Lecture 12: Signal Transduction III

Question 1

GPCR molecular level

Answer 1

-difficult to work with bc integral membrane protein
-hard to isolate to study bc no like water
-difficult to crystallize

Question 2

bovine rhodopsin

Answer 2

-1st GCPR structure determined in 2000
-binds retinol
-responds to light in eye
-one of easiest GPCR to work with

Question 3

B2 adrenergic receptor structure

Answer 3

-took 7 years after bovine rhodopsin

Question 4

GPCR timeline (GPCRdb)

Answer 4

-bovine first
-B2 7 years later
-now we have a lot more (1,102)

Question 5

GPCR family

Answer 5

-largest class of btransmembrane proteins conserved thru evolution

Question 6

Human GPCRs (not including olfactory)

Answer 6

~360
~120 orphans (ligand unknown)
~700 (~35%) drugs target > 135 GPCRs
*not targeting olfactory ones with drugs

Question 7

Heterotimeric G-protein signaling

Answer 7

SLIDE SMTH

Question 8

G-protein cycle

Answer 8

-association with agonist bound GPCR increases GDP release from Ga
-one agonist-bound GPCR can activate 10-100 G proteins
-intrinsic GTPase activity converts GTP to GDP

Question 9

G-protein cycle Steps

Answer 9

1. RESTING (GDP and Ga, GB, Gy)
2. Ligand binding & nucleotide exchange
3. Ga-dissociation (GB and Gy)
4. ACTIVE (GTP on Ga on effector) = SIGNALING
5. GTPase
6. GTP hydrolysis
7. Ligand-dissociation and trimer formation (Ga, GB, Gy)

Question 10

Association with AGONIST bound GPCR INCREASES

Answer 10

DISSOCIATION OF GDP from Ga

Question 11

Why does GTP bind

Answer 11

-lots of GTP inside the cell
-protein work is getting rid of GDP

Question 12

guanine-nucleotide exchange (GEF) activity

Answer 12

-dissociation of GDP from Ga by agonist-bound GPCR

Question 12

GTPase activity

Answer 12

converts GTP to GDP

Question 13

one agonist-bound GPCR can activate

Answer 13

10-100 G proteins

Question 14

GPCR is the GEF

Answer 14

true

Question 15

monomeric vs trimeric?

Answer 15

-Ga, GB, Gy in trimeric?

Question 16

Mechanism of GPCR activation of timeric G protien

Answer 16

1. agonist binding
2. G protein coupling and nucleotide exchange (GTP to GDP)
3. activated G protein subunits regulate effector proteins (ATP to cAMP)
4. GTP hydrolysis (lose phosphate) and inactivation of Ga protein
5. Reassembly

Question 17

GPCR structure

Answer 17

-7 transmembrane receptors (7TM)
-helical loops
-binding site in middle
-couple to G-proteins –> downstream signal transduction

Question 18

G-protein crystallization

Answer 18

-must cut off 7TM loops for purification

Question 19

7TM structure pathway

Answer 19

-3 intracellular loops
-3 extracellular loops
-N and C terminus

Question 20

Where does GTP bind (trimeric)

Answer 20

-at the edge of GaRAS
-GaAH folds over and encloses it

-all intracellular

Question 21

Where does ligand bind to GPCR

Answer 21

-extracellular domain

Question 22

G protein activation conformational changes

Answer 22

1. nucleotide free
2. GTP binds under GaRAS
3. GaAH encloses GTP
4. G subunit uncoupling
5. dissociation of subunits

Question 23

Where do effectors bind?

Answer 23

G subunits after dissociation

Question 24

Internalization/Desensitization: GPCR signal termination

Answer 24

1. Phosphorylation of INTRAcellular loops
2. Desensitization (bind B-arrestin)
3. Internalization (recruitment to clathirin-coated pits)
4. Recycling/Degradation
5. Arrestin can also mediate intracellular signaling

Question 25

GPCR kinases (GRK)

Answer 25

-phosphorylate GPCR

Question 26

G-protein inavtication: signal termination

Answer 26

1. GTPase converts GTP to GDP
   -facilitated by either RGS or GAP

Question 27

RGS

Answer 27

G-protein signaling proteins
-promotes GTPase activity

Question 28

GAP

Answer 28

-GTPase-accelaerating protien
-promotes GTPase activity

Question 29

Signaling pathway

Answer 29

1. GPCR
2. G protein
3. effector
4. 2nd messenger
5. kinases, channels, etc

-signal amplifies as goes through pathway

Question 30

Gs and Gi/o signaling pathway

Answer 30

-adenylate cyclate
-cAMP
-PKA

Question 31

Gs signaling

Answer 31

-stimulates adenylate cyclase
-in response to hormones or neurotransmitters

Question 32

Gi

Answer 32

-inhbits adenylate cyclase
-couple to other GPCRs with different ligands

Question 33

adenylate cyclase

Answer 33

-ATP to cAMP
-integral membrane
-many transmembrane segments and cytoplasmic domain

Question 34

cAMP

Answer 34

-second messenger
-activates PKA (protein kinase A)
-broken down by phosphodiesterases to 5’-AMP

Question 35

Adenylate cyclase structure

Answer 35

1. transmembrane domain
2. helical domain inside cell
3. catalytic domain

Question 36

catalytic domain

Answer 36

-ATP conversion
-regulation of ATP conversion dependent on Ga subunit bound

Question 37

Protein Kinase A (PKA)

Answer 37

-mediates most cAMP signaling
-binding of cAMP to R-subunits results in dissociation of C-subunits from R-subunits

Question 38

r-subunits

Answer 38

-PKA regulatory subunits
-2

Question 39

C-subunits

Answer 39

-PKA catalytic subunits
-2

Question 40

PKA substrates

Answer 40

-enzymes in glycolysis and glycogenolysis
-ion channels
-CREB

Question 41

CREB (cAMP Response Element Binding)

Answer 41

-transcription factor
-phosphorylated form binds CRE and rapidly changes transcription levels

Question 42

activated PKA

Answer 42

-can enter nucleus