Lecture 4 - GPCRs Flashcards
GPCRs: what are they, what percentage of drugs target GPCRs, what is their structure, what do they interact with, and what do they do?
G-protein coupled receptors - The largest class of cell-surface receptors
~30% of therapeutically useful drugs act via GPCRs
Seven TM a-helices
‘heterotrimeric G proteins’ (cytoplasmic surface)
Activated G protein stimulates the next component of the pathway - signal transduction
Heterotrimeric G proteins: how are they related to GCPRs, why are they heterotrimeric, what do each of its subunits do, what are they, and how do they become active?
NOT part of receptor - distinct proteins that associate with it as a lipid anchor attached to the membrane but can move independently
“3 non-identical subunits” (a, b & g )
α - Has GTPase activity, G protein dissociates from it when GTP is bound, bg associates when GDP is bound after a hydrolyses it
β - stay tightly associated with g – bg complex
γ - stay tightly associated with b – bg complex
Guanine nucleotide-binding proteins: bind GDP or GTP - a subunit binds GDP/GTP: GTP bound form is active
GPCR activation: what do they do and is it an example of signal amplification?
GPCR activation causes a conformational change – activated receptor stimulates GDP-GTP exchange (active receptor acts as a GEF)
Yes, one GPCR can activate many G proteins
GPCR deactivation: how does it work, what types of deactivation are there, and what do they do?
Phosphorylation reduces the receptor’s ability to respond even when the ligand is still present
2 types:
* Homologous (only activated receptors are switched off – specialised kinase targets active conformation of GPCR)
- Heterologous (receptors switched off whether activated or not) – kinase activated downstream in the signalling pathway feeds back onto receptors
Heterotrimeric G proteins: what are the types and what do they do?
Gₛ: Gaₛ activates adenylyl cyclase - produces the second messenger cyclic AMP (cAMP)
Gᵢ/ₒ: alpha subunits inhibit some adenylyl cyclases; bg subunits regulate ion channels or certain signalling enzymes
Gq: activates phospholipase Cb - produces two second messengers: inositol 1,4,5 trisphosphate (IP3) and diacylglycerol (DAG)
G₁₂/₁₃: regulates actin cytoskeleton via Rho family small GTPases
Other G-interacting proteins - b-arrestins: what do they do and hoe are they recruited?
Signalling proteins that bind to activated GPCRs - they have several effects:
* Prevents more G proteins from being activated (desensitization)
* Targets the receptor for removal from the membrane (internalization by endocytosis)
* Recruits additional signalling proteins to the receptor – impact on cellular response
* Mediate “homologous desensitization” (inactivation of activated GPCRs)
Recruited following GPCR phosphorylation by ”G Protein Coupled Receptor Kinases” (GRKs) – only phosphorylate the active conformation of the receptor
GRKs: what are they, what do they do, and what do b-arrestins do when bound to receptors?
GRKs associated with membrane (lipid anchors or lipid binding domains)
They are S/T kinases - they only phosphorylate the activated conformation of the GPCR
Phosphorylated receptors specifically bound by b-arrestins:
* Block interaction with G proteins
* Promote GPCR endocytosis
* Recruit signal termination enzymes
* May recruit additional signalling proteins (e.g. components of MAPK cascade, other kinases) – nature of signal changes but signalling can continue, even after endocytosis
GPCR interaction with b-arrestin
Signalling through bg complexes
Signalling through bg complexes: what does it do, does it completely dissociate from the membrane, and what types of G protein are they most important for?
bg complex dissociates after GPCR activation and regulates binding proteins (ion channels and signalling enzymes) by activating specific target proteins (includes Class IB PI3K as well)
It still stays associated with the membrane (lipid anchor)
More important for certain classes of G protein – particularly for Gi/o family
– ion channel signalling
KACh – a cardiac K+ channel:
- Opens in response to cholinergic stimulation (parasympathetic NS – vagus nerve)
- Causes hyperpolarization of cardiomyocyte - reduction in cardiac output
Adding purified bg to isolated membrane patches leads to channel opening: no second messenger generation required
GBG
Voltage-gated Ca2+ channels in nerve cells are inhibited by Gbg; opiates suppress pain-related nerve signals by activating Gi-coupled GPCRs
KACh potassium channel regulation
Class IB PI-3 kinase
Heterodimer: 110kDa catalytic subunit (p110g) and a regulatory subunit (p101 or p84) that binds to bg
Allows activation of powerful PI3-K signalling pathway downstream of GPCRs; important in the immune system
What do different alpha subunits do and what are two major targets of alpha subunits?
Physical interaction of a subunit with target proteins causes a change in protein activity - may be activation (enzyme) / opening (ion channel) or inactivation/closure
Different alpha subunits regulate different targets
Two major targets of G protein alpha subunits:
- Adenylyl cyclase (activated by Gsa-GTP; inhibited by Gia-GTP)
- Phospholipase C beta (activated by Gqa-GTP)
Ga GTPase activity: how speedily does it occur, what interactions enhance them, what happens after the hydrolysis of GTP, what do they do, and what are additionally needed for it to complete its function?
Ga subunits have intrinsic GTPase activity; occurs fairly slowly
Enhanced by interaction with other proteins - GAP functions:
* Targets of Ga-GTP
* RGS proteins (regulator of G Protein Signalling)
After hydrolysis of GTP, the heterotrimer reassembles and signalling ceases
Ensures efficient termination of signalling: impaired signalling can cause over-stimulation of signalling pathways leading to disease
‘Off switches’ are often needed to terminate downstream signalling - phosphatases, enzymes that remove second messengers, etc
Signalling through alpha subunits
Gq G protein signalling: what do they activate, what do they do, and what products are formed?
Gq a-GTP activates the signalling enzyme Phospholipase C b (PLC b) - catalyses the hydrolysis of the membrane lipid PI(4,5)P2
Products are both second messengers: inositol(1,4,5)trisphosphate (IP3) and diacylglycerol (DAG)
IP3: what does it do, where is it located, how does it activate a secondary messenger, and what does the secondary activator do
Opens an intracellular ligand-gated ion channel: IP3 receptor which releases Ca²⁺ into the cytoplasm - signal amplification
In the membrane of intracellular Ca²⁺ stores
[Ca²⁺] is normally low (10-100nM range); increased [Ca²⁺] acts as another second messenger, relaying the signal, binding to many proteins and regulating them
- Gqa-GTP recruits PLCb
- PI(4,5)P2 hydrolysis releases IP3 and DAG
- IP3 binds receptors (ligand-gated ion channels) in the Ca²⁺ store membrane
- Ca²⁺ released into the cytoplasm
- Activation of Ca²⁺-sensitive proteins
Calmodulin: what is it and what does it do?
Low molecular weight, acidic, calcium-binding protein
Mediates the Ca²⁺ regulation of a wide range of physiological processes throughout eukaryotic organisms
The general function of calmodulin/Ca2+ ion
Calcium binding exposes hydrophobic patches on calmodulin, these patches collapse around the amphipathic helix on the target protein (eg CaM kinase).
The binding of Ca²⁺-calmodulin to CaM kinase displaces an inhibitory peptide from the active site; activated CaMK phosphorylates specific protein targets on S/T (amplification)
Calcium oscillation and waves: what is its signal like, how quickly is it moved around, and what happens with local calcium?
Oscillatory - fixed amplitude, variable frequency, these oscillations may merge to form a Ca²⁺ wave
Ca²⁺ released and very rapidly removed from the cytoplasm
Local Ca²⁺ does not remain high for prolonged periods: deleterious if not
IP3 removal
Achieved by sequential dephosphorylation, or by further phosphorylation (followed by sequential dephosphorylation)
Ca²⁺ removal: why is it necessary and what is the process behind it?
Calcium buildup is deleterious
- Na⁺/Ca²⁺ cross-membrane exchange
- Plasma membrane Ca²⁺ ATPase PMCA pump extrusion
- Sarcoendoplasmic Reticulum Calcium ATPase SERCA pump store reuptake
- Binding to buffering proteins
- Mitochondrial uptake
DAG: what is it,
Diacyl glycerol
Hydrophobic: remains in membrane.
Can diffuse within the membrane.
Stimulates several binding proteins, including protein kinase C (PKC; several isoforms; also requires elevated cytoplasmic Ca2+)
Cytoplasmic Ca2+ elevated due to release from internal stores by IP3 – the other branch of the PLC signalling pathway
Protein kinase C signalling
Occurs on cytoplasmic leaflet of plasma membrane: platform for signal organisation
