GPCRs Flashcards
how many transmembrane domains do GPCRs have?
7 a helices
which TM domain sits next to binding pocket of gpcrs?
TM3 centrally located next to binding pocket, crucial for ‘transduction’ of ligand binding
important GPCR structures for binding
TM3
extracellular N terminus
other TMs
when is a GPCR activated
when a ligand is bound
how are GPCR classes distinguished
by structural features of the extracellular domains defining the ‘ligand’ binding site
how are protease-activated receptors activated in platelets
- N terminus is cleaved and acts as ligand to activate itself
- causes activation of platelets -> bind to exposed collagen
what are G proteins
- Guanine nucleotide-binding proteins
- GTPase family
G proteins function and regulation
- molecular switches inside cell to transmit signals from extracellular
stimuli - Regulated by ability to bind and hydrolyse GTP (‘on’) to GDP (‘off’)
G proteins structure
heterotrimeric complexes made up of ⍺, β, and 𝛾 subunits
mechanism of action of GPCRs
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
how are GPCRs controlled
- 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
GiαGPCR function
- a-adrenergic R
- negative feedback in neuronal synapses
- decrease insulin from pancreas
Gqα GPCR function
- a-adrenergic
- smooth muscle contraction
- vasoconstriction
Gsα GPCR function
β-Adrenergic
β1- increase heart rate
β2 - smooth muscle relaxation
Gtα GPCR function
Rhodopsin
Vision
G13a function
platelet activation
Golfa GPCR function
sense of smell
- specific subunit and cell type determines response using same signalling as other cell types
how do effectors work
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
how does direct activation of an ion-channel occur
- Similar mechanism as ligand-gated channels
- Slow to open or close
- Stay open or closed for longer - minutes rather than milliseconds
How do activated G proteins regulate the activities of enzymes that control the levels of second messengers
- 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
example of melanoma link to GPCR mutation
most uveal melanoma have mutations in Gq subunit -> blocking of GTP hydrolysis so subunits are always active -> permanent signal transduction -> growth
what factors contribute to the diversity of receptors
- Specificity of the ligand-binding domain
- the G protein α subunit
- the effector within a particular cell type
how come organisms can respond to different environmental stimuli
GPCR diversity
how are the effect of a ‘stimulus’ on cell function determined
by the receptor and signalling molecules expressed in that cell
advantage of determining effect of stimulus on cell function using GPCR knowledge
Allows us to specifically target certain receptors for therapeutic gain
advantage of determining effect of stimulus on cell function using GPCR knowledge
Allows us to specifically target certain receptors for therapeutic gain
what are effectors and examples
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
what are second messengers
⇒ small molecules that carry signals inside cells
Activated G proteins regulate the activities of enzymes that control the levels of second messengers
cAMP second messenger system
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
isoforms
forms of a protein produced by different genes/ same gene but alternative splicing
β2 Adrenoceptor regulation of metabolism in liver and skeletal muscle - how does it work?
Binding of single Epinephrine molecule to a receptor sets off a signalling cascade resulting in the phosphorylation/activation of enzymes controlling glycogen metabolism
4 ways to switch off GPCR signalling
- Agonist dissociating from receptor
- GTPase activity of Gαs
- cAMP breakdown by phosphodiesterase
- Dephosphorylation of enzymes
difference between cAMP and cGMP second messenger signalling
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)
how are second messengers encoded
conc
fequency of changes in conc
what determines local conc of 2nd messengers
- production
- diffusion
- removal
- site of production
what is production of cAMP regulated by
adenylyl cyclase
what is breakdown of cAMP regulated by
phosphodiesterase
how can effector ion channels be regulated
directly (βγ subunits) or indirectly by 2nd messengers and their effectors
phospholipase CB function
→ cleaves lipids in membrane
2nd messengers generated by receptor regulated lipase
- water soluble and diffuse through cytoplasm e.g IP3 → IP3 receptor (Ca channel)
- hydrophobic molecules that remain in membrane e.g DAG → PKC
IP3 main functions
main functions are to mobilize Ca2+ from storage organelles and to regulate cell proliferation and other cellular reactions that require free calcium
lipid kinases function
add phosphate groups to lipids
purpose of having different isoforms of PLC and PLK
- activated by differnt molecules
- expressed in different tissues
- difference in regulatory domains
protein kinase C structural/functional features
- 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
where on PKC does alpha unit bind
carboxyl group
where do b/g subunits bind
pH unit
PKC activation via DAG
DAG binding → dissociation of intramolecular pseudosubstrate domain from active site = opens binding site for target protein
