Lecture 12: Signal Transduction III Flashcards

1
Q

GPCR molecular level

A

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

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2
Q

bovine rhodopsin

A

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

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3
Q

B2 adrenergic receptor structure

A

-took 7 years after bovine rhodopsin

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4
Q

GPCR timeline (GPCRdb)

A

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

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5
Q

GPCR family

A

-largest class of btransmembrane proteins conserved thru evolution

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6
Q

Human GPCRs (not including olfactory)

A

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

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7
Q

Heterotimeric G-protein signaling

A

SLIDE SMTH

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8
Q

G-protein cycle

A

-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

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9
Q

G-protein cycle Steps

A
  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)
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10
Q

Association with AGONIST bound GPCR INCREASES

A

DISSOCIATION OF GDP from Ga

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11
Q

Why does GTP bind

A

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

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12
Q

guanine-nucleotide exchange (GEF) activity

A

-dissociation of GDP from Ga by agonist-bound GPCR

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12
Q

GTPase activity

A

converts GTP to GDP

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13
Q

one agonist-bound GPCR can activate

A

10-100 G proteins

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14
Q

GPCR is the GEF

A

true

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15
Q

monomeric vs trimeric?

A

-Ga, GB, Gy in trimeric?

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16
Q

Mechanism of GPCR activation of timeric G protien

A
  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
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17
Q

GPCR structure

A

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

18
Q

G-protein crystallization

A

-must cut off 7TM loops for purification

19
Q

7TM structure pathway

A

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

20
Q

Where does GTP bind (trimeric)

A

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

-all intracellular

21
Q

Where does ligand bind to GPCR

A

-extracellular domain

22
Q

G protein activation conformational changes

A
  1. nucleotide free
  2. GTP binds under GaRAS
  3. GaAH encloses GTP
  4. G subunit uncoupling
  5. dissociation of subunits
23
Q

Where do effectors bind?

A

G subunits after dissociation

24
Q

Internalization/Desensitization: GPCR signal termination

A
  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
25
Q

GPCR kinases (GRK)

A

-phosphorylate GPCR

26
Q

G-protein inavtication: signal termination

A
  1. GTPase converts GTP to GDP
    -facilitated by either RGS or GAP
27
Q

RGS

A

G-protein signaling proteins
-promotes GTPase activity

28
Q

GAP

A

-GTPase-accelaerating protien
-promotes GTPase activity

29
Q

Signaling pathway

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

-signal amplifies as goes through pathway

30
Q

Gs and Gi/o signaling pathway

A

-adenylate cyclate
-cAMP
-PKA

31
Q

Gs signaling

A

-stimulates adenylate cyclase
-in response to hormones or neurotransmitters

32
Q

Gi

A

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

33
Q

adenylate cyclase

A

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

34
Q

cAMP

A

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

35
Q

Adenylate cyclase structure

A
  1. transmembrane domain
  2. helical domain inside cell
  3. catalytic domain
36
Q

catalytic domain

A

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

37
Q

Protein Kinase A (PKA)

A

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

38
Q

r-subunits

A

-PKA regulatory subunits
-2

39
Q

C-subunits

A

-PKA catalytic subunits
-2

40
Q

PKA substrates

A

-enzymes in glycolysis and glycogenolysis
-ion channels
-CREB

41
Q

CREB (cAMP Response Element Binding)

A

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

42
Q

activated PKA

A

-can enter nucleus