G protein-coupled receptors

Question 1

What are G Protein-Coupled Receptors (GPCRs)

Answer 1

• Largest class of human cell surface receptors.
• Involved in smell, taste, vision, hormone detection.
• Bind a huge array of molecules.
• Characterised by 7 transmembrane domains
• The N-terminus of a GPCR is always on the outside of a cell

Question 2

List some drugs that target GPCR’s

Answer 2

Question 3

How many pharmaceuticals drugs target GPCRs?

Answer 3

50% of pharmaceuticals

Question 4

How many of the top selling drugs target GPCRs?

Answer 4

25% of top selling drugs

Question 5

Name some GPCR ligands

Answer 5

• Light (photons).
• Taste and smell molecules.
• Pheromones.
• Histamines (allergies).
• Hormones e.g. oxytocin, adrenaline.
• Any many many more!

Question 6

Describe GPCR taxonomy

Answer 6

•Schiöth and Fredricksson (2003, 2005): Performed a phylogenetic analysis of the human genome and divided the GPCR super-family into 5 distinct families:

1. Glutamate-like GPCRs
2. Rhodopsin-like GPCRs
3. Adhesion-like GPCRs
4. Frizzled-like GPCRs
5. Secretin-like GPCRs

Question 7

What is the other way of naming GPCR families?

Answer 7

• Family A (Rhodopsin-like): Ligands ranging from light, small molecules, peptides to proteins
• Family B (Secretin-like): Peptide hormones characterised by large extracellular domain and disulfide bonds
• Family C (Glutamate-like): Bind to ions and small molecules. This family are obligate dimers that have two receptors on the outside joined by disulfide bonds
• Adhesion family: Ligands are mostly unknown but they undergo proteolytic cleavage
• Frizzled family: Bind Wnt proteins

Question 8

What is the full name for G-Proteins?

Answer 8

•Guanine nucleotide binding proteins e.g. GTP (analogous to ATP) and GDP (analogous to ADP).

Question 9

TRUE or FALSE: GPCRs signal via heterotrimeric G proteins (3 different subunits).

Answer 9

TRUE

• α – 16 types, β – 5 types, γ – 12 types
• α subunit binds guanine nucleotides
• β and γ form an obligate heterodimer

Question 10

Describe the mode of action of G-Proteins

Answer 10

• G proteins act as molecular switches.
• In the inactive state they are bound to GDP.
• Activation of the G protein results in release of GDP.
• This GDP is then replaced by GTP producing an active G protein.

Question 11

Explain G-Protein activation

Answer 11

1. Ligand binds to the extracellular side.
2. Changes the conformation of the receptor.
3. This recruits the G protein.
4. At this point the G protein is inactive (GDP-bound).
5. Binding to the receptor induces GDP release; GDP is replaced by GTP.
6. GTP-bound Gα separates from the GPCR and Gβγ.
7. Gα goes on to activate signalling cascades.

An active GPCR can activate many G proteins (signal amplification)

Question 13

What are the function of GAPs?

Answer 13

They are GTPase activating proteins (GAPs).

• Enhance (increase) the GTPase activity of the G protein (hydrolyses GTP to DGP).
• Cause inactivation.
• E.g. Regulator of G protein Signalling (RGS) proteins.

Question 14

What are the funtion of GEFs?

Answer 14

•Guanine nucleotide exchange factors (GEFs).

–Accelerate the dissociation of GDP from the G protein, allowing it to be replaced by GTP.

–Enhance G-protein activation.

–E.g. Accelerator of G protein Signalling (AGS) proteins.

Question 15

What is the function of Chlorea toxin?

Answer 15

It inactivates GTPase and stops the G-protein becoming inactive (signalling is always on)

Question 16

What is the function of pertussis toxin?

Answer 16

It locks the G-Protein in the inactive state (signalling is always off)

Question 18

G-Protein taxonomy: There are 4 families are based on sequence homology

List the 4 families

Answer 18

–G_s →stimulates adenylate cyclase.

–G_i/o →inhibits adenylate cyclase.

–G_q/11 →stimulates PLCβ.

–G_12/13 →activates RhoGEFs.

Question 19

TRUE or FALSE: Gβγ cannot signal

Answer 19

FALSE

Gβγ can also signal

Question 20

What is Adenylate cyclase?

Answer 20

•Membrane bound protein (doesn’t span the membrane).

• Stimulated by Gs
• Inhibitated by Gi
• Acts as an enzyme.
• Converts ATP to cAMP (cyclic AMP).

cAMP is a second messenger

Question 21

What is the function of cAMP?

Answer 21

•Activates protein kinase A (PKA).

–Phosphorylates a whole range of downstream targets.

• Also activates cyclic nucleotide-gated ion channels and exchange proteins activated by cAMP (EPACs).
• Broken down by cAMP phosphodiesterases.

Linked to vasodilation and also colour blindness

Question 22

What is protein kinase A?

Answer 22

•Holoenzyme that contains 4 subunits (2 regulatory and 2 catalytic).

The regulatory domains block the active sites of the catalytic domains

Question 23

How is protein kinase is affected by a low concentration of cAMP?

Answer 23

The 4 subunits remain together.

Question 24

How is protein kinase is affected by a high concentration of cAMP?

Answer 24

4 AMP molecules bind to the regulatory subunits and causes a conformational change which results in the release of the catalytic subunits.

Question 25

Describe Protein Kinase A activity

Answer 25

•Phosphorylates target proteins.

–Increases lipolysis (by activating lipases)

–Reduces glycogen synthesis (by inactivating glycogen synthase).

–Increases glycogen breakdown (a cascade reaction beginning with phosphorylase kinase activation).

Question 26

Describe transcriptional regulation by protein kinase A

Answer 26

• Also regulates transcription via cAMP response element-binding protein (CREB).
• cAMP and CREB mediated transcriptional responses.
• Much slower effects than direct modulation of protein activity.

Question 27

Describe the properties of Phospholipase C (Gq)

Answer 27

• This family of proteins cleave phospholipids

PLC family contains 13 enzymes divided into 6 subfamilies.

•PLCβ is essentially only activated by Gq.

Question 28

What is the function of PLCβ?

Answer 28

PLCβ is able to cleave a phospholipid called phosphatidylinositol 4,5-bisphosphate (PIP2).

Question 29

Waht does cleavage of PIP₂ by PLCβ produce?

Answer 29

–Produces diacyl glycerol (DAG) which remains in the membrane

–Produces 1,4,5-trisphosphate (IP₃) which is soluble and goes into the cytosol of the cell.

Question 30

Draw a diagram to show the relationship between PIP₂, PLCβ DAG and IP₃

Answer 30

Question 31

What is the functional role of IP_3?

Answer 31

• Diffuses to the ER and activates IP₃ receptors.
• IP₃ opens Ligand-gated Ca²⁺ channels.
• DAG and Ca²⁺ activate protein kinase C (PKC).
• Acts like PKA to phosphorylate target proteins.

e.g. receptors for desensitisation, transcription factors etc.

Question 32

What are the four different ligands for the CRH receptors?

Answer 32

• CRH
• Urocortin 1
• Urocortin 2
• Urocortin 3

Question 33

Which tyoe of GPCR does CRH bind to?

Answer 33

Family B (peptide hormone) GPCRs

Charcterised by large extracellular N-terminal domains that help to bind the ligand

Question 34

Give an example of receptors that bind multiple ligands

Answer 34

Question 35

Affinity is generated by key residues that bind strongly to a molecule. How does affinity vary among ligands?

Answer 35

Although ligands have similar sequences, they can bind to specific residues (with varying affinities) within the protein and activate it in different ways