Module 2 - Wally

Question 1

GPCRs - background

Answer 1

About 1000 in genome

Activate heterotrimeric G protein

Question 2

GPCRs and G-proteins

Answer 2

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

Question 3

G Proteins

Answer 3

Alpha subunits: 16 subtypes - GS, G1-3, Gq, Golf, etc with different functions

Question 4

Gs

Answer 4

Stimulatory, activates cAMP through adenyl cyclase (a membrane localised enzyme) > takes AMP &raquo_space; cAMP > activates kinases and phosphorylation > 2nd messenger cascade

Question 5

Gq

Answer 5

Activates Phospholipase C to activate IP3 and release Ca2+ > activate Protein Kinase C

Question 6

GPCR defects and disease

Answer 6

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

Question 7

Vasopressin/ADH

Answer 7

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

Question 8

Mutation of vasopressin V2 receptor

Answer 8

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

Question 9

Constitutively active receptors

Answer 9

Have mutations, forces rcs into active state in absence of hormone binding
Constant signalling may lead to disease

Question 10

Rhodopsin

Answer 10

Detects photons of light, allows vision

Mutation&raquo_space; constitutively active = continuous signalling, wear out retinal cels = retinitis pigmentosa

Question 11

Adrenaline

Answer 11

Mutation leads to cancer

Question 12

Gain of function

Answer 12

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

Question 13

Cholera - defects in G proteins

Answer 13

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&raquo_space; active alpha subunit, continuous cAMP, increases Na+ pumping and water therefore watery diarrhea

Question 14

Adenomas of pituitary and thyroid

Answer 14

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

Question 15

GPCR desensitisation

Answer 15

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

Question 16

GRKs

Answer 16

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

Question 17

Characteristics of GRKs

Answer 17

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

Question 18

GRK phosphorylation

Answer 18

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.

Question 19

Radioactive phosphate

Answer 19

Loaded cultured cells with radioactive phosphate P32&raquo_space; plasmids that expressed rcs at cell surface&raquo_space; incorporated into ATP&raquo_space; radioactive ATP&raquo_space; stimulate cells with agonist&raquo_space; phosphorylated rc > load onto gel and lyse, immunoprecipitation > elute > SDS gels

Question 20

Angiotensin receptor

Answer 20

Cutting off of tail = no phosphorylation, required for arrestin binding

Question 21

Receptor internalisation

Answer 21

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

Question 22

Measuring receptor internalisation

Answer 22

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

Question 23

Arrestins

Answer 23

Regulators of GPCRs
Binds to them to prevent further assoc with G proteins - desensitisation
4 families - 2 in retina
2 (Beta arrestins)

Question 24

Structure of arrestins

Answer 24

410 a.a
N terminal: interacts with phosphorylated GPCRs
Hinge: R175, phosphorylation-sensitive trigger
C-terminal: interacts with clathrin/AP2 and causes internalisation

Question 25

Arrestins as scaffolds

Answer 25

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

Question 26

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

Answer 26

To inactivate GRKs

Question 27

Describe the various steps involved in desensitisation of GPCRs.

Answer 27

Receptor phosphorylation, internalisation and down-regulation. Explain each of these

Question 28

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

Answer 28

A mutation of the alpha subunit of a G protein that prevents GTP binding

Question 29

Angiotensin, Gq/11

Answer 29

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&raquo_space; ER&raquo_space; releases Calcium

Question 30

Angiotensin constitutive receptors

Answer 30

Primary agonist = Sar1-AngII
then Sar1,Ile4-AngII
Sar1,Ile8-AngII
Sar1,Ile4,Ile8-AngII

Question 31

Angiotensin constitutive receptors

Answer 31

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**

Question 32

Activation of MAPK via EGF

Answer 32

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&raquo_space; growth, phosphorylate other substrates and activates TFs in nuclei.

Question 33

ErbB2 (HER2)

Answer 33

preferred dimerisation partner for other ErbB receptors

Good target for inhibition

Question 34

Overexpression of ErbB2

Answer 34

Cancer
To block - block cell surface expression, or receptor at cell surface
Herceptin

Question 35

Renin-angiotensin system

Answer 35

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

Question 36

Angiotensin II

Answer 36

Important in cardiovascular. Blood constriction and water/salt balance. Causes thirst. Potent growth factor for heart. Controlled by AT1 receptor

Question 37

AngII hypertrophy

Answer 37

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