G-Proteins

Question 1

G-proteins full name

Answer 1

guanine nucleotide binding regulatory proteins

Question 2

What are G-proteins?

Answer 2

they are the go-between proteins and act as transducers between receptors and effectors

Question 3

Two classes of G-proteins have been described

Answer 3

• heterotrimeric G-proteins (large proteins), membrane proteins, consist of alpha, beta and gamma subunits
• monomeric G-proteins (small proteins), not membrane-associated, but bind GTP and GDP

Question 4

The structure of heterotrimeric G-protein

Answer 4

• consists of alpha, beta and gamma subunits
• in the inactive state, 3 subunits are associated together and the alpha subunit binds to GDP (guanosine diphosphate)
• beta and gamma units are tightly bound with each other

Question 5

G-proteins - the switch between active and inactive states

Answer 5

• G-proteins are membrane proteins, anchored to the membrane through a fatty acid chain
• in the inactive state, 3 subunits are associated together and the alpha subunit binds to GDP
• agonist binding to a receptor causes a conformational change which encourages the receptor to bind to the G-protein complex
• this coupling causes the GDP bound to the alpha subunit to be exchanged for GTP
• the G-alpha(GTP) subunit dissociates from the receptor and from the beta and gamma subunits and interacts with the target protein, leading to the generation of second messengers
• in the meantime, the alpha subunit constitutive GTPase hydrolyses GTP to GDP
• this hydrolysis process is the turn-off signal that induces G-alpha to disengage from the effectors
• finally the G-alpha-GDP subunit reunites with the beta and gamma subunits

Question 6

Summary of G protein activation

Answer 6

G-proteins are membrane proteins comprised of 3 subunits: alpha, beta and gamma

• in the inactive state, 3 subunits are associated together and the alpha subunit binds to GDP (GDP bound G-alpha))
• agonist-receptor binding causes receptor conformational change, which encourages the receptor to bind to the G-protein complex
• the receptor-G-protein coupling promotes the exchange of GDP for GTP on the G-alpha subunits
• the G-alpha(GTP) dissociates from the receptor and from beta and gamma subunits, and interacts with the target protein (e.g. adenylyl cyclase), leading to the generation of second messengers
• the cycle is completed by the hydrolysis of GTP to GDP by the G-alpha constitutive GTPase, resulting in the re-association of G-alpha(GDP) with the beta and gamma subunits

Question 7

GPCR signalling cascade

Answer 7

receptor (sensory receptors, peptide receptors, hormone receptors, neurotransmitter receptors) –> G-protein –> effector (enzymes - adenylyl cyclase, phospholipase A, phospholipase C; ion channels) –> 2nd messenger (cyclic nucleotides, products of inositol lipids, calcium) –> 2nd effector (enzymes - kinases, phophatases; ion channels)

Question 8

The main G-proteins subtypes and their functions

• G-alpha-s

Answer 8

G-alpha-s
Associated receptors
- many amine and other receptors (e.g. catecholamines, histamine, serotonin)
Main effectors
- stimulates adenylyl cyclase, causing increased cAMP formation
Notes
- activated by cholera toxin, which blocks GTPase activity, thus preventing inactivation

Question 9

The main G-proteins subtypes and their functions

• G-alpha-i

Answer 9

G-alpha-i
Associated receptors
- as for G-alpha-s, also opioid, cannabinoid receptors
Main effectors
- inhibits adenylyl cyclase, decreasing cAMP formation
Notes
- blocked by pertussis toxin, which prevents dissociation of alpa-beta-gamma complex

Question 10

The main G-proteins subtypes and their functions

• G-alpha-o

Answer 10

G-alpha-o
Associated receptors
- as for G-alpha-s, also opioid, cannabinoid receptors
Main effectors
- limited effects of alpha-subunit (effects mainly due to beta-gamma subunits)
Notes
- blocked by pertussis toxin
- occurs mainly in nervous system

Question 11

The main G-proteins subtypes and their functions

• G-alpha-q

Answer 11

G-alpha-q
Associated receptors
- amine, peptide and prostanoid receptors
Main effectors
- activates phospholipase C, increasing production of second messengers inositol triphosphate and diacylglycerol

Question 12

Characteristics of GPCR and G-protein interactions

General

Answer 12

1. Multiple coupling of receptors to different G-protein families
2. Divergence of signal
3. Bidirectional control of target enzyme
4. Convergence of signals
5. Specificity of receptor-G-protein interactions

Question 13

Characteristics of GPCR and G-protein interactions

1. Multiple coupling of receptors to different G-protein families

Answer 13

1. Multiple coupling of receptors to different G-protein families
   e.g. Beta-ARs couple to both Gs (classical) and Gi proteins
   Gs-mediated activation if PKA causes phosphorylation of beta-AR which then switches the receptor coupling to Gi and simulates the mitogen-activated protein (MAP) kinase

see PKA signalling pathway

Question 14

Characteristics of GPCR and G-protein interactions

2. Divergence of signal

Answer 14

2. Divergence of signal: Both alpha and beta-gamma subunits are signalling mediators

The specific functions of the beta-gamma complex are not yet known, but it may be involved in:

• activating potassium channels
• inhibiting voltage-gated calcium channels
• activating GPCR kinases
• activating mitogen-activated protein kinase cascade

Question 15

Characteristics of GPCR and G-protein interactions

3. Bidirectional control of a target enzyme

Answer 15

3. Bidirectional control of a target enzyme
   - the heterogeneity of G-proteins allows different receptors to exert opposite effects on a target enzyme (e.g. adenylyl cyclase)

• in the intestine and bladder, M3 receptor activation causes smooth muscle contraction
• the ratio of M2:M3 = 70:30
• M2 receptor doesn’t seem to play a role in the control of smooth muscle activity
• what is the function of M2 receptor in the intestine and bladder?
  
  • see diagram
• M2 receptor inhibits cAMP formation, thus inhibiting cAMP-dependent relaxation of smooth muscle

Question 16

Characteristics of GPCR and G-protein interactions

4. Convergence of signals

Answer 16

4. Convergence of signals: different receptors act on the same family of G-proteins

e. g. alpha-2-adrenergic, adenosine A1, muscarinic M2, opioid receptors stimiulate G-alpha-i/o, which leads to:
- inhibition of AC
- activation of K channel (via G-alpha-i and or/ beta-gamma)
- inhibition of calcium channels (via Go)

Question 17

Characteristics of GPCR and G-protein interactions

5. Specificity of receptor-G protein interactions

Answer 17

5. Specificity of receptor-G-protein interactions

• Hundreds of GPCRs use a family limited range of G-proteins.
• How do they get specificity?
• Studies by antisense oligos-directed against G-alpha, beta, and gamma subunits show that the selectivity of receptor-effector coupling is determined by the specific combination of alpa, beta, and gamma heterotrimers
• e.g. carbachol acts on M1 muscarinic receptor in basophilic lymphocytes to stimulate PLC via an alpha-q/11-beta-1/4-gamma-4 complex

Question 18

Small GTPases (small G-proteins)

Answer 18

• monomeric G-protein
• also called the Ras GTPase
• more than a hundred proteins in this superfamily
• can loosely be divided into: Ras, Rab, Arf, Ran, Rheb, Rho and Rap families
• they regulate a wide variety of processes in cells, including growth, cellular differentiation and proliferation, cell survival, angiogenesis, vesicle transport, hypertrophy and cancer etc.
• G-alpha-12/13 couples to the activation of Rho (see diagram)

Question 19

Diseases associated with G-protein mutations

Answer 19

• Albright hereditary osteodystrophy (AHO) - Gs loss
• Pseudohypoparathyroidism type 1a - Gs loss
• Testotoxicosis - Gs gain/loss
• McCune Albright syndrome - Gs gain (mosaic)
• Somatotroph adenomas with acromegaly - Gs gain
• Ovarian and adrenocortical tumours - Gi gain

Question 20

Diseases associated with G-protein mutations

• Ovarian and adrenocortical tumours

Answer 20

Ovarian and adrenocortical tumours

• G-alpha-i subunit mutations are present in 300% of ovarian sex cord tumours and in adrenocortical tumours
• activating G-alpha-i mutations result in constitutively activated signal transduction; this may lead to tumourigenesis

Question 21

Diseases associated with G-protein mutations

• McCune Albright syndrome

Answer 21

• G-alpha-s mutations occur in either Arg201 or Gln227 residues
• the mutations disrupt the intrinsic GTPase activity, thus the G-alpha-s gets stuck on the “on” position
• if the mutation affects the skin cells, it causes darker than normal pigment (mosaic)
• if the mutation affects bone cells, it causes weakness and fractures
• in hormone-producing cells, the mutation causes excess release of hormones

Question 22

Diseases associated with G-protein mutations

• Albright hereditary osteodystrophy (AHO)

Answer 22

Patients who inherit a heterozygous G-alpha-s mutation develop AHO, a syndrome characterised by one or more of these clinical features:
- short stature, subcutaneous ossifications, centripetal obesity, depressed nasal bridge, and mental or developmental retardation

Question 23

Diseases associated with G-protein mutations

• Pseudohypoparathyroidism type 1a

Answer 23

• in addition to the AHO phenotype, patients who maternally inherit G-alpha-s mutations also develop resistance to various hormones that stimulate G-alpha-s/cAMP in their target tissues
• in contrast, patients who paternally inherit G-alpha-s mutations develop only the AHO phenotype

Question 24

Rho-GTPases and cancer

Answer 24

Over expression, rearrangement, point mutations or alternative splicing at RHO proteins can cause a variety of different types of tumours/cancers

see Rho signalling pathway

Question 25

Rho GTPases as therapeutic targets in cancer

Answer 25

Different strategies to interfere with Rho-GTPase signalling pathway - Rho-spatial regulation - Rho-GEF interaction inhibition - Rho-nucleotide interaction inhibition - Effector inhibitors - cell migration, cell cycle, apoptosis, gene transcription

Question 26

Common drugs used in large G-protein research

Answer 26

- GTP/GDP analogs: Gpp[NH]p, GTP-gamma-S - Pertussis toxin (PTX): catalyses the ADP-ribosylation of the Gi, Go alpha subunits which interferes GDP exchange for GTP - Cholera toxin (CTX)-induces ADP-ribosylation of an Arg residue on G-alpha-s-protein and inhibits GTPase activity, resulting in persistent G-protein activation