PCOL3022 Lectures 4 & 5: GPCRs

The structure of GPCRs (number of transmembrane domains, residues, terminals)

- 7 transmembrane domains / anti-clockwise alpha-helices
- 25-35 hydrophobic residues
- N-terminal faces the extracellular side
- C-terminal faces the cytoplasmic side

How many transmembrane domains are there in GPCRs?

7

How many residues are there in GPCRs?

25-35 hydrophobic residues

Functions of GPCRs

- Sensing extracellular signals (sensory such as light, taste, odor, etc)
- Nearly all NTs interact with GPCRs (information is converted into 2nd messenger metabolite in the intracellular side)
- They are major drug targets (Involved in more than 60% of drugs)
- Activation of messenger pathways (Binding of ligands triggers cascade complex to produce biological effects and physiological responses to stimuli)
- Protein phosphorylation (Ligand binding causes conformational shifts => GRK adds phosphate groups to proteins which alters function)

Identify the classes of GPCRs

Glutamate
Rhodopsin
Adhesion
Fizzled/taste 2
Secretion

Function of GluR

- Restricted to small molecules (GABA)

Structure of GluR Class

- Venus fly trap domain
- The N-terminus of the Glutamate receptor forms two lobes separated by a cavity (Venus fly trap domain)
- Function as dimers (covalently-linked via Cys in membrane)

Describe how the ligand activates GluR

- Glutamate binds in the cavity
- The lobes will trap the ligand like a Venus flytrap

Function of Rhodopsin Class

- Interaction with light causes cis/trans isomerism in ligand
- This activates ligand

- This GPCR protein is most abundant in rod cells (eyes)

Structure of Rhodopsin Class

- Ligand site is deep within the extracellular mouth
- Mouth then "closes" around ligand

Describe how the ligand activates Rhodopsin proteins

- After the ligand binds deep inside the extracellular mouth, the pore will close
- This traps ligand in high concentration of wet volume
- This increases receptor affinity to the ligand

What are the exceptions in the Rhodopsin class?

- Growth hormones: They are large peptide receptors that have an extracellular domain that binds peptides (i.e. glycoprotein hormones)

- Protein-activated receptors (PARS): Requires hydrolysis via protease for the ligand to bind and cause activation
(Thrombin binds to N-terminal where the sequence of amino acids don't signal --> Thrombin hydrolyses in the N-terminal and creates a ligand, PAR+ for the receptor)

Describe the signalling mechanism of GPCRs

- The g protein is made up of three subunits -- alpha (α), beta (β) and gamma (γ)

- At resting state, these subunits are covalently bonded

- Once the ligand binds and activates the GPCR, GPD is replaced with GTP at intracellular sites

- Alpha subunit separates from the beta and gamma subunit to signal at the membrane

- Beta and gamma subunit also diffuses away to signal via the membrane

- Hydrolysis (via catalytic α-subunit): Enzyme hydrolyses GTP to GDP and all subunits for re-assembly of the complex

G-Protein α Subunit Families

- Each GPCR has selectivity for an α-subunit type
- α family determines the type of signal

s (Stimulatory) - Increases cAMP formation

i/o (Inhibitory) - Decreases cAMP formation, activate or inhibit ion channels

q (Usually excitatory) - Stimulates phospholipase C then protein kinase C, and mobilises Ca2+

Gi coupled GPCRs influence GIRK and VGIC

- Major transducers: GIRKs, VGICs

- GIRK: Beta and gamma binds to GIRK => more K+ flow out => inhibits action potential

- Ca+ VGIC: Beta and gamma interacts with Ca+ VGICs => reduce Ca2+ influx => inhibit NTs release

- G alpha i: Inhibits adenyl cyclase from producing cAMP => reduces cAMP => inhibits second messenger cascade

G-protein βγ Subunit Signalling

- Acts in membrane
- Includes modulating adenyl cyclase (cAMP formation)
- Inhibits N-type Ca2+ channels (inhibits NT release)
- Activates phospholipase C (many processes)
- Activates GIRK K+ channels (inhibits action potential activity)
- Activates PI3 kinase (many processes)

Are G-α-q Coupled GPCRs excitatory or inhibitory?

Excitatory

G-α-q GPCR mechanism

- Gq activates phospholipase C + inositol triphosphate activation from membrane lipids
- Leads to the release of Ca2+ (stimulatory) from intracellular stores

G-α-S function/mechanism

- Activates adenyl cyclase
- Increases cAMP cascade

G-α-i function/mechanism

- Inhibits adenyl cyclase
- Reduces cAMP cascade

3 ways of GPCR signal amplification

1. Increased ligand-receptor affinity
2. Increased receptor numbers
3. Increase efficiency of transduction (GPCRs pretty efficient already)

What happens with increased ligand affinity?

- Improves signal detection
- Results in slow dissociation

- Therefore, high speed signalling requires low affinity ligands
- The workaround for this low affinity is signal amplification

GPCR activity is [...] compared to LGICs

Slower

- GPCR modulation is slower compared to inotropic receptors
- Mediates fast synaptic transmission

What is intrinsic efficacy?

- The ability of a receptor and drug to produce a maximal response

- Some drugs have high intrinsic efficacy than others (e.g. methadone > morphine > buprenorphine)
- Buprenorphine has a lower max response because its intrinsic efficacy is so low

What will higher intrinsic efficacy produce?

- Produce a greater % receptor response
- Methadone has the greatest intrinsic efficacy compared to morphine (e.g. methadone > morphine > buprenorphine)

What does "Effective number of receptors" mean?

- How many receptors are able to be activated

Effects of Regulations of GPCRs

- Reduces the number of receptors
- Therefore, reduces the signalling efficacy

Process of Regulation of GPCRs

1. βγ subunits dissociate
2. GPCR is phosphorylated by GRK (a kinase)
3. Now a substrate for β-arrestin
4. β-arrestin drags the receptor to clathrin-coated pits which eventually become vesicles that ares sent to the endosome
5. From the endosome, the receptor may:
(a) eventually return to the membrane;
(b) be hydrolysed;
(c) may signal again from endosome

- This is an intrinsic feedback process

Signalling can also occur in the [...] for GPCRs

- Some receptors can still signal from within the endosome
- Surface signal usually dominates

Regulation of GPCRs lead to...

- Desensitisation and tolerance in the receptor cells
- Decreased responsiveness (instrinsic feedback)

Which is long term/short term: Desensitisation, tolerance

- Tolerance is long term
- Desensitisation can happen after one dose

T/F: Once a GPCR has undergone endocytosis, it cannot be reused

False. GPCRs can be recycled

GPCRs structure and selectivity

- Closely related GPCRs have highly conserved structures, especially within binding pockets
- But these receptors can have vastly different physiological effects

(e.g. the adrenoreceptors)

(e.g. D2/D3 receptors:
D2/D3 antagonists are antipsychotics;
D2 mediates worst side effects;
Receptors are very similar and no selective antagonists exist yet;
Selective antagonists could probably be developed)

A problem with GPCRs as drug targets

- Hard to discriminate pharmacologically
- Binding pockets don't always differ too much (such as D2 vs. D3 receptor)

Selectivity from Signal Bias

- E.g. opioid receptors
- Only one receptor is involved (μOR)
- Different agonists have different combinations of G-protein and arrestin signalling
- Structural bias not yet understood
- Established in many GPCRs

Differences in downstream signalling lead to [...] bias

Signal bias

What does 'biased signalling' mean?

- Different agonists induce differing conformations and initiate different signalling cascades

Comparison example of biased signalling

- Endomorphin-2 produces moderate G-protein signal and strong arrestin signal
- Methadone produces strong G-protein signal and strong arrestin signal

Orthosteric agonism

- Agonist binds in endogenous ligand's binding pocket

Allosteric agonism

- Ligand binds in alternative area ("allo" means "other")
- Can be positive, neutral, or negative

Why is selectivity high for allosteric modulators of GPCRs?

- The binding site is remote from the orthostatic site
- There is more divergence in allosteric binding sites, meaning the greater potential for selective PAMs that can be used therapeutically
- E.g. Ca-sensing receptor PAM (positive allosteric modulators) -- increases agonist affinity and/or efficacy -- helps increase the sensitivity of receptor -- treats hyperthyroidism

[...] will produce signalling which can be increased by [...]

- Orthosteric agonism will produce signalling which can be increased by positive allosteric modulator (PAM)

- A negative allosteric modulator (NAM) will decreased GPCR signalling

Are greatest structural constraints for drug manufacture at the orthosteric or allosteric site?

Orthosteric

Selectivity from Dimerisation

- Most GPCRs exist as dimers
- Can possibly develop selective drugs from certain heterodimer combinations
- Unclear if these heterodimers do exist in vivo
- E.g. GABAB receptor requires both GABA1 and GABA2.
GABA1 is responsible for binding GABA,
While GABA2 is responsible for transduction of signal

DREADDs

Designer Receptor Exclusively Activated by Designer Drugs

What are DREADDs?

- Proteins manipulated to react specifically with small molecules which act as chemical actuators, but which were not previously recognized by these proteins

Example of DREADDs

- Created mutant MR (mutated at TM3, TM5)
- Doesn't respond to MR agonists
- Responds to novel agonist CNO, which doesn't act on any other GPCRs

Optogenetics example of DREADDs

Channelrhodopsin (ChR2) activates in response to light