Signal Transduction III: GPCR-mediated signaling

Question 1

Describe the general GPCR structure, and their importance in therapeutics.

Answer 1

• There are 7 transmembrane (7TM) receptors
• Structure: membrane helices, trimeric G protein, alpha, beta, gamma regions
• Couple to G-proteins–> downstream signal transduction
• GPCRs are the most targeted himan protein family for drug development

Question 2

Why are GPCRs considered GEFs for hetero-trimeric G proteins?

Answer 2

• GPCRs are considered Guanine Nucleotide Exchange Factors for hetero-trimeric G proteins because they facilitate the exchange of GDP for GTP on the Galpha subunit, therby activating them and enabling the transmission of signals intracellularly.

How does this process work?
1. Inactive State: Heterotrimeric G proteins ixist in an inactive state bound to GDP. This complex has 3 subunits: alpha, beta, and gamma
2. Ligand Binding: when a ligand binds to a GPCR on the cell surface, it induces a conformational change in the receptor.
3. Activation of G protein: The activated GPCR can interact with inactive G protein, prmoting the release of GDP from the alpha subunit
4. GTP Binding: The release of GDP allows GTP to bind to the alpha subunit, activating it. This GTP-bound form of the G protein can then dissociate from the beta and gamma subunits, allowing both the GTP-bound alpha and the beta-gamma complex to interact with downstream effectors.
5. The activated G proteins then transmit the signal to various cellular pathways, affecting physiological responses.

Question 3

How does GPCR signaling turn off at a receptor and G protein level?

Receptor desensitization/internalization

Answer 3

1. Phosphorylation by G protein-coupled receptor kinases (7 types)
2. Binding of Beta-arrestin- desensitization (3 subtypes)
3. Recruitment to Clarthrin-coated pits- internalization
4. Recycling or degradation
5. Arrestin can also mediate intracellular signaling

Question 4

Describe Gs and Gi role in cAMP-mediated signaling.

Answer 4

• Adenylate cyclase catalyzes the conversion of ATP to cAMP.
• Human enzyme–> integral membrane protein with may transmembrane segments and a cytoplasmic domain.
• Stimulated by Galphas in response to a variety of hormones or neurotransmitters
• Inhibited by Galphai of G proteins that couple to other GPCRs with different ligands
• cAMP: second messenger with numerous signaling roles
• A major role= activates PKA
• Broken down by phosphodiesterases
• cAMP–> 5’-AMP

Question 5

Describe the GPCR tree of life

Answer 5

GPCRs represent the largest known class of biologicaly omportant transmembrane proteins conserved througout evolution.
* >350 human GPCRs (not counting olfactory GPCRs ) are considered druggable
* ~120 are orphans– meaning the endogenous ligand is not known.
* ~700 (~35%) of FDA-approved drugs target >135 GPCRs

Question 6

What is the pharmaceutical relevance of GPCR?

Answer 6

They can act as antagonists, agonists, full agonists, partial agonist, inverse agonists, and postitive, and negative action.

Question 7

What are some challenges/opportunities with GPCR biologics?

Answer 7

• Antibodies can target the ligand binding site or other allosteric regions of GPCRs
• Antibodies can stabilize GPCR conformations, allowing for complex pharmacology (positive and negative modulators, inverse agonists)
• Antibodies can activate the diverse signals downstream of GPCRs and be engineered to selectively activate certain pathways.
• Antibodies can disrupt protein interactions in the same cell or in cis including blocking higher order GPCR oligomer formation.

Question 8

How do different GPCRs couple to different trimeric G-proteins and alternative signal pathways?

Answer 8

GPCR–> G-protein–> Effector–> 2nd Messenger–> Kinases, Channels, etc.

Gs–> Adenylate Cyclase–> cAMP–> PKA

Gq–> Phopholipase C-Beta–> [Diacylglycerol][IP3]

Diacylglycerol–> PKC
IP3–> IP3 receptor–> Ca2+

Question 8

What is the mechanism of GPCR activation of trimeric G protein?

Answer 8

1. The agonist binds to the receptor–> Nucleotide free
2. G protein coupling and nucleotide exchange (bye bye GDP) –> GTP goes in
3. Activated G protein subunits (beta and alpha) regulate effector proteins–> Uncoupling
4. GTP hydrolysis and inactivation of Galpha protein–> Dissociation of Beta and Alpha subunits (effectors bind here)