of aldosterone and cortisol
|
Aldosterone is physiological ligand for MR cortisol also binds MR
There is 100x more cortisol than aldosterone 11-β-hydroxysteroid dehydrogenase 2 converts cortisol to cortisone Licorice inhibits this This causes hypertension |
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Use of progesterone drugs (3)
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Hormonal contraception prevention of endometrial hyperplasia in HRT
Maintenance of gravid uterus/endometrium |
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3 options for the consensus sequence
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1. Most: AGGTCA
2. Steroid: AGAACA 3. Estrogen: AGGTCA |
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What does the P box consist of
|
2 coordinating cysteines
consensus sequence in between, plus a few aa's after Steroids: CGSCKV Estrogen: CEGCKA other: CEGCKG |
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what amino acids are L and I
|
leucine
isoleucine |
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what is it called when you go backwards through these steps:
1. physiology 2. ligand 3. receptor 4. gene |
reverse endocrinology
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all of the estrogen in all tissues
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breast: all good, except HRT
uterus: HRT, tamoxifen bad; raloxifene, fulvestrant good blood clot: all bad LDL and HDL: HRT, tam, ral good colorectal cancer: HRT good, rest unknown menopause: HRT good, rest bad bone: all good, except fulvestrant |
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what do PXR, CAR, VDR and TR do
|
induce 3a4
|
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2 things inhibited by lxr
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1. PPAR alpha beta oxidation of FAs
2. 3a4 |
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significance of rifampicin
|
activates PXR
|
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tamoxifen vs. raloxifene
|
tamoxifen is good for clot prevention
raloxifen is better for uterine cancer and LDL/HDL (tam unknown) |
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which receptor has a dimerization arm in its ectodomain
|
EGFR
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EGFR dimerization process
|
tethered monomer: 4 domains. domain 2 has a dimerization arm that is buried in domain 4.
extended dimer: ligand binds to domains 1 and 3, inducing a conformational change that pulls 2 out of association with 4 and dimerization arm is exposed. relationship between 1 and 2 is identical. the relative orientation of these domains does not change. |
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role of fibroblast growth factor
|
muscle and cartilage development
|
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which tyrosines of RTKs are phosphorylated first
|
activation lip tyrosines
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significance of phosphorylating activation lip tyrosines
|
tyrosine kinase is poorly active prior to this
|
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which Erbb does not bind growth factors
|
Erbb2
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significance of the fact that Erbb2 does not bind growth factors
|
constitutively active formation
|
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3 facts about heterodimers containing Erbb2
|
more potent mitogens
internalize slowly bind more ligands |
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definition of cytokine and growth factor
|
small secreted protein
both: differentiation Cytokine: growth GF: proliferation, motility |
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role of CSF-1
|
macrophage stimulation
|
|
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4 Erbb ligands
|
heregulin
neuregulin EGF TGF-alpha |
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What happens to EGFR and HER2 knockout mice (4)
|
Die mid-gestation/birth/postnatal day 20
Aberrant epithelial cells skin/lung/intestine brain development defect Progressive neurodegeneration |
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how Her2 is different from other Erbbs
|
does not bind GFs
constitutively active heterodimers containing it: More potent mitogens internalize slowly Bind more ligands |
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PDGF roles
|
fibroblast motility and proliferation
|
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which Erbb dimers cannot form
|
2-2 homodimers
3-3 homodimers 3-4 heterodimers |
|
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what do SH2 domains bind (and what determines specificity)
|
phosphorylated tyrosines
positions +1, +2, +3 C-term are most important |
|
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what do SH3 domains bind (and what determines specificity)
|
5-6 prolines in a row
variable loops that flank the hydrophobic pocket of SH3 (part of SH3 structure) |
|
|
EGFR pathway
|
1. Ligand binds (2 EGF bind to 2 EGFR)
2. Receptor dimerization 3. Kinase activation: Transphosphorylation on activation lip tyrosines 4. Phosphorylation of additional tyrosine residues 5. GRB2 binds phosphorylated tail via SH2 domain 6. Sos binds to GRB2 via SH3 tail on GRB2 7. Sos uses GEF activity to activate Ras 8. Ras hydrolyses GTP 9. Ras dissociates from Sos 10. Ras activates MAPKKK (Raf) 11. Activates MAPKK (Mek1) 12. Activates MAPK (ERK1/2) 13. MAPK dimerizes and translocates to the nucleus 14. MAPK phosphorylates TFs 15. Turns on or off genes involved in cell proliferation |
|
|
what type of molecules are in the MAPK cascade
|
serine-threonine kinases
|
|
|
Drosophila eye analogue of EGFR
|
sevenless
|
|
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what is sevenless
|
drosophila eye analogue of EGFR
|
|
|
ligand of sevenless
|
bride of sevenless (Boss)
|
|
|
sevenless signalling in drosophila
|
R8 secretes ligand (boss)
when it binds receptor (sevenless) on target cell, the target cell becomes R7 |
|
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what class of molecule is ras
|
G protein
|
|
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what kind of protein performs its role in signalling when bound to GTP
|
G protein
|
|
|
example of a GEF
|
SOS
|
|
|
do SH2 and SH3 have enzyme activity
|
no
|
|
|
Approximate # of aas in SH2 domain
|
100
|
|
|
Approximate # of aas in SH3 domain
|
60
|
|
|
structure of SH3
|
2 beta sheets at right angles
binding surface has hydrophobic residues that contact pro residues in target hydrophobic pocket flanked by variable loops that determine binding specificity |
|
|
This is the structure of what
2 beta sheets at right angles binding surface has hydrophobic residues that contact residues in target hydrophobic pocket flanked by variable loops that determine binding specificity |
SH3 domain
|
|
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role of Sos
|
EGFR Pathway
GEF for Ras |
|
|
role of Ras
|
EGFR pathway
G protein that activates Raf |
|
|
role of MAPK (ERK1/2)
|
EGFR pathway
dimerizes and phosphorylates transcription factors |
|
|
what class of molecule is mek1 (MAPKK)
|
serine threonine kinase
|
|
|
what domain does Grb2 have
|
SH2
SH3 |
|
|
what domain does Sos have
|
proline rich (ie. binds SH3)
|
|
|
genes that are activated by EGFR signalling
|
early response genes
ex. c-fos, c-jun genes that propel the cell through the cell cycle |
|
|
physiological result of EGFR signalling
|
proliferation (ex. skin cells after a wound)
|
|
|
Due to amino acid specificity of surrounding residues, Grb2 binds tyrosine #__ of EGFR
|
1068
|
|
|
during which period in the cell cycle are cells responsive to external signals that determine whether it progresses through the cell cycle or not
|
G1 until R point
|
|
|
7 hallmarks of cancer
|
1. self sufficiency in growth signals
2. resistant to anti-growth signals 3. resistant to apoptosis 4. limitless replicative potential 5. angiogenesis 6. genetic instability 7. tissue invasion and metastasis |
|
|
oncogene vs. tumor suppressor gene
|
gain of function in proto-oncogene
proto-oncogenes are proteins involved in cell proliferation (and other cancery stuff) tumor suppressor genes are anti-cancer genes. when loss-of-function mutation occurs, cancer can result |
|
|
types of mutations that convert oncogene into proto-oncogene
|
gain of function
1. constitutive activity 2. amplification 3. chromosomal translocation to a new promoter |
|
|
common oncogene
|
RTK
|
|
|
Autocrine receptor signalling in cancer
|
Cells generate their own mitogenic signal (GF)
|
|
|
what is it called when cells generate their own mitogenic signal
|
autocrine signalling
|
|
|
what mutation often occurs in Ras
|
loss GTPase activity (ie. locked in active conformation)
|
|
|
Which are the critical positions in Ras for GTPase activity
|
Gly 12, Gln 61
|
|
|
problems with anti-receptor antibodies for treating cancer
|
1. When the mutation causing the tumour is a deletion in the ligand binding domain/ectodomain (because this is where these agents bind)
2. Due to size may not be efficiently distributed to cells at the centre of a cancerous mass 3. Costly to produce (protein) 4. Need to be administered IV |
|
|
MOA Iressa
|
binds ATP binding site and prevents trans-phosphorylation of the receptor (works best in patients with mutation in tyrosine kinase domain)
|
|
|
in which patients does Iressa work
|
patients with mutation to tyrosine kinase domain
|
|
|
what is it called when a gene moves to a new promoeter
|
chromosomal translocation
|
|
|
what is it called when the number of copies of a gene in the genome are increased
|
amplification
|
|
|
2 viral oncogenes that tell us something about cancer-causing mutations
|
1. v-Erbb: avian erythroblastosis viral oncogene analogue of Erbb1 in which the ligand binding domain is truncated. The result is ligand-independent signalling. a similar protein is expressed in neuroblastoma.
2. v-sis: viral oncogene homologous to B chain of PDGF. when infected with the virus, large amounts of v-sis are produced. They bind to PDGFR in an autocrine loop |
|
|
why HER2 overexpression can cause cancer
|
need less GF because you wont have other receptors bound to GF looking around for dimerization partner - they will find one right away
|
|
|
3 antibodies that can block RTKs and their MOAs
|
Cetuximab (Erbitux)
Erbb1, domain 3, blocks GF binding trastuzumab (Herceptin) Erbb2, domain 4, precise MOA unknown, may lock receptor into place Pertuzumab (Omnitarg): Erbb2, domain 2, prevents dimerization |
|
|
Die mid-gestation/birth/postnatal day 20
Aberrant epithelial cells skin/lung/intestine brain development defect Progressive neurodegeneration |
EGFR or HER2 knockout mice
|
|
|
where is insulin made
|
beta cells
islets of langerhans |
|
|
what type of receptor is InsR
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RTK
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draw the insulin synthetic pathway
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ok (Anger, lec 4, slide 4)
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what is cleaved of off insulin during its synthesis and released with insulin into the bloodstream
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C peptide
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overall effect of insulin
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reduced blood glucose
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glucose transporter on pancreatic beta cells
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GLUT2
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regulation of insulin release
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enters pancreatic beta cells through GLUT2
ATP increase K channels close depolarization VG Ca channels open Ca enters vesicles release |
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which subunit of InsR binds insulin
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alpha
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which subunit of InsR is autophosphorylated
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beta
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2 domains that bind phospho-tyrosine
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SH2
PTB |
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PTB domain
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binds phospho-tyrosine
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precursor of PIP2 and PIP3
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phosphatidylinositol
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PH domain
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pleckstrin homology
recognizes PIP2 or PIP3 |
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Insulin signalling pathway
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1. Autophosphorylation of the β subunits
2. Tyrosine kinase activity is stimulated 3. IRS1 recruited to receptor via PTB domain 4. IRS1 phosphorylated on tyrosine residues 5. PI3K is recruited to IRS1 via SH2 6. PI3K converts PIP2 to PIP3 7. PDK1 and AKT are recruited to the PM through their PH domains 8. PDK1 phosphorylates AKT/PKB and activates it a. AKT phosphorylates GSK3, inactivating it a. This prevents GSK3 from phosphorylating and inactivating GS b. AKT also promotes translocation of GLUT a. Glucose uptake |
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4 modular protein interaction domains and what they bind
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SH2: p-Tyr
SH3: 5-6 prolines pleckstrin homology: PIP3 PTB: p-Tyr |
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enzymes that convert PIP2 to PIP3 and vice versa
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PIP2 --> PIP3 = PI3K
PIP3 --> PIP2 = PTEN |
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basic definition of diabetes
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high blood sugar
failure of insulin production/secretion/signalling |
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proportion of diabetics who are type 2
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90-95
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etiology type 1 diabetes
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autoimmune destruction of pancreatic beta cells
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definition of type 2 diabetes
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insulin resistance
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Mechanism of insulin resistance - what is it NOT (2)
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failure of insulin binding to receptor
defect in insulin production |
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mechanism of insulin resistance
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Inhibitory phosphorylation of IRS proteins
1. phosphorylation of serine residues of IRS1 2. Overexpression of phosphatases (that dephosphorylate tyrosine residues) Many different independent events are involved |
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Hypothalamic-pituitary axis of IGF-I
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Hypothalamus: GHRH
Pituitary: GH Liver: IGF-I |
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2 effects of IGF-I
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increased chondrogenesis
increased tissue growth |
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IGF-I levels correlate with
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Birth weight (levels during pregnancy)
gain in height (levels during growth) knockouts are lethal in mice |
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IGF-I levels throughout life
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Increase from infanthood to 20s
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IGF-I excess = ____ (condition/syndrome)
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Gigantism/acromegaly
closing of epiphyseal plates: gigantism if before acromegaly if after |
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common etiology of gigantism
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pituitary tumor
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signs of gigantism
signs of acromegaly |
gigantism: height, feet/hands, obesity
acromegaly: big limbs, facial features |
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glucose transporter in muscle and adipose
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GLUT4
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hormone pathway that leads to IGF1 production
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hypothalamus: GHRH
pituitary: GH liver: IGF-1 |
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4 examples of cytokines and 4 Erbb ligands
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prolactin
interleukins interferons EPO heregulin neuregulin EGF TGF-alpha |
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what does prolactin do
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During pregnancy induces epithelial cells in the mammary gland to differentiate into the acinar cells that produce the milk proteins
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Role of interleukins
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Proliferation and functioning of T and B cells
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role of interferons
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antiviral response
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where is EPO made
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kidney
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what stimulates kidney cells to make EPO
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low plasma O2 (suggests to low a level of RBCs)
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% of erythrocytes in blood (number and what this is called)
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45%
hematocrit |
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hematocrit
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% of blood that is erythrocytes
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why EPO used as doping agent
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increases amount of O2 that can get to muscles
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indication of EPO drugs
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anemia in
HIV cancer kidney disease |
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3 effects of EPO on RBCS
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prevents apoptosis
promotes proliferation promotes differentiation |
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why EPO knockouts are lethal
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they dont make RBCs
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2 differences of cytokineR signalling compared to RTK signalling
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1. JAK protein does the phosphorylation rather than the receptor itself
2. Cytokine signalling is more direct from the receptor to the nucleus |
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STAT stands for
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signal transduction and activation of transcription
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what does JAK phosphorylate first
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itself and the receptor
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Signalling pathway of CytR
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1. ligand binds
2. conformational change activates JAK 3. JAK phosphorylates itself and the receptor 4. STAT is recruited to the phoshporylated receptor by its SH2 domain 5. JAK phosphorylates STAT 6. STAT proteins dimerize 7. this exposes a nuclear localization signal 8. STAT proteins bind to genes and induce transcription |
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termination of EPO signal
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SHP1 phosphatase deactivates JAK2 by dephosphorylating a specific tyrosine
recruited via SH2 domain |
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SHP1
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phosphatase that deactivates JAK2 by dephosphorylating a specific tyrosine
recruited via SH2 domain |
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why do SHP1 knockouts die
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excess production of RBCs and WBCs
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dimerization of the 3 major receptor types (angers)
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EGFR: dimerization after ligand binding
InsR: constitutive dimer CytR: constitutive dimer |
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tyrosine kinase activity of the 3 major receptor types (Angers)
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EGFR: Intrinsic
InsR: intrinsic CytR: needs to associate with JAK |
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what drug does this
binds ATP binding site and prevents trans-phosphorylation of the receptor (works best in patients with mutation in tyrosine kinase domain) |
Iressa
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stoichiometry of the Angers receptors
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EGFR: 2 ligand per dimer
InsR: 1 ligand per dimer EpoR: 1 ligand per dimer |
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This is the beginning of life for ___
When they enter the secretory pathway they are transmembrane proteins They enter the membrane and then a protease cleaves off the ectodomain, which can now act extracellularly |
GFs
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draw the box diagram with Kr, alpha Kr, etc.
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ok
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look at the equation for constitutive activity. what does each parameter determine. what is the alpha for full agonist, partial, pure antagonist, inverse agonist
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Kr: basal activity
Kd: drug binding alpha: whether drug is agonist, antagonist... full ag: alpha <<1 partial ag: alpha < 1 antag: alpha = 1 inverse ag: alpha > 1 |
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Hyperfunctioning thyroid adenomas
molecular basis how to treat |
normally, the anterior pit produces TSH
binds to TSHR on thyroid gland AC --> cAMP produces T3, T4 these hormones have negative feedback on the release of TSH in the case of adenoma, neoplastic thyrocytes are activated independently of TSH --> hyperthyroidism --> negative feedback on TSH release --> silencing of thyroid function in extra-adenomatous tissue Mutations result in constitutive activity of TSHR molecular basis: single amino acid substitutions an inverse agonist would be used to treat this |
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what factors regulate CFTR
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PKA and PKC dependent phosphorylation
ATP hydrolysis |
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compare and contrast the molecular structure of 3 superfamilies/classes of ligand-gated ion channels
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1. Cys loop receptors (pentamers, 4 TMs)
2. Glutamate receptors (tetramers, 3 TMs) 3. P2X receptors (trimers, 2TMs) |
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What ions are able to permeate nicotinic receptors? What is the approximate reversal potential of these nicotinic receptor channels
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equal permeability to Na and K
Ena = +70mV Ek = -85mV channel: Erev = -15mV |
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2 regions that agonists of VG Na channels could bind
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1. voltage sensor (activation gate S4) - drug would stabilize open state
2. bind in pore - stabilize open state |
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3 major roles of Cl channels
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1. Regulate Cl- homeostasis
2. Modulate membrane potential profile/duration: for heart myocytes, depolarize diastolic potential and hyperpolarize during the action potential 3. regulate cell volume and organelle volume |
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Compare and contrast the differences in pore helices between biological channels (like K+ channels) and anionic Cl- channels. What role do these pore α helices play in ion channel function
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pore alpha helices determine ion specificity of the channel
Cl channel 1. both monomers of the dimer can form a functioning channel on their own 2. 2 alpha helices 3. antiparallel 4. amino terminal end of the helices orient toward the ion in the binding pocket K channel 1. monomers must arrange into a tetramer to form a functioning pocket 2. 4 alpha helices 3. parallel (barrel stave) 4. negative carboxyl end points toward the ion helices point in opposite directions |
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which transporters function to transport very large molecules
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ABC transporters
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function of ABC transporters
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transport of very large molecules across cell membrane
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Discuss the structure and function of gap junction channels (6 marks)
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Gap junctions mediate chemical and electrical communications between myocytes this is important for AP propagation and coordinated chamber contraction
One gap junction is composed of 2 connexons (AKA hemichannels) Hemichannels are homo- or hetero- hexamers of connexin proteins |
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equation for IC50 for binding of probe
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IC50 = Ka (1 + [P]/Kp)
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all about cholera toxin
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consists of A subunit and B homopentamer
A subunit's C terminal alpha helix protrudes into the central pore of the B pentamer binds gangliosides on luminal surface of intestinal cells internalized A1 fragment enzymatically cleaved enters cytosol catalyzes ADP ribosylation of Arg 201 on alpha subunit of Gs GTPase activity is blocked Gas is constitutively active Gas activates AC AC: ATP --> cAMP cAMP dependent kinase Na pump Na into GI tract diarrhea In the ADP ribosylation reaction NAD+ --> nicotinamide |
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what is the equation for Q
D + R ----[Kd]---> DR ----[E]---> S ---> Q |
Q = Qm S/(1+S)
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look at the folded page in the book
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ok
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equation of IC50 for binding of probe
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IC50 = Ka (1+[P]/Kp)
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how to demonstrate that sites labelled by a radioligand are identical to ones associated with a particular effect
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using the radioligand as the antagonist
plot Q vs. agonist at various concs of antagonist Are they parallel? dr=D(A=x)/D(A=0) plot log(dr-1) vs. antagonist slope should be linear and equal to 1 |
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important facts from the cube
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R + G: free R has low affinity for A
free G has high affinity for N RG: bound R has high affinity for A bound G has low affinity for N GTP (N) promotes R, G uncoupling (low affinity for agonist state) Agonists promote coupling When GTP is present it wins the battle Therefore, all receptors are converted to lower affinity Kh indicates K (affinity) of the agonist for the high affinity RG complex (not GTP bound) Kl indicates K (affinity) of the agonist R when it is not bound to G (GTP bound) KL/Kh correlates with intrinsic activity KL is higher than Kh so as KL/Kh decreases, KL is approaching KH a is intrinsic activity it refers to the ability of DR complex to produce Q (proportionality constant) To show intrinsic activity on a graph of y: axis = physiological effect x-axis: log [agonist] The one with the higher Qmax has the higher intrinsic activity |
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Alzhiemer's
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Cholinergic deficit treated with acetylcholineesterase inhibitors M2 receptors are presynaptic: binding of Ach to these receptors inhibits further release of Ach
M1 receptors are post synaptic Ideal treatment for alzhiemers would be agonist of M1 and antagonist of M2 in AD mutations, there is no GTP shift = no efficacy |
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model that accounts for reduced receptor number
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Rt in EC50 equation
the one with e |
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model that accounts for constitutive activity
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the one with the alpha
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process of desensitization
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1. GRK binds and phosphorylates the activated receptor
2. β arrestin binds the phosphorylated receptor 3. This promotes the accumulation of the receptor in clathrin coated vesicles 4. They are pinched off and internalized 5. So it will no longer signal 6. Once it is internalized it can either be taken to the endosome and taken apart or it can be dephosphorylated and recyled back to the membrane 7. In a living cell this is going on all the time so you have different levels of steady state receptor |
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Acromegaly
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Normally GHRH stimulates AC
End of pathway = secretion of growth hormone Tumor of pituitary AC constitutively active Excess secretion of GH Mutation where Gs GTPase activity is lost |
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read last box in wells chart
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ok
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