G-proteins

Question 1

GPCR structure and coupling(5)

Answer 1

1. Structure consists of 7 transmembrane segments,
2. N terminus = extracellular, C terminus = cytoplasm.
3. Often have lipid modifications on the C terminal tail.
4. Actually function as a dimer, most as homodimer but some as heterodimer.
5. As its name suggests, the receptor is coupled to a heterotrimeric G protein that signals downstream.

Question 2

GPCR ligands

Answer 2

hormones

Question 3

what is function of lipid modifications to GPCRs?

Answer 3

keep them near the plasma membrane

Question 4

which 2/3 subunits of GPCRs function as one unit?

Answer 4

beta and gamma

Question 5

RGS proteins

Answer 5

regulators of G protein signaling, analagous to GAPs. Proteins speed up GTP hydrolysis, help with spatial resolution and signal duration

Question 6

What is the GEF analog in G protein signaling

Answer 6

The GPCR itself

Question 7

GDIs

Answer 7

Guanine nucleotide dissociation inhibitor, block GDP release and prevent GPCR from being activated

Question 8

agonist

Answer 8

molecule that binds GPCR and stimulates its downstream signaling

Question 9

antagonist

Answer 9

molecule that binds GPCR and inhibits its downstream signaling

Question 10

partial vs full agonist

Answer 10

whether agonist stimulates maximum or partial signaling (may have to do with how well it binds/specific conformation).

Question 11

neutral antagonist

Answer 11

acts via competitive inhibition; i.e. it does not physically prevent the GPCR from signaling, but it blocks the agonist from binding. GPCR can still have constitutive signaling.

Question 12

inverse antagonist

Answer 12

binds the GPCR and physically prevents it from signaling by locking it in an inactive state => no constitutive signaling.

Question 13

allosteric modulators

Answer 13

bind to a site on the GPCR elsewhere from the ligand binding site and allosterically enhance or inhibit agonist binding.

Question 14

cholera toxin mechanism of action

Answer 14

ADP-ribosylates Gαs and blocks GTP hydrolysis, so Gαs is overactive (effect = more AC activity)

Question 15

pertussis toxin mechanism of action

Answer 15

ADP-ribosylates Gαi and blocks ability of receptor to trigger release of GDP from Gαi, so Gαi not active and cannot inhibit AC (effect = more AC activity)

Question 16

features of G protein cycle (4)

Answer 16

1. Directionality: GDP must be released before GTP can bind, and GTP is always hydrolyzed to GDP + P
2. Amplification: one receptor can activate multiple G proteins, and activated G proteins can activate multiple downstream targets
3. Bifurcation: Gα and Gβγ both can signal, contributing to complexity of signaling
4. Timing: GDP release is rate limiting step; GTP hydrolysis can also contribute to timing and can be sped up by RGS proteins.

Question 17

Canonical GPCR signaling pathway

Answer 17

1. Upon ligand binding (hormone/agonist), conformational changes in the GPCR promote release of GDP from the Gα subunit.
2. GDP release enables the Gα subunit to immediately “pick up” a GTP.
3. Gα in its GTP-bound state can no longer associate with the βγ subunit (note: the portion of βγ that has downstream targets is the same portion that binds Gα at “rest”; thus when βγ is released, its active site is free to interact with downstream effectors).
4. Gαs stimulates adenylyl cyclase (AC) activity (increase in cAMP) while Gαi inhibits AC activity. Gαq stimulates PLCβ (less important). Gαt (transducin) stimulates PDE.
   Increase in cAMP activates PKA (lecture 7).

Question 18

GPCR densensitization mechanism

Answer 18

Cytoplasmic tail of GPCR has many phosphorylation sites (PKA can phosphorylate the tail).
Highly phosphorylated tails inhibit Gα association with receptor, and furthermore when β-arrrestin binds the highly phosphorylated tail, Gα is completely sterically hindered from binding to the receptor.
β-arrestin can further desensitize the signal by promoting endocytosis.

Question 19

T/F. Endocytosed signaling pathway is independent of G proteins

Answer 19

True - scaffold proteins with PDZ domains bind to PDZ motif on C terminus of GPCR and recruit more proteins

Question 20

What happens to GPCR signaling when the receptor is endocytosed?

Answer 20

Endocytosis can lead to degradation and more permanent regulation of GPCR signaling, or sometimes, an endocytosed GPCR can signal down a different pathway like MAP kinase. It’s complicated and depends on the integration of many signals.