Lecture 4 - GPCRs

Question 1

GPCRs: what are they, what percentage of drugs target GPCRs, what is their structure, what do they interact with, and what do they do?

Answer 1

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

Question 2

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?

Answer 2

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

Question 3

GPCR activation: what do they do and is it an example of signal amplification?

Answer 3

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

Question 4

GPCR deactivation: how does it work, what types of deactivation are there, and what do they do?

Answer 4

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

Question 5

Heterotrimeric G proteins: what are the types and what do they do?

Answer 5

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

Question 6

Other G-interacting proteins - b-arrestins: what do they do and hoe are they recruited?

Answer 6

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

Question 7

GRKs: what are they, what do they do, and what do b-arrestins do when bound to receptors?

Answer 7

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

Question 8

GPCR interaction with b-arrestin

Question 9

Signalling through bg complexes

Question 10

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?

Answer 10

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

Question 11

– ion channel signalling

Answer 11

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

Question 12

GBG

Answer 12

Voltage-gated Ca2+ channels in nerve cells are inhibited by Gbg; opiates suppress pain-related nerve signals by activating Gi-coupled GPCRs

Question 13

KACh potassium channel regulation

Question 14

Class IB PI-3 kinase

Answer 14

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

Question 15

What do different alpha subunits do and what are two major targets of alpha subunits?

Answer 15

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)

Question 16

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?

Answer 16

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

Question 17

Signalling through alpha subunits

Question 18

Gq G protein signalling: what do they activate, what do they do, and what products are formed?

Answer 18

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)

Question 19

IP3: what does it do, where is it located, how does it activate a secondary messenger, and what does the secondary activator do

Answer 19

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

Answer 20

• 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

Question 21

Calmodulin: what is it and what does it do?

Answer 21

Low molecular weight, acidic, calcium-binding protein

Mediates the Ca²⁺ regulation of a wide range of physiological processes throughout eukaryotic organisms

Question 22

The general function of calmodulin/Ca2+ ion

Answer 22

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)

Question 23

Calcium oscillation and waves: what is its signal like, how quickly is it moved around, and what happens with local calcium?

Answer 23

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

Question 24

IP3 removal

Answer 24

Achieved by sequential dephosphorylation, or by further phosphorylation (followed by sequential dephosphorylation)

Question 25

Ca²⁺ removal: why is it necessary and what is the process behind it?

Answer 25

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

Question 26

DAG: what is it,

Answer 26

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

Question 27

Protein kinase C signalling

Answer 27

Occurs on cytoplasmic leaflet of plasma membrane: platform for signal organisation