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BIO319 - CELLULAR SIGNALLING > GPCRs, G proteins and second messengers > Flashcards

GPCRs, G proteins and second messengers Flashcards

1
Q

What is the difference between classical and molecular pharmacology?

A

Classical pharmacology is the addition of diff amounts of a drug and seeing what happens
- doesn’t tell you a lot about the mechanism of the drug
Molecular pharmacology is the study of the receptor which is responding to the drug

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2
Q

What are the 4 main sub-types of receptors? Incl e.g. in each

A
  1. Ion channel
    - Acetylcholine receptor
  2. GPCR
    - Beta-adrenergic receptors
  3. Tyrosine Kinase
    - epidermal growth factor receptor
  4. Steroid/ nuclear receptors
    - nuclear receptor subfamily 3 (NR3)
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3
Q

What are G protein -coupled receptors?

A

> a large group of receptors
7 transmembrane helices
interact with a variety of effect molecules
activated by transducer proteins - G proteins (guanine nucleotide binding proteins)

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4
Q

What is an agonist?

A

An agonist is a chemical that binds to a receptor and activates the receptor to produce a biological response

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5
Q

What does the secondary/ tertiary structure of GPCRs look like?

A

Primary seq folds to form 7 hydrophobic transmembrane helices

  • hydrophilic NH2 & COOH
  • barrels up
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6
Q

Describe the GPCR cycle.

A
  1. All subunits are bound together. The alpha subunit has GDP bound to is (inactive state)
  2. A hormone (first messenger) triggers the complex by binding to the receptor
    - a conformational change occurs
    - reveals a site in the alpha subunit that is able to bind to GTP
    - cannot carry both GTP & GDP
  3. Has a high affinity for GTP therefore GTP binds to the area and knocks off GDP
    - the bound GDP was keeping the alpha subunit bound to the beta and gamma
  4. GTP bound alpha subunit dissociates
  5. G-alpha subunit is able to bind to its effector
    - triggers the second messenger
    - second messenger carries on the message
  6. Hydrolysis of GTP to GDP
    - sometimes regulator of G-protein signalling (RGS) speeds this up
  7. G-alpha GDP can then bind back to the GPCR
    CYCLE STARTS OVAAA
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7
Q

What part of the GPCR/ G-protein does most of the signalling and determines activity?

A

Galpha subunit

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8
Q

Name the 4 main families of Galpha subunits and the effector responses they are involved in.

A

Gs alpha subunit:

  • g alpha stimulatory
  • Increase cAMP levels by activating adenylyl cyclase. This converts ATP to AMP then cyclises it.
  • cAMP activates PKA and CREB.

Gi alpha subunit:

  • inhibitory G alpha.
  • Decreases cAMP levels.
  • Opposite of Gs alpha subunit.

Gq/11 alpha subunit:

  • activates beta-type phospholipase C (PLC-β) enzymes.
  • PLC splits PIP2 into IP3 and DAG.
  • DAG activates PKC and IP3 mobilises calcium.

G12/13 alpha subunit:
- triggers Rho activation (Rho is a small monomeric G protein) and other small G proteins.

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9
Q

Name some biological functions of G-alpha subunits.

A
  • responsible for smell & taste (~1000 types of receptors)
  • neurotransmission
  • perception of light
  • chemotaxis
  • exocytosis
  • oncogenesis
  • therefore is a massive drug target, >30% of all drugs target GPCRs due to the large biological functions
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10
Q

What are the differences between the 3 families of GPCR?

A

The GPCR families are divided by ligands. Between families, they are functionally homologous but are not sequentially homologous as they diverged so long ago

  • family A is the biggest
  • family B is usually peptide receptors & there are around 15 of them
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11
Q

What are common structural motifs seen in GPCRs?

A
  • 7 hydrophobic transmembrane regions
  • separated by 3 loops outside (extracellular) and 3 loops inside (intracellular)
  • extracellular N-terminus
  • intracellular C-terminus
  • the size of the N &; C termini &; various loops differ
  • Certain a.a. motifs are similar in the same families but not between families (family A have a D.R.Y a.a. motif)
  • extracellular glycosylation sites and disulphide bonds between loops
  • intracellular phosphorylation and palmitoylation sites
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12
Q

How are you able to compare the motifs of GPCR’s in family A?

A

Using the Ballesteros-Weinstein numbering system

  • choosing the most conserved residue at each predicted transmembrane region
  • this residue is then point zero
  • proline is the most conserved in all of family A and so is labelled 7.5
  • going up/ down from that residue goes up (7.51/ 7.49)
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13
Q

Do most drugs act as antagonists or agonists? Name an agonist drug.

A

Most drugs are antagonists.

Salbutamol = an agonist

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14
Q

Name the key types of receptors within GPCR’s.

A
  • biogenic amine receptors
  • thrombin receptors
  • peptide receptors
  • glycoprotein hormone receptors
  • metabotropic glutamate receptors
  • family B peptide receptors
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15
Q

Explain the ternary complex model of activation for GPCRs.

A

> GPCRs are in an equilibrium, sometimes they are on and sometimes they are off
an agonists job is to increase probability of the GPCR being in the active conformation
the presence of agonist stabilises the active conformation of the GPCR thereby increasing the level of signal occurring inside the cell past a functional threshold

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16
Q

What do RAMPs do?

A

Class of proteins that interact with and modulate the activities of several Class B GPCRs

  • binds to receptor
  • changes preference for the hormone
  • allows it to signal
    e. g. the receptors for secretin and glucagon.
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17
Q

Do agonist and antagonist have to bind in the same site?

A

No. They can have different binding sites.

- importnat to maintain specificity

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18
Q

Where do vasopressin and oxytocin bind?

A
  • interact within the transmembrane domains and extracellular domains of GPCRs (family A)
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19
Q

What is the orthosteric binding site?

A

The same binding site/ normal binding site.

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20
Q

Which family of GPCRs has a venus fly trap effect formed by its disulphide bonds?

A

Family C GPCRs, receptor for glutamate

  • buried in the middle between the TM (transmembrane) bundle and the binding site are loads of disulphide bonds
  • venus fly trap effect formed
  • glutamate then binds causing the G protein to dissociate
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21
Q

Where can an antagonist drug bind to in GPCRs?

A

Both orthosteric and allostertic sites

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22
Q

What structure is seen in most Family B GPCR’s?

A

hook on a stick broad structure
- there is no sequence homology
2 phase process
- the stick binds first, then the hook activates

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23
Q

Can you locate a precise region as to where the G protein will bind to intracelluarly?

A

No. It is hard to define a precise region. Tends to involve membrane proximal regions of C terminus, intracellular loop 2/3.
- also, splice variants may differ in their G protein selectivity and which Galpha subunit they want to bind to

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24
Q

What happens to the affinity for the agonist once Galpha binds back to the receptor?

A

The affinity for the hormone/agonist increases, once Galpha binds
- turning it into an active receptor once more

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25
Q

What happens once the receptor is activated?

A

A conformational change increases the affinity of the receptor for the G protein.
Receptor dimerisation can occur in some receptors:
- e.g. GABA receptors can form dimers, if heterodimer formed, diff affinities and signalling compared to if a homodimer is formed
- some forms of dimerisation has one GPCR and one “something else”
- such as CGRP receptor and adrenomedullin receptors which are GPCR’s and RAMPs

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26
Q

What effect do agonists have on GPCRs with respect to the ternary complex model?

A
  • An agonist increases the probability of GPCRs in a population to be active.
  • Causes 9 out of 10 GPCRs to be active.
27
Q

Can GPCRs be active in the absence of an agonist? Why?

A

Yes, due to ternary complex model. There is always 1 in 10 GPCR which is active randomly.

28
Q

What is seen on the different levels of structure of a GPCR?

A

Secondary:

  • 7 transmembrane helices, N-terminal EC domain, C-terminal IC domain.
  • 3 EC loops, 3 IC loops.

Tertiary:
- Barrel where antagonists and agonists bind.

Quaternary:

  • Sites of glycosylation.
  • Dimerisation.
  • GPCR coupled to a heterotrimeric G protein.
29
Q

Give an example of a GPCR - G protein effector response.

A

Changing heart rate via the beta-1-adrenergic receptor.

- Agonist is adrenaline.

30
Q

Describe the complexity of effector responses.

A

Very complex and tightly regulated.

- There are many downstream signalling pathways which are activated, e.g. MAPK.

31
Q

Why are GPCRs typically targeted for drug design?

A
  • Have a large biological function and involved in many processes.
  • They have a large extracellular part which is easily targeted.
32
Q

How many types of each subunit in a G protein are there?

A

Alpha subunit:

  • There are 4 different types.
  • 16 genes and splice variants for this subunit.

Beta subunit: 5
Gamma subunit: 12

33
Q

What are all the different alpha subunits and what do they activate/inhibit?

A
  • s: activate adenylyl cyclase.
  • i/o/z (inhibitory): inhibit adenylyl cyclase and interact with ion channels.
  • t/g: stimulate cGMP-PDE (cGMP phosphodiesterase).
  • q: activate PLCβ.
  • 12/12: regulate small rho-type G proteins.
34
Q

Name the different structural motifs in a G protein and what they are formed from.

A
  • β-propeller region (β subunit).

- Coiled coil region (gamma subunit).

35
Q

What is the role of the alpha subunit?

A
  • Binds the nucelotide guanine.
  • Has GTPase activity.
  • Interacts with the receptor.
  • Interacts with the effector.
  • Localises the GPCR and G protein to the membrane.
36
Q

How does the alpha subunit allow for membrane localisation?

A

It is myristoylated post-translationally.
- As myristoyl is a 14 carbon lipid chain, it embeds into the hydrophobic tails of the membrane, allowing for membrane localisation.

37
Q

What is CTX and PTX and where are thye found?

A

CTX: site sensitive to cholera toxin.
PTX: site sensitive to pertussis toxin.
- They are found on the primary sequence of the alpha subunit.

38
Q

What is the role of the beta subunit?

A
  • Promotes interaction with the receptor.
  • Allows for membrane localisation.
  • Targets receptor kinases to the membrane.
  • Dampens the signal by interacting with the alpha subunit.
  • Has some role in regulating effectors.
39
Q

How does the beta subunit allow for membrane localisation?

A
  • It is post-translationally modified with lipid chains, e.g. farnesyl and geranylgeranyl.
  • Work in the same way as myristoylation.
40
Q

What effectors are involved with the beta subunit?

A
  • PLCβ2.

- Tyrosine kinases

41
Q

How have G proteins been studied?

A
  • Looking at the effect of toxins cholera toxin and pertussis toxin.
  • Using non-hydrolysable GTP analogues to constitutively switch on the alpha subunit.
  • Using fluoroaluminate ion as a substitute for the last phosphate on GTP to see activation.
  • Using antibodies and molecular biology techniques, e.g. mutagenesis.
42
Q

What was observed in G proteins when using toxins and non-hydrolysable GTP analogues?

A

Cholera toxin:

  • This causes ribosylation of Gs alpha subunit.
  • Causes constitutive activation and signalling of cAMP.

Pertussis toxin:

  • Ribosylation of Gi subunit is caused.
  • Causes uncoupling of the receptor and inactivation of Gi, so the expression can’t be switched off and there is excessive signalling.

GppNHp:

  • This is a GTP which can’t be deactivated.
  • Shows prolonged activation of the G protein, showing the GTPase activity if important.
43
Q

Describe the specificity of the receptor - G protein interaction. Why is this a problem?

A

It forms non-specific interactions.

- This is a problem as it means co-immuno precipitant experiments can’t be done to see what binds.

44
Q

How is the interaction between receptor and G protein investigated?

A
  1. Photolabelling experiments.
    - A GTP analogue is photolabelled and incubated with receptor and G protein.
    - Look at which Galpha subunit lights up.
  2. Antisense mRNA experiments.
    - Use an antisense mRNA to a specific subunit to silence it between 20 and 70%.
45
Q

What was the result of photolabelling experiments?

A
  • It was seen that alpha2 receptor agonists only stimulate inhibitory alpha subunits, as only these light up.
46
Q

What was the result of the antisense mRNA experiments?

A
  • It was concluded that the Ach receptor only works through the alpha01 splice variant in GH3 cells and that only β1 is involved.
47
Q

What was done in the antisense mRNA experiment? Reagents etc

A
  • GH3 cells were used.
  • CCh is an agonist for Ach. CCh was used.
  • > Blocking alpha01 variant blocked CCh response.
48
Q

What did the receptor - G protein interaction experiments allude to?

A

Allude to a role for sub-cellular compartmentalisation.

49
Q

What are G proteins with respect to cell signalling?

A

They are the link between ligand-receptor activation and cell signalling.

50
Q

What is the second messenger?

A

The effect of G proteins.

51
Q

What is the role of a second messenger and how does it achieve this?

A

Changes cellular function via cross-talk between pathways such as:

  • Changing membrane potential through affecting ion channels.
  • Phosphorylating proteins, e.g. tyrosine kinases.
  • Causing gene expression, e.g. steroid receptors.
52
Q

Give some examples of second messengers and their effectors.

A

cAMP:
- Regulates PKA. This phosphorylates serine and threonine residues of proteins involved in the regulation of glycogen metabolism.

Calcium:

  • Regulates calmodulin dependent processes, e.g. smooth muscle contraction.
  • Work by binding 4 calcium = active.

IP3:
- Mobilises calcium.

DAG:

  • Activates PKC.
  • Works in the same way as PKA but affects transcription and learning and memory.

cGMP:

  • Activates PKG.
  • Works in the same way as PKA but affects smooth muscle relaxation.

NO:
- Activates guanylyl cyclase which converts GTP to cGMP.

Arachidonic acid:
- Activates some forms of PKA.

53
Q

What are phosphodiesterases (PDEs) and why are they important in drug design?

A

They are an enzyme which breaks cGMP into GMP.
- They allow for the effect of the agonist to be prolonged as inhibitors of PDEs keep cGMP present, therefore increasing the signalling response.

54
Q

How many different adenylyl cyclase isoforms are there?

A

9 membrane bound (AC1-9), 1 soluble form (sAC)

55
Q

What is the difference between AC isoforms?

A
  • Have different affinities.

- Require different subunits for activation.

56
Q

How is a cell able to have different signalling pathways at the same time due to cAMP production by 2 different receptors?

A

Compartmentalisation.
- There are islands of PDE which breaks down cAMP and therefore keep the signalling by cAMP where it is required.

  • Localisation of PKA via AKAPs (A-kinase anchoring proteins).
57
Q

Name a role of cGMP.

A

In vasodilation.

58
Q

What are the types of guanylyl cyclases? Include examples

A

Membrane bound.
- e.g. ANPa and ANPb receptors.
Soluble.
- e.g. haem containing enzymes activated by NO.

59
Q

How is intracellular calcium controlled?

A
  1. Agonist binds to Galpha q/11.
  2. IP3 and DAG is made by the action of PLC.
  3. IP3 interacts with receptors on the sarcoplasmic reticulum to release calcium.
  4. DAG activates receptor activated calcium channels to cause an influx.
  5. Storage operated calcium channels and voltage operated calcium channels are activated and cause an influx.
60
Q

What are the different isoforms of PLC and what are they regulated by?

A

PLCβ:

  • Regulated by G proteins.
  • PLCβ1 mainly by Gq/11 alpha.
  • PLCβ3 mainly by Gβγ.

PLCγ
- Regulated by tyrosine phosphorylation.

PLCδ
- Thought to be regulated by calcium.

PLCε
- Thought to be regulated by Rho, Rap and Ras.

61
Q

What is cross talk?

A

Interaction between second messengers.

62
Q

What is known about cross talk?

A

Not much.

  • Can occur at a number of levels.
  • May be inhibitory, facilitatory or synergistic.
63
Q

Summarise second messengers.

A
  • They cause cascades and effects.
  • They are usually GPCR mediated.
  • They are cell and time specific.
  • They cross talk.

BIO319 - CELLULAR SIGNALLING (15 decks)

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