Module 2 - Wally Flashcards
GPCRs - background
About 1000 in genome
Activate heterotrimeric G protein
GPCRs and G-proteins
Alpha and beta-gamma subunits of G Protein. Complex interacts with receptor, ligand binds and conformation change in rc causes conformation change in alpha subunit - displaces and binds to GTP - G protein can signal
To turn off: Alpha subunit has GTPase activity, can hydrolyze GTP to GDP and turn off
G Proteins
Alpha subunits: 16 subtypes - GS, G1-3, Gq, Golf, etc with different functions
Gs
Stimulatory, activates cAMP through adenyl cyclase (a membrane localised enzyme) > takes AMP »_space; cAMP > activates kinases and phosphorylation > 2nd messenger cascade
Gq
Activates Phospholipase C to activate IP3 and release Ca2+ > activate Protein Kinase C
GPCR defects and disease
Receptor mutation: signalling increase/decrease
G protein modification effects: increase/decrease, GTP binding and/or hydrolysis
G protein mutation: increase/decrease, effects on GTP binding and/or hydrolysis
Vasopressin/ADH
Hormone, released from thalamus into pituitary gland to blood and primarily kidneys
Vasopressin V2 - normally couples to Gs and activates cAMP > makes water channels enter nephron of kidney and moves water from urine back into body
Mutation of vasopressin V2 receptor
Reduces receptor function. Vasopressin may or may not bind, and rc isn’t activated. cAMP isn’t activated therefore no water channel at the surface of nephron = water flows out of urine = DIABETES INSIPIDUS
Constitutively active receptors
Have mutations, forces rcs into active state in absence of hormone binding
Constant signalling may lead to disease
Rhodopsin
Detects photons of light, allows vision
Mutation»_space; constitutively active = continuous signalling, wear out retinal cels = retinitis pigmentosa
Adrenaline
Mutation leads to cancer
Gain of function
Can occur without mutation
Activates autoABs against GPCRs
e.g Grave’s disease - thyroid enlarges and TSH produced - large ECD seen as “unknown”. AB binds to TSH and force into active conformation > excess TH
Cholera - defects in G proteins
Cholera - bacteria modifies G protein via toxin , sticks ADP-ribose onto Gs alpha subunit in cells lining gut. Modifies on Arg201 and alters function, can’t induce hydrolysis and turn off»_space; active alpha subunit, continuous cAMP, increases Na+ pumping and water therefore watery diarrhea
Adenomas of pituitary and thyroid
Gas protein with patients with agromegaly (excess GH). Causes cancer - somatic (developed during lifetime) due to overactivation of Gas and too much GH
Mutation in Arg201
GPCR desensitisation
Protective mechanism, waning of a response to continuous or repeated stimuli i.e. coffee smell
Turning-off receptor-mediated signal transduction pathways
Receptor phosphorylation, internalisation, down-regulation
GRKs
6 in total. 1 is in retina, 4 in testes and the rest are everywhere. Regulates GPCRs by phosphorylating their intracellular domains after G proteins have been released and activated.
N-terminal: 180a.a , structurally homologuous, receptor recognition and binding
Catalytic domain: adds phosphate group to serine and threonine residues in activated rcs
C-terminal: variable size, modified by lipid attachment, membrane association, site of interaction with regulatory proteins
Characteristics of GRKs
Phosphorylate GPCRs only when they are activated (agonist-occupied)
Interact with rcs at sites distal from sites of phosphorylation
Phosphorylate serines and threonine in 3rd cytoplasmic loop/carboxyl-terminus
GRK phosphorylation
Receptor at cell membrane interacts withj G protein, ligand comes and binds, alpha subunit peels off and binds GTP, betagamma sits and GRK binds to it - lipid assoc. and binds to membrane or to phospholipids in rc. Once recognises active rc, adds pi groups to serine and threonine. Arrestins then bind to rc and sterically hinder activation of further G proteins.
Radioactive phosphate
Loaded cultured cells with radioactive phosphate P32»_space; plasmids that expressed rcs at cell surface»_space; incorporated into ATP»_space; radioactive ATP»_space; stimulate cells with agonist»_space; phosphorylated rc > load onto gel and lyse, immunoprecipitation > elute > SDS gels
Angiotensin receptor
Cutting off of tail = no phosphorylation, required for arrestin binding
Receptor internalisation
AngII binds to AT1A, phosphorylated by GRK, recruits arrestins > binds to rc, interact with clathrin, bends membrane and fuses, buds off due to dynamin and moves down to endocome, generate low pH and dissociation > cell either degrades or recycles back to surface
Measuring receptor internalisation
Receptor at membrane, add radioactive AngII 125, binds to rc and incubate. At 20 minutes, only a few left at surface due to vesicles, use acid strip (acid on outside of cells, lower pH and AngII falls off rc). Some are trapped inside and collected into tube. Small amount of radioactivity at 20 mins. Add NaOh to burst cells and collect. Radioactivity/Total = Internalisation index
Arrestins
Regulators of GPCRs
Binds to them to prevent further assoc with G proteins - desensitisation
4 families - 2 in retina
2 (Beta arrestins)
Structure of arrestins
410 a.a
N terminal: interacts with phosphorylated GPCRs
Hinge: R175, phosphorylation-sensitive trigger
C-terminal: interacts with clathrin/AP2 and causes internalisation
Arrestins as scaffolds
Scaffold bind other proteins and allow them to signal
Receptor at surface, phosphorylated by GRK, arrestin binds and has a protein that has to come off, once bound to rc it recruits diff proteins that have diff cell signalling and activation roles
Which of the following is not a function of arrestins?
- To bind phosphorylated receptors
- To sterically hinder G protein activation
- To inactivate GRKs
- To associate with clathrin
- To promote receptor internalisation
To inactivate GRKs
Describe the various steps involved in desensitisation of GPCRs.
Receptor phosphorylation, internalisation and down-regulation. Explain each of these
Which of the following would not be expected to produce enhanced G protein-mediated cell signalling?
- A receptor mutation that causes constitutive activity
- Autoantibodies that bind the receptor and mimic agonist binding
- Modification of the alpha subunit of a G Protein that inhibits its intrinsic GTPase activity
- Mutation of the receptor to prevent GRK-mediated phosphorylation
- A mutation of the alpha subunit of a G protein that prevents GTP binding
A mutation of the alpha subunit of a G protein that prevents GTP binding
Angiotensin, Gq/11
Angiontensin rc bind to Gq, once activated it binds with phospholipase C - cuts lipids in membrane to convert to IP3 and DAG. IP3 activates protein kinase C»_space; ER»_space; releases Calcium
Angiotensin constitutive receptors
Primary agonist = Sar1-AngII
then Sar1,Ile4-AngII
Sar1,Ile8-AngII
Sar1,Ile4,Ile8-AngII
Angiotensin constitutive receptors
Constitutive rcs show basal but not AngII-stim phosphorylation - R* is /=/ RP
SIIA phosphorylates WT but not N111 mt - R* /=/ RP
AngII internalises N111 mts - R1 /=/ RP
SIIA does not promote WT internalisation - R1 /=. RP
SIIA cause MAPK activation of WT rc - R* /=/ R**
Activation of MAPK via EGF
EGFR monomer in membrane (transmembrane protein). Has tyrosine-kinase domain on C-terminus of cell. When EGF binds to rc = dimerisation and transphosphorylate. Addition of Pi to kinase domain and to a.a. tyrosines in tail of receptor. Acts as a beacon through SH2 domain on GRB2, recognises phosphorylation event and its SH3 domain is recognised by Sos. This activates Ras and changes conformation so it binds GTP and is activated. Activates RAF which pi’s MEK, upstream activator of MAPK»_space; growth, phosphorylate other substrates and activates TFs in nuclei.
ErbB2 (HER2)
preferred dimerisation partner for other ErbB receptors
Good target for inhibition
Overexpression of ErbB2
Cancer
To block - block cell surface expression, or receptor at cell surface
Herceptin
Renin-angiotensin system
Angiotensinogen from liver, renin released from kidneys and cuts to make Angiotensin I. converted to angiotensin II via angiotensin converting enzyme. Peptide, only works when bound to T1 and T2 cell surface receptor.
Cardiac functions through T1
Angiotensin II
Important in cardiovascular. Blood constriction and water/salt balance. Causes thirst. Potent growth factor for heart. Controlled by AT1 receptor
AngII hypertrophy
involves MAPK and EGFR, highjacks EGFR pathway.
Activate metalloproteinase (ADAM) which clips EGF ligand from cell surface. Receptor dimerises and transphosphorylates, recruits proteins and leads to signalling
