Park

Question 1

Answer 1

Question 2

Answer 2

Question 3

Describe full cycle of g-protein coupled signaling w/ respect to alpha/beta/gamma subunits

Answer 3

Question 4

How many potential alpha subunits? Beta subunits? Gamma subunits?

Answer 4

20 alpha
6 beta
12 gamma
(in humans)

Question 5

Useful terminology:
1. first messenger
2. receptor
3. transducer
4. primary effector
5. second messenger
6. secondary effector

Answer 5

1. first messenger == ligand
2. receptor == GPCR
3. transducer == G-alpha
4. primary effector == enzymes activated by G-alpha
5. second messenger
6. secondary effector

Question 6

G-alpha signaling:
cAMP

Answer 6

Question 7

G-alpha signaling:
phosphoinositol

Answer 7

Question 8

G-alpha signaling:
arachidonic

Answer 8

Question 9

The adenylate cyclase
pathway

Answer 9

Gs
 Activation of GPCR
 GDP/GTP exchange
 Activation of AC
 Synthesis of cAMP
 Activation of PKA (Protein Kinase A)
 PKA translocates to nucleus
 PKA phosphorylates CREB
(cAMP response element
binding, a transcription
factor)
 CREB recruits CBP (CREB
binding protein)
 Both bind to CRE site
(cAMP resonse element)
 Transcription is now activated

Question 10

PLC Pathway

Answer 10

Gq

Question 11

Function of PLC-beta

Answer 11

converted to:

diacylglycerol
–activates protein kinase C

IP3
– Releases Ca2+ of ER

Question 12

Three types of ligands

Answer 12

Agonists
Ligands that shift the equilibrium in favor of the active state
(example: adrenalin, light).

Inverse agonists
Ligands that shift the equilibrium in favor of inactive states (example:
11-cis-retinal in rhodopsin, MPEP: mGluR5).

Neutral antagonists that do not affect the equilibrium, but can block
agonists to activate the GPCR
(example: carvedilol, a non-selective beta blocker).

Question 13

[rhodopsin] on disc

Answer 13

(8 mM Rhodopsin on disc)

Question 14

How does GPCR achieve “FAST” and
“SENSITIVE” responses?

Answer 14

High concentration

clustering of GPCR and its transducers for downstream effects. Largely amplified signal

Question 15

Is G_alpha the only subunit used for signaling?

Answer 15

βγsubunit can directly regulate channel properties (SHORT RANGE SIGNAL)

beta_arrestin signaling

Question 16

beta_arrestin signaling

Answer 16

GPCR activation/internalization by β-arrestin sequesters
signaling molecules of other pathways.

Question 17

Complexity of GPCR signaling

Answer 17

• βγsubunit can directly regulate channel properties (short-
  range signal).
• β-arrestin regulates GPCR signaling in many ways: (1)
  desensitization (blocking the binding of Gαto GPCR,
  recruiting 2nd messenger-degrading enzymes), (2) GPCR
  internalization (removing GPCR from the cell surface), and
  (3) transducing other signaling such as MAPK pathway.
• GPCRs serve as a scaffold of many signaling molecules.

Question 18

Common Lipid modifications

Answer 18

Acylation
(Gαsubunit)
myristic acid (myr)
palmitic acid (palm)

Prenylation
(Gγsubunit)
farnesol (fa)
geranylgeraniol (gg)

Question 19

prenylation

Answer 19

The CAAX box motif signals postranslational prenylation
C = cysteine
A = aliphatic (nonpolar and hydrophobic) residue
X = any residue
if x = L (leucine), C will be geranylgeranylated
if X = not L, C will be farnesylated

Question 20

Lipid-attachment of heterotrimeric G-proteins

Answer 20

Most Gαsubunits are associated with membrane via acylation.
Gβγsubunit is always associated with membrane via Gγ-prenylation.

Question 21

G protein activation (10 steps)

Answer 21

1. GPCR becomes activated by an agonist
2. The activation changes the structure of the GPCR (outward movement of TM5-
   TM6)
3. GPCR interacts with G protein α-subunit carrying GDP
4. The interaction induces a major displacement of the α-helical domain of Gα,
   which reveals the nucleotide-binding pocket and causes an exchange of GDP
   with GTP
5. The Gαand Gβγsubunits dissociate
6. The activated GPCR acts as an enzyme (GEF), and turns over many G protein α-
   subunits
7. The Gαsubunit with GTP attached is the activator of a target enzyme
   (amplification)
8. Activation of the target lasts as long as GTP is bound
9. Activation terminates when GTP is hydrolyzed to GDP
10. Hydrolysis is accelerated by a GTPase Activating Protein complex, termed GAP

Question 22

Name 2 applications of GPCRs

Answer 22

Optogenetics (Channel Rhodopsin) and
Pharmacogenetics (DREADDs)

Question 23

Which structure in G protein is flexible?

Answer 23

helical domain of G-alpha. Allows for exchange of GDP to GTP

Question 24

Answer 24

Question 25

DREADD intracellular signaling

Answer 25

Question 26

cell specific DREADD protocols

Answer 26

Question 27

Phototransduction summary

Answer 27

Flash Photolysis
* Rhodopsin is completely inactive in the dark (inverse agonist!)
* A single photon can cause isomerization of 11-cis retinal
* Rhodopsin forms an active conformation called R*
* 1 R* can activate 100 transducins/sec
* Activation of T consists of GDP/GTP exchange at Tα
* Tα-GTP activates PDE by removal of PDEγ
* PDE/Tα-GTP rapidly hydrolyzes thousands of cGMP/sec
* Depletion of cytoplasmic cGMP closes CNG channels in the PM
Return to Dark:
* R* is inactivated by phosphorylation, and arrestin binding
* Opsin recombines with 11-cis retinal to for rhodopsin
* PDE/Tα-GTP are inactivated by GAP
* cGMP is resynthesized by guanylate cyclase
* CNG channels re-open

Question 28

three distinct signaling events mediated by Beta-arrestin upon GPCR activation

Answer 28

• β-arrestin regulates GPCR signaling in many ways: (1)
  desensitization (blocking the binding of Gαto GPCR,
  recruiting 2nd messenger-degrading enzymes), (2) GPCR
  internalization (removing GPCR from the cell surface), and
  (3) transducing other signaling such as MAPK pathway.

cell survival/apoptosis
chemotaxis
dopaminergic behaviors
cardiac contractility

Question 29

Gαi can both

Answer 29

1. Inhibit adenylate
   cyclase
2. Activates cAMP
   phosphodiesterase