L6: Structure and Function of GPCRs

Question 1

What is the structure of GPCRs?

Answer 1

• 7 transmembrane domains - helices of hydropathic amino acids - cross the phospholipid plasma membrane in a barrel-like conformation
• extracellular N-terminal domain - glycosylation sites
• 4 extracellular loops - some maintained by disulphide (Cys:Cys) links
• 4 intracellular loops - G protein interaction
• intracellular C-terminal domain - G protein interaction and regulatory phosphorylation sites

Question 2

How many transmembrane domains do GPCRs have?

Answer 2

7 transmembrane domains - helices of hydropathic amino acids - cross the phospholipid plasma membrane in a barrel-like conformation

Question 3

Which structure of GPCRs do G proteins interact with?

Answer 3

• 4 intracellular loops - G protein interaction
• intracellular C-terminal domain - G protein interaction and regulatory phosphorylation sites

Question 4

What are the classes of GPCRs?

Answer 4

• class A: rhodopsin like (light detector in the eye)
• class B: secretin R-like (secretin/glucagon receptor family)
• class C: GABAbR / mGluR-like (metabotropic glutamate receptor/calcium sensor family)
• Frizzled Rs (Wnt ligand)

Question 5

What are the examples of class A GPCRs?

Answer 5

• visual pigments (rhodopsin)
• neurotransmitter receptors
• peptide receptors
• glycoprotein hormone receptors (LH and FSH)
• protease-activated receptors (thrombin receptors)

Question 6

What are the structures that define class A GPCRs?

Answer 6

• short extracellular N-terminal tail, ligand binds between TM helices (amines) or to extracellular loops
• several strongly conserved motifs (DRY motif under TM3)
• often palmitoylated in proximal C-terminal (attachment of a fatty acid to Cys) tail

Question 7

What are the ligands of class B GPCRs?

Answer 7

• calcitonin
• corticotropin-releasing factor (CRF)
• glucagon
• parathyroid hormone (PTH)
• pituitary adenylate cyclase-activating peptide (PACAP)

all 25-30 amino acids long

Question 8

What are the structures that define class B GPCRs?

Answer 8

extended extracellular N-terminal tail, which contributes to binding of ligands (peptide hormones)

Question 9

What are the receptors of class C GPCRs?

Answer 9

• metabotropic glutamate (mGlu) receptor
• gamma-aminobutyric acid type B (GABAb) receptor
• calcium-sensing receptor

Question 10

What are the structures that define class C GPCRs?

Answer 10

Very large extracellular N-terminal tail, fully responsible for ligand binding (“Venus Fly-Trap Domain”)

Question 11

Where does the ligand bind in the beta-2-adrenergic receptor in GPCRs?

Answer 11

Agonist binds between several TM domains with specific residues in TM III, V and VI

Question 12

Where does the ligand bind in the NK1-R in GPCRs?

Answer 12

Substance P binds to extracellular regions and the top of TM domains.

Question 13

Which structures of GPCRs are well defined and which are not?

Answer 13

Only helical TM domains are defined, not the structure of extended extracellular or intracellular regions

Question 14

When does computer-modelling assessment of chemical structure of GPCRs work well?

Answer 14

Only works well with small drugs, where the ligand binding site is very well defined and discrete

Question 15

What are the conformational changes in GPCRs upon activation? example of light-mediated activation of rhodopsin

Answer 15

• unusual because it has a captive ligand (retinal) bound to TM VII Lys296 of rhodopsin
• light causes cis to trans isomerisation of retinal and therefore conformational change in rhodopsin structure (retinal sticks out)
• isomerisation of retinal moves TM V and twists TM VI

Question 16

What are the conformational changes in agonist-activated beta-2-adrenergic receptor upon its activation?

Answer 16

When agonist binds, it induces separation of inner ends of TM V and TM VI to allow G protein access. Major conformational changes induced in alpha helical domain of Gs alpha-subunit, access enabled.

Question 17

Explain G protein activation cycle

Answer 17

(ADD PICTURE from L6, slide 16)
1) Resting (GDP-bound) state
2) Ligand Binding and Nucleotide exchange. Activates alpha-G
3) GTP binds instead of GDP which cause alpha-G dissociation
4) active (GTP-bound state)
5) Activation of GTPase
6) GTP hydrolysis, degradation
7) Ligand-dissociation and G-protein Trimer formation
8) back to resting state

Question 18

What are the properties of G protein alpha subunit?

Answer 18

• GTP/GDP-binding subunit with intrinsic GTPase activity
• principal role in intracellular signalling
• 4 main families

Question 19

What are the 4 main families of G protein alpha subunit and their function?

Answer 19

• G-alpha-s - activates adenylyl cyclase to produce 2nd messenger cAMP
• G-alpha-i / G-alpha-o - inhibit adenylyl cyclase
• G-alpha-q / G-alpha-11 - activate phospholipase C to produce dual 2nd messengers IP3 (leads to Ca2+ mobilisation) and DAG
• G-alpha-12 / G-alpha-13 - activate small G protein Rho to cause cytoskeletal changes

Question 20

What is the role of G-alpha-s G protein family?

Answer 20

activates adenylyl cyclase to produce 2nd messenger cAMP

Question 21

What is the role of G-alpha-i / G-alpha-o G protein family?

Answer 21

inhibit adenylyl cyclase

Question 22

What is the role of G-alpha-q / G-alpha-11 G protein family?

Answer 22

activate phospholipase C to produce dual 2nd messengers IP3 (leads to Ca2+ mobilisation) and DAG

Question 23

What is the role of G-alpha-12 / G-alpha-13 G protein family?

Answer 23

activate small G protein Rho to cause cytoskeletal changes

Question 24

What are the properties of G protein beta and gamma subunit?

Answer 24

• have signalling roles, eg: various beta subunits regulate K+ channels and some isoforms of AC and PLC
• specific roles are generally poorly understood

Question 25

How are **GPCRs** switched off?

Answer 25

(ADD PICTURE from L6, slide 19) ADD MORE INFO Process of switching off GPCR is **homologous desensitisation** by the co-ordinated action of **GRKs** (G Protein-Coupled Receptor Kinases) and **arrestins** (arrest signalling). **GRK** phopshorylates **serines** and **threonines** in the tail of receptor, provides **strong binding site** for **arrestin**. Binding of **arrestin** to the tail of **GPCR** prevents further **G-protein access**.

Question 26

What is the **basal state** of switching off of **GPCRs**?

Answer 26

**arrestin** folded up - only modest affinity for GPCRs

Question 27

What increases affinity to **arrestins** during switch off of **GPCRs**?

Answer 27

**GRK** phosphorylation of **GPCR tail**

Question 28

What causes **conformational changes** in **beta-arrestin** during switch off of **GPCRs**? What is the result of this conformational change?

Answer 28

**Phospho-GPCR** invades polar core of **arrestin**; conformational change, which newly exposes regions that bind strongly to **intracellular loops of GPCR**, and thereby **obstruct G protein access**.