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Signalling
Fichas del conjunto (46)
- 1Examples of target proteins in a signalling pathway- metabolic enzymes
- gene regulatory protein
- cytoskeletal protein - 2Types of receptors1) cell surface
2) IC
e.g. ion channel coupled receptors
GPCRs
Enzyme linked - 3Synaptic signallingfast
low affinity receptor
high concentration of local ligand - 4Endocrine signallingSlow
high affinity receptor
low concentration of local ligand - 5Responses to ACh (heart muscle cell/salivary gland cell/skeletal muscle cell)Heart muscle - decreased rate and force of contraction
Skeletal muscle - contraction
Salivary gland - secretion - 6Intracellular signalling complexes1) on scaffold protein
2) on activated receptor
3) on phosphoinositide docking sites - 7EC domain of RTKsVaried
e.g. cysteine-rich/fibronectin-like/immunoglobin-like - 8RTK activationLigand dimerises and facilitates dimerisation of receptors.
IC kinase domains cross-phosphorylate each other - 9Analysis exploiting dimerisation1) inactivation of one IC domain will poison the other = dominant negative
2) addition of homodimerisation domain will lead to constitutive activation - 10HSPGs-multifunctional ECM components
- Protein core (tethered/secreted/TM)
- Sugar chains = modified mainly by sulfation (code that creates binding sites for specific protiens) - 11What does SH2 bind to?1 binding site for phospho-tyrosine
1 for amino acid (glutamic acid, glutamic acid, isoleucine) - 12RAS activation-SH2 domain of GBR2 binds to docking sites
-SH3 domain binds to SOS(GEF) and couples receptor to inactive RAS
-SOS promotes GDP-dissociation from RAS leading to GTP binding and SOS dissociation - 13FRET-2 fusion proteins are produces attaches to different coloured GFP so that excitation of the first protein will emit light that excites the second protein if they are close enough together. If emitted light corresponds to emission of seconds protein they are interacting
- 14Leprechaunsim-insulin receptor mutation
- fatal in first 2yrs
- low subcutaneous fat and muscle
- abnormal skin
-excess hair growth - 15Rabson-Mendenall syndrome- insulin receptor mutations
- survival to second decade
- skin/teeth abnormality
- hair overgrowth
- pineal hyperplasia - 16Type-A insulin resistanceSurvival to middle age
- 17Insulin productionMade in the B-cell of the pancreas and secreted to the bloodstream
Requires a lot of processing from pro-insulin to insulin - 18Immediate effects of insulinGlucose uptake from the blood to muscle cells and adipocytes
- 19Effects due to long-term exposureincreased expression of liver enzymes that synthesise glycogen and adipocyte enzymes that synthesise triacylglycerols
- 20Insulin receptoralpha (EC) and beta (TM and IC) subunits are initially formed as a single polypeptide and then cleaved
Disulphide bonds hold the dimers together - 21Receptor activation - RAS pathway- binding to the alpha subunit causes cross-phosphorylation of the IC kinases
- the receptor then phosphorylates IRS which contains a phosphotyrosine domain (therefore binds to site on receptor)
- IRS acts as a docking site for proteins e.g. GRB2 to activate RAS pathway - 22PI3-kinase2 subunits - p85 (SH2 domain)
p110 (kinase domain) - 23RAS-independent signalling (inositol pathway)-PI3-Kinase binds to IPS and phosphorylates docking site on PKB (membrane bound phosphoinositol)
At the membrane PKB is phosphorylated by kinases e.g. PKD1
PKB undergos a confirmational change and becomes active - 24PKB effectsphosphorylates GSK3 meaning glycogen synthesis increases (GSK3 inhibits glycogen synthesis)
Causes Glut4 translocation to membrane
PEPCK increases glycogen synthesis and is activated when FOXO binds to IRS. PKB inhibition of FOXO (phosphorylation) means PEPCK is inhibited - 25How do we know the exact mechanism of regulation for some targets?Promoter analysis
- 26qPCR- make cDNA from tissue
- Perform PCR in presence of fluorescent dye
- measure fluorescence after each cycle - 27Promoter analysis (promoter bashing)putative binding sites found by comparison with genes known to bind same TF
Promoter is bound to a reporter gene so level of reporter gene is a measure of promoter activity
Construct is altered (deletions) and gene activity measured - 28NO-gaseous
-free radical
-half-life = 5-10s - 29NO in CNS-cNOS close to NMDA receptors so it responds to Ca2+ implicating synaptic plasticity
e.g. glutamate activates receptors which stimulate NO pathway
NO feedsback to stimulate glutamate production (LTP) - 30NO in immune systemkills bacteria and parasites
iNOS in inflam. cells uses NO as cytostatic and cytotoxic agent
When overproduced in lungs and intestine can develop arthritis, asthma, crohns disease
Role in tumour apoptosis/necrosis - 31NO in vasculatureANS releases ACh which acts on endothelial cells which releases NO causing vasodilation
inhibits platelet aggregation and vascular SM proliferation (=anti-atherosclerotic) - 32NO ProductionACh creates NOS which forms NO from arginine
NO binds to guanlyl cycline causing production of cGMP. cGMP produces cGMP dependent protein kinase which leads to myosin light chain phosphatate activating mysosin light chain - 33cNOS- constituitively active
- Enzyme expression controlled by Ca2+/calmodulin
- short-lives
-picomoles of NO - 34nNOS-type of cNOS
- neural
- cytosolic
-found in CNS, ENS, NANC neurons and retina - 35eNOS-type of cNOS
-endothelial
-bound at cell membrane
- expressed in endothelium, osteoblasts, osteoclasts, cardiac myocytes, platelets - 36iNOS- inducible expression, constituitive enzyme activity
-long-lived effects
-nanomoles of NO
- transcriptionally induced in response to pathological material (LPS, IFN-g)
expressed in macrophages, kupffer cells, neutrophils, fibroblasts, vascular SM, endothelial cells - 37Nitroglycerine100 year old treatment for angina. Breaks down into NO in vivo and causes vasodilation
(also active component in dynamite) - 38Build up of cGMP is counteracted by.....phosphodiesterase-5
- 39Viagrainhibits the activity of phosphodiesterase-5 which means excess cGMP builds up, BVs relax to get increased blood flow
- 40Signalling molecules that bind to nuclear receptorssteroid hormones, thyroid hormones, retanoids
- 41Nuclear receptorsHeld in inactive state by inhibitory proteins.
Binding to ligand - confirmational change
Interact with co-receptors to direct transcription
DNA binding domain of each receptor contains 2 repeats of c4 zinc fingers - 42DNA response elements bound by homodimerse.g. GRE/ERE - steroid horomes bind as symmetric homodimers on inverted repeat DNA site
Homodimeric receptors = in cytoplasm in absence of ligand (anchored by inhibitors e.g. hsp90) Translocate to nucleus on binding (lose inhibition) - 43DNA response elements bound by heterodimerse.g. VDRE, TRE, RARE bound by thyroid hormones and retinoid heterodimers (with RXR) to direct repeat sites
Heteromeric dimers = exclusively in the nucleus. Absence of ligand = HDAC recruited. Presence of a ligand - HAT recruited - 44FLp- DNA recombinase
- fusion of estrogen LBD to FLP in lab is sufficicent to the recombinase ligand-dependent - 45Early primary response to steroid hormones1-6hrs
receptor complexes activate primary response genes
Induced synthesis of a few different proteins - 46Delayed secondary response to steroid hormones6-48hrs
Primary protein shuts off primary response gene
Different primary protein turns on secondary response genes
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- Fichas sobre Signalling, creado por lucypickles el 05/06/2015.
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