cell signalling

Question 1

describe general structure of GPCR

Answer 1

• 7 TM spanning regions: a helices of 24AAs
• amino terminus outside cell, carboxy terminus inside cell
• extracellular domain has lots of diversity
• intracellular domain has little diversity

Question 2

why is does the diversity of the intracellular and extracellular GPCR sequence differ?

Answer 2

• extracellular domain has lots of diversity due to range of potential extracellular ligands
• intracellular domain does not require this, as not so many intracellular ligands (second messengers)

Question 3

what do GPCRs respond to?

Answer 3

neurotransmitters: ACh, glutamate, GABA
peptide and non-peptide hormones: glucagon, adrenaline
large glycoproteins: thyroid stimulating hormone

Question 4

explain how G proteins are ‘molecular switches’

Answer 4

inactive state: GDP bound
GDP for GTP exchange activates protein
intrinsic GTPase activity on a subunit regulates it

Question 5

what is a GEF?

Answer 5

guanine nucleotide exchange factor. stimulates GDP for GTP exchange

Question 6

how do activated GPCRs act as GEFs?

Answer 6

GEF activity is caused by small sequence of AAs usually obscured. upon GPCR activation, TM 6 displacement exposes them

Question 7

what are the Gs, Gi and Gq proteins responsible for?

Answer 7

Gs- activation of adenylyl cyclase
Gi- inhibition of AC
Gq- activation of phospholipase C

Question 8

how are cholera and pertussis toxins used to distinguish between subtypes of GPCR?

Answer 8

cholera toxin inhibits Gs- constant AC activity

pertussis toxin inhibits Gi, reversing suppression of AC

Question 9

what is the basis of GPCR specificity?

Answer 9

molecular variation of a-subunits and the selectivity of each for receptors and effectors

Question 10

what is the difference between tyrosine kinase-linked receptors and serine/threonine kinases?

Answer 10

tyrosine kinase linked receptors have the enzyme bound, not part of primary sequence

Question 11

what processes do receptor tyrosine kinases control?

Answer 11

cell growth, movement, and differentiation

Question 12

what are the main ligands for receptor tyrosine kinases?

Answer 12

growth factors; EGF and NGF

Question 13

what is the extracellular domain of an RTK responsible for?

Answer 13

ligand recognition and binding

Question 14

what is the transmembrane domain of an RTK responsible for?

Answer 14

information transmission

Question 15

what is the intracellular domain of an RTK responsible for?

Answer 15

enzymic activity; adds phosphate onto tyrosine

Question 16

how do RTKs display ligand induced oligomerisation?

Answer 16

binding of the ligand induces a conformational change, this results in receptor dimerisation, allowing intracellular kinase domains to act on each other

Question 17

how does NGF activate the RTK? what type of ligand is this?

Answer 17

NGF is a bivalent ligand - each component binds to one part of the receptor, bringing them closer together

Question 18

how does EGF activate the RTK? what type of ligand is this?

Answer 18

EGF is a monovalent ligand, it binds to the receptor, initiating a shape change which activates the RTK by activating two adhesive domains

Question 19

how does clustering of RTK activate the kinase?

Answer 19

it triggers information transfer across the plasma membrane

Question 20

how does phosphorylation activate an RTK?

Answer 20

under basal condtions, the substrate binding site is occluded by the regulatory domain, which has a hydroxyl group at one end. the hydroxyl group prevents access to the kinase domain. the phosphorylation opens the substrate binding site, activating the enzyme

Question 21

what property of the regulatory domain allows the kinase to become active?

Answer 21

the dynamic activity, the kinase is activated by autophosphorylation in the split second the regulatory domain flips out

Question 22

how do RTKs cause a cellular response?

Answer 22

via protein recruitment, phosphorylation of intracelular proteins/molecules recruits second messengers to initiate signalling cascades

Question 23

explain how phospholipase C is activated by EGF-receptor tyrosine kinase?

Answer 23

EGF causes dimerisation of the receptor, phosphorylation of multiple intracellular domains activates kinase. PLC catalyses production of IP3. PLC has a binding site for SH2, brings it from the cytosol to the plasma membrane. phosphorylated tyrosines are binding site for recruitment of signalling proteins

Question 24

explain how SH2 domain permits recruitment of functional proteins to a target?

Answer 24

binding of SH2 to protein requires phosphotyrosine plus additional adjacent residues; the SH2 domain is attached to an enzyme or additional binding sequence, permitting recruitment of functional proteins to a target

Question 25

explain how PLC(B) and PLC(y) differ

Answer 25

PLCB is associated with GPCR signalling, it contains catalytic domains which cause PIP2 to split. it interacts with G protein via C2 regions at C-terminus
PLC(y) interacts with RTKs, it has no C2 regions, but many SH2 domains to allow recruitment to the phosphorylated intracellular domain of the RTK

Question 26

how is GPCR signalling terminated?

Answer 26

• there are serine/threonine sites on the C-terminal cytoplasmic tail
• GPCR kinase phosphorylates them, stopping intracellular domains from interacting with G proteins
• this creates binding site for B-arrestin, which brings clathrin
• when assembled, it is invaginated by the cell and degraded by lysosome or recycled
• results in receptor desensitisation

Question 27

how does a ligand gated ion channel respond to ligand?

Answer 27

• in the basal state, there is a kink in the transmembrane region
• this straightens out upon activation to allow influx or efflux of ions