GPCRs

Question 1

how many transmembrane domains do GPCRs have?

Answer 1

7 a helices

Question 2

which TM domain sits next to binding pocket of gpcrs?

Answer 2

TM3 centrally located next to binding pocket, crucial for ‘transduction’ of ligand binding

Question 3

important GPCR structures for binding

Answer 3

TM3
extracellular N terminus
other TMs

Question 4

when is a GPCR activated

Answer 4

when a ligand is bound

Question 5

how are GPCR classes distinguished

Answer 5

by structural features of the extracellular domains defining the ‘ligand’ binding site

Question 6

how are protease-activated receptors activated in platelets

Answer 6

• N terminus is cleaved and acts as ligand to activate itself
• causes activation of platelets -> bind to exposed collagen

Question 7

what are G proteins

Answer 7

• Guanine nucleotide-binding proteins
• GTPase family

Question 8

G proteins function and regulation

Answer 8

• molecular switches inside cell to transmit signals from extracellular
  stimuli
• Regulated by ability to bind and hydrolyse GTP (‘on’) to GDP (‘off’)

Question 9

G proteins structure

Answer 9

heterotrimeric complexes made up of ⍺, β, and 𝛾 subunits

Question 10

mechanism of action of GPCRs

Answer 10

Inactive state – GDP bound to the alpha subunit
1. Ligand binding = conformational change in receptor that activates G-protein
2. GDP released and ⍺ subunit separates from other subunits and binds GTP – now active
3. Binds to target protein in membrane to elicit a response within the cell

Question 11

how are GPCRs controlled

Answer 11

• act as timers
• Duration of signalling by activated trimeric G protein is regulated by rate of GDP hydrolysis by Gα
• RGS proteins stimulate GTPase activity in the ⍺ subunit

Question 12

GiαGPCR function

Answer 12

• a-adrenergic R
• negative feedback in neuronal synapses
• decrease insulin from pancreas

Question 13

Gqα GPCR function

Answer 13

• a-adrenergic
• smooth muscle contraction
• vasoconstriction

Question 14

Gsα GPCR function

Answer 14

β-Adrenergic
β1- increase heart rate
β2 - smooth muscle relaxation

Question 15

Gtα GPCR function

Answer 15

Rhodopsin
Vision

Question 16

G13a function

Answer 16

platelet activation

Question 17

Golfa GPCR function

Answer 17

sense of smell
- specific subunit and cell type determines response using same signalling as other cell types

Question 18

how do effectors work

Answer 18

include enzymes that create 2nd messengers and ion channels whose gating is regulated either directly (β𝛾 subunits) or indirectly by 2nd messengers and their effectors

Question 19

how does direct activation of an ion-channel occur

Answer 19

• Similar mechanism as ligand-gated channels
• Slow to open or close
• Stay open or closed for longer - minutes rather than milliseconds

Question 20

How do activated G proteins regulate the activities of enzymes that control the levels of second messengers

Answer 20

• Second messengers are small molecules that carry signals inside cells
• e.g Hydrophobic lipids confined to the membrane in which they are generated
• Small soluble molecules that diffuse through the cytoplasm (cAMP, cGMP)
• Calcium ions

Question 21

example of melanoma link to GPCR mutation

Answer 21

most uveal melanoma have mutations in Gq subunit -> blocking of GTP hydrolysis so subunits are always active -> permanent signal transduction -> growth

Question 22

what factors contribute to the diversity of receptors

Answer 22

• Specificity of the ligand-binding domain
• the G protein α subunit
• the effector within a particular cell type

Question 23

how come organisms can respond to different environmental stimuli

Answer 23

GPCR diversity

Question 24

how are the effect of a ‘stimulus’ on cell function determined

Answer 24

by the receptor and signalling molecules expressed in that cell

Question 25

advantage of determining effect of stimulus on cell function using GPCR knowledge

Answer 25

Allows us to specifically target certain receptors for therapeutic gain

Question 26

advantage of determining effect of stimulus on cell function using GPCR knowledge

Answer 26

Allows us to specifically target certain receptors for therapeutic gain

Question 27

what are effectors and examples

Answer 27

Effectors of trimeric G proteins include enzymes that create 2nd messengers and ion channels whose gating is regulated either directly (βγ subunits) or indirectly by 2nd messengers and their effectors

Question 28

what are second messengers

Answer 28

⇒ small molecules that carry signals inside cells Activated G proteins regulate the activities of enzymes that control the levels of second messengers

Question 29

cAMP second messenger system

Answer 29

10 isoforms of receptor - Activated by G⍺s, inhibited by G⍺i 1. Ligand binds to receptor activating G protein 2. α subunit moves and binds to adenylate cyclase in the membrane 3. activated enzyme catalyses formation of a cAMP from ATP 4. cAMP (2nd messenger) activates Protein kinase A 5. PKA phosphorylates/activates protein 6. Initiates a response within the cell

Question 30

isoforms

Answer 30

forms of a protein produced by different genes/ same gene but alternative splicing

Question 31

β2 Adrenoceptor regulation of metabolism in liver and skeletal muscle - how does it work?

Answer 31

Binding of single Epinephrine molecule to a receptor sets off a signalling cascade resulting in the phosphorylation/activation of enzymes controlling glycogen metabolism

Question 32

4 ways to switch off GPCR signalling

Answer 32

1. Agonist dissociating from receptor 2. GTPase activity of Gαs 3. cAMP breakdown by phosphodiesterase 4. Dephosphorylation of enzymes

Question 33

difference between cAMP and cGMP second messenger signalling

Answer 33

in cGMP - Enzyme is guanylate cyclase which can be receptor bound or ‘free’ in the cytoplasm - Converts GTP to 3’, 5’-cyclic guanosine monophosphate (cGMP)

Question 34

how are second messengers encoded

Answer 34

conc fequency of changes in conc

Question 35

what determines local conc of 2nd messengers

Answer 35

- production - diffusion - removal - site of production

Question 36

what is production of cAMP regulated by

Answer 36

adenylyl cyclase

Question 37

what is breakdown of cAMP regulated by

Answer 37

phosphodiesterase

Question 38

how can effector ion channels be regulated

Answer 38

directly (βγ subunits) or indirectly by 2nd messengers and their effectors

Question 39

phospholipase CB function

Answer 39

→ cleaves lipids in membrane

Question 40

2nd messengers generated by receptor regulated lipase

Answer 40

1. water soluble and diffuse through cytoplasm e.g IP3 → IP3 receptor (Ca channel) 2. hydrophobic molecules that remain in membrane e.g DAG → PKC

Question 41

IP3 main functions

Answer 41

main functions are to mobilize Ca2+ from storage organelles and to regulate cell proliferation and other cellular reactions that require free calcium

Question 42

lipid kinases function

Answer 42

add phosphate groups to lipids

Question 43

purpose of having different isoforms of PLC and PLK

Answer 43

- activated by differnt molecules - expressed in different tissues - difference in regulatory domains

Question 44

protein kinase C structural/functional features

Answer 44

- PKCs (protein kinase C) are Ser/Thr kinases - activated by DAG (C1 domain), and Ca2+ (C2 domain) - allows binding to lipids in membrane - carboxyl group -> alpha unit binds

Question 45

where on PKC does alpha unit bind

Answer 45

carboxyl group

Question 46

where do b/g subunits bind

Answer 46

pH unit

Question 47

PKC activation via DAG

Answer 47

DAG binding → dissociation of intramolecular pseudosubstrate domain from active site = opens binding site for target protein