G-Protein Beta-Gamma Subunits

Question 1

G-protein activation cycle

Answer 1

Activation of a GPCR results in release of GDP from the G-alpha. The nucleotide-free-G-alpha quickly gets occupied by GTP, which is abundant in a cell. The resulting conformational change of the G-alpha results in its dissociation from the receptor and G-beta-gamma subunits. The intrinsic GTP-ase activity of G-alpha converts GTP to GDP, causing re-association with the receptor and G-beta-gamma.

Question 2

Beta-gamma subunits

Answer 2

• the propeller structure is the most conserved amongst the beta subunits
• binding of G-alpha does not noticeably alter the structure G-beta-gamma
• functional differences between different beta-gamma combinations are not well understood

Question 3

G-beta-gamma synthesis, lipidation and membrane targeting

Answer 3

G-alpha, G-beta and G-gamma are synthesised at cytoplasmic ribosomes and they do not have hydrophobic transmembrane sequences like that of GPCRs. They become membrane anchored due to lipidation after translation. G-beta and G-gamma are folded separately and heterodimerised before G-gamma is iso-prenylated, thus also anchoring the unmodified G-beta to ER membrane. Association of G-beta-gamma with palmitoylated or myristoylated G-alpha is necessary for the transport of G-alpha-beta-gamma to the plasma membrane.

Question 4

How long from G-beta synthesis to membrane?

Answer 4

Method: Pulse-chain experiments help us monitor incorporation of radioactively labelled methionine into newly synthesised proteins. Radio-activity in the gel measured using scintillators. SDS us used to separate the G-beta-gamma assembly, so that ARC9 (antibody) can bind the free beta subunit.
Observation: G-beta-gamma association occurs in 2.5 min in the cytoplasm based on recovery using SDS. They reach membranes in about 15 min and plasma membrane in about 60 min.

Question 5

Not all beta and gammer subunits can dimerise to form G-beta-gamma

Answer 5

• except beta-s, all other beta subunits can assemble irreversibly with all gamma subunits.
• beta-s has a significantly increased coupling selectivity for gamma-2 and gamma-7
• coupling of beta-5 to gamma-2 is weak and reversible
• the divergence in the sequence of beta-5 from all other beta subunits (only 50% sequence similarity is the likely reason for these peculiarities

Question 6

Gβγ - effectors

Answer 6

Channels
o GIRK – G-protein coupled inward rectifying channels
o Voltage-dependent Ca2+ Channels

Enzymes
o Adenylyl Cyclase
o GRK – G-protein receptor kinase
o PLC β, MAPK, PI3 Kinase …

Other proteins
o Dynamin, RGS, IP3 R etc…

Binding of Gβγ with its
effectors (GIRK2 and GRK2)
are mediated by the beta-propeller region of the Gβ
subunit

Question 7

Does Gβγ increase or decrease the open probability of GIRK Channels?

Answer 7

GIRK = G-protein coupled Inwardly Rectifying potassium (K) channels

Activating Gαi coupled receptors leads to separation of the βγ subunit away from
the αi subunit. The βγ subunit interacts with G protein-gated GIRK channels,
increasing their open probability.

βγ subunit binds to cytoplasmic channel domain of GIRK channels

Question 8

Modulation of adenylyl cyclase (AC) by βγ subunits

Answer 8

Of the 8 membrane bound isoforms of AC, 3
are inhibited and 5 are stimulated
(synergistically with Gαs) by Gβγ.
Stimulation requires the binding of the
“hotspot” in the Gβ propeller structure.
Mutating this region results in inactive Gβγ (in
terms of ability to activate AC5).

Question 9

AC:Gβγ binding is facilitated by both the N-terminal and

the propeller structure of β subunit

Answer 9

BiFC assay shows that
both N-terminal of Gβ and
the hotspots are required
for binding to AC.

Question 10

Why study Gβγ?

Therapeutic potential

Answer 10

o Inhibition of chemotaxis and inflammation using compounds that block Gβγ -
interactions with its effectors (e.g., PI3-kinase γ). One such compound, Gallein, binds
to “hotspots” of Gβγ

o The figures show the inhibition of carrageenan-induced paw edema

o Targeting Gβγ may have therapeutic potential

o However, there is potentially a major problem with this strategy. Can you think of it?

Question 11

Classical models of GPCR : G-Protein coupling?

Answer 11

Are G-proteins pre-coupled to inactive GPCRs, or does it only occur upon agonist binding?
Pre-coupling model vs.
Collision-coupling (a.k.a. ligand-induced coupling) model

Question 12

Does radioligand binding data answer the question?

Are G-proteins pre-coupled to inactive GPCRs, or does it only occur upon agonist binding?
Pre-coupling

Answer 12

o Competitive radioligand-binding assay
o Gpp(NH)p is a non-hydrolysable GTP
analogue. It prevents Gα and Gβγ
from interacting with receptors
o Beta-adrenergic receptor is used
o [3H]-DHA is radioactive antagonist
o HBI is an agonist

Receptor exists in two states.
o When it is G-protein coupled, it has high
affinity for agonist.
o When it is uncoupled, it has low affinity
for agonist.
o Question: Does this mean the receptors
and G-proteins are pre-coupled?

Question 13

Immunoprecipitation shows both

pre-coupling and collisional-coupling

Answer 13

o Solubilised brain immuno-precipitated
using using 8730 (anti-Gαi
) and 9072 (antiGαo) antibodies.
o The precipitate showed somatostatin
analogue (MK 678) binding activity,
suggesting pre-coupling between
somatostatin receptors and Gα.
o Interestingly, the MK 678 binding
increases in anti-Gαo-precipitate when the
cells are pre-treated with an agonist (DTrp8-SRIF).
o This shows pre-coupling to Gαi and agonistinduced coupling to Gαo – perhaps this is
origin of G-protein coupling selectivity?

Question 14

FRET shows no pre-coupling and evidence for collision coupling

Answer 14

FRET between fluorescent α2A-adrenergic receptor and Gγ increases upon agonist (NE)
addition. The basal FRET (FYFP/FCFP) value of 0.2 was separately shown to be non-specific.
This implies there is no pre-coupling between receptors and G-proteins. Dependence of Gprotein activation time on the density of G-protein also indicates collision coupling.

Question 15

BRET study shows significant pre-coupling

Answer 15

o BRET between tagged β2-adrenergic receptor and Gγ measured every 0.05 seconds
before and after agonist (ISO) addition.
o Non-zero basal FRET indicates pre-coupling.
o The fast increase in BRET (~300ms) after agonist addition could be because of loosely
formed receptor:G-protein complex that then quickly change their conformation.
o Thus, pre-coupling model explains the very fast G-protein coupled signalling

Question 16

Summary of classical

GPCR-G-protein coupling models

Answer 16

Pre-coupling model
o Explains selectivity between
receptors and g-proteins
o Explains fast (~300 ms) response

Collision coupling model
(a.k.a. ligand induced coupling)
o Explains receptor reserve and
signal amplification

Question 17

Single Molecule Tracking of

GPCR and G-protein to detect coupling

Answer 17

Fluorescently label α2A-AR and Gαi
o Obtain video of the molecular
diffusion (motion)
o Detect centre of spots and
generate tracks of their paths
o Analyse: How often do the
molecules “collide”
o Analyse: After collision, do they codiffuse (move together)? If so, for
how long?
o Analyse: How fast do the
molecules diffuse (move)?
o Analyse: Do the molecules spend
more time in certain locations?

Question 18

Is coupling is permanent?

Answer 18

No

o α2A-AR and Gαi undergo transient collisions even without agonist.
o Collisions are sometimes “productive”, resulting in complexes that last ~1 second on
average.
o Agonist stimulation increase the number of “productive” collisions.
o The collisions primarily occur at membrane hotspots (formed by clathrin pits or
cytoskeletal pockets).

Question 19

Current understanding on

GPCR : G-protein coupling

Answer 19

o GPCRs : G-protein interaction is driven by affinity
o High affinity before ligand addition will resemble the pre-coupling model
o Low affinity before ligand addition and high affinity after ligand addition will
resemble ligand-induced coupling model
o GPCRs : G-protein specificity is determined by their mutual affinities
o Signalling speed is likely mediated by formation of membrane “hotspots”
o Signal amplification due to large concentrations of molecules in the
hotspots
o So, both pre-coupling and collision coupling models are universally incorrect
– the answer is most likely a bit of both! Affinity driven coupling.

Question 20

Conclusions

Answer 20

o Post-translational modifications (of Gα and Gγ) and assembly of Gαβγ
is necessary for their transport to the plasma membrane
o Gβγ are crucial components of the GPCR signalling pathway. Its
effectors include ion channels, enzymes and many other proteins
o The classical models of pre-coupling and collision coupling between
GPCRs and G-protein are extreme cases. Recent evidence suggests
that it is affinity driven coupling and it may be supported by
membrane hotspots
o Single molecule techniques such as Single Molecule Tracking and
Fluorescence Correlation Spectroscopy can provide more definitive
understanding of coupling