GPCRs

Question 1

7. ​Draw a diagram of how G proteins cycle between active and inactive states; describe the general class of enzymes that regulate this process (GAPs and GEFs).

Answer 1

• GAPS are GTPase activating proteins. remove phosphate to go from GTP→ GDP.
• GEFS are guanine nucleotide exchange factors. turn on G protein to go from GDP→ GTP.

see diagram

Question 2

8. Draw a diagram of how positive and negative feedback loops (with and without delayed responses) affect gene expression.

Answer 2

Positive Example: Enzyme binds to its ligand that leads to increases activity.

Negative Example: Signal activates an enzyme which causes phosphorylation and inhibits signaling.

• see diagram

Question 3

9. Describe the structural characteristics of G-protein coupled receptors (GPCRs) and their corresponding G-proteins (a, b, g subunits).

Answer 3

● G-protein bound to GDP when not bound to a ligand

● Trimeric g-protein - Alpha, beta, and gamma subunits

● Binding of signaling molecule causes change in alpha subunit, acts as GEF = GDP -> GTP,
○ causes phosphorylation GDP to GTP

○ GDP -> GTP activates BOTH the alpha & Beta Gamma subunit

● Alpha subunit
○ GTP binding by alpha subunit causes conformational change for binding

○ Target protein is activated when activated alpha subunit is bound to GTP

○ Alpha active till (hydrolysis) of GTP -> GDP; GDP inactive

○ Hydrolysis of GTP to GDP inactivates alpha subunit,

○ Inactive subunit re-associates with complex, which inactivates the complex and forms the inactive G protein

● Beta Gamma Subunit
○ Activated beta gamma released to act on target protein

Question 4

11.List the types of second messenger molecules used in signaling pathways (cAMP, IP3, Ca+2).

Answer 4

● G-Proteins regulate activity of ​adenylyl cyclase​ to make cAMP
○ Adenylyl cyclase - regulated by g-proteins and calcium
○ cAMP - second messenger whose concentrations can rise and fall in seconds
○ PKA (cAMP dependent Protein Kinase) can phosphorylate Ser/Thr on target proteins

■ Inactive PKA - four subunits (2 catalytic and 2 regulatory). binding cAMP the regulatory subunits dissociate, releasing and activating the catalytic subunits

■ PKA-directed responses can be immediate or may take time to develop

● Translocation to nucleus and the phosphorylation of
transcription factors
○ CRE: cAMP response element, DNA site in
promoter location of gene

○ CREB: CRE-binding protein; phosphorylated by
PKA

○ CBP: CREB-binding protein; recruited by CREB and
needed for transcription

● It can regulate ion channels, GEFs, transcription, and
enzyme activities
○ (Adenylyl cyclase → cAmp → PKA)

● G-Proteins trigger PLCBeta (phospholipase C) and phospholipid signaling

● Alpha subunit - activates Phospholipase C enzyme which cleaves lipids

● Activated PLCB acts on PIP2 and generate IP3 and releases the lipid molecule diacylglycerol
○ IP3 is a secondary messenger that is now soluble in the cytoplasm and can diffuse in cytosol (water soluble)

■ Can go on to interact with calcium channels

■ Calcium is stored in organelles in high concentrations
■ When IP3 binds to the gated Ca2+ channels which leads to release calcium in cytoplasm
● Can bind to calcium sensitive proteins
● Calmodulin is calcium receptor that can
modulate target proteins

○ Calcium Calmodulin-dependent protein kinases (CaM Kinases) mediate many of the calcium signals in the cells
○ As calcium levels rise, calmodulin is activated and binds to CaM Kinase which causes self phosphorylation which will let it phosphorylate other proteins.

■ As calcium diminishes, the CaM kinase is still phosphorylated but will have diminished effects (memory device)
○ Diacylglycerol is also a secondary messenger that is soluble in the membrane (lipid soluble)
■ At the same times as IP3 is binding to calcium channels it can recruit Protein Kinase C
● Protein Kinase C needs calcium to be activated
● Phospholipase C (PLC) → IP3 & Diacylglycerol → Ca2+ & PKC → CaM Kinase