G Proteins

Question 1

G Protein Definition

Answer 1

Guanosine nucleotide binding proteins

Question 2

GPCR details

Answer 2

G Protein coupled receptors

1. Plasma membrane receptor = 7 transmembrane helical segments
2. G protein active with GTP
3. Effector enzyme in plasma membrane activated by G protein

Question 3

Generic pathway for G proteins

Answer 3

1st messenger - Allosteric transition for GPCR - G protein activated - G protein binds to effector protein - causes change in concentration for second messenger - binds to target

Question 4

Two big functions of GPCRs

Answer 4

Detect hormones/growth factors

Aid in smell/ taste

Question 5

Beta adrenergic receptor

Answer 5

Base form for GPCRs

Mediates epinephrine effects = adrenaline

Question 6

Where are beta adrenergic receptors located?

Answer 6

Muscle, liver, adipose tissue

On membrane

Question 7

G Protein structure

Answer 7

3 subunits:

alpha = binding site for GTP or gdp = GTPase functionality

GPCR is transmembrane component not G protein

Beta

Gamma

When gdp bound = heterotrimeric

Question 8

When is Gs accurate?

Answer 8

Stimulated G protein = activated = GTP

Question 9

AC Enzyme function

Answer 9

Adenylyl Cyclase = integral plasma membrane protein

Synthesizes cAMP from ATP = removes PPi

Stimulated by Galpha protein

Question 10

ATP structure reminders

Answer 10

Two aromatic rings with 2 N each, one has nh2 offshoot,
Glucose ring with oh x2
3 po4 groups

Nitrogen
Glucose
Phosphorous

= NGP = nasty gal pal = energizes me the whole night !!!

Question 11

cAMP structure reminders

Answer 11

Same as atp but has lost PPi

P interacting w glucose rings = comrades

= nitrogens, glucose, phosphorous

Nasty gal pal with piss taken out of her by AC = air conditioning has ruined her amazing hair that she spent a lot of time on and she has to put a jacket on

Question 12

AMP structure reminders

Answer 12

Like cAMP but p is not interacting with glucose ring

Nitrogen, glucose, phosphorus

cAMP minus the c = comrades

Question 13

Cyclic nucleotide phosphodiesterase

Answer 13

cAMP -> AMP via hydrolysis

Question 14

What does cAMP activate?

Answer 14

Protein kinase A
= PKA

Memory: the camp at peak A = best mountain camp around = fight or flight

Question 15

Why is PKA important?

Answer 15

Key to phosphorylation of important ser or thr residues of proteins for fight or flight response

Question 16

How many cAMPs to activate PKA?

Answer 16

4

2 per regulatory subunit

Question 17

PKA structure details

Answer 17

Bound to AKAP = a kinase anchoring protein

Dimer of two R subunits = regulatory subunits = dimerization domain is amino terminal attached to AKAP

Each r subunit has 1 catalytic subunit
When inactive = atp binding site blocked by R subunits

Conformational change caused by cAMP = C subunits available for ATP binding = transfer of P to side chain of a ser or thr in residue of target protein

Question 18

PKA consensus sequence

Answer 18

X-r-r/k-x-s/t-b

X=any, r=arg, k=lys, s/t= ser or thr, b= basic

Ex in rage at rack
Ex stabs

Question 19

FRET

Answer 19

Fluorescence resonance energy transfer

Fret proportional to (1/d^6) between fluorescent proteins

Lower fluorescence if close enough

Question 20

FRET for detecting cAMP in cells

Answer 20

BFP gene with R unit of PKA

GFP gene with C unit of PKA

More cAMP = more blue bc units further apart

Less cAMP = more green bc units close together

Question 21

FRET for PKA activity in cells

Answer 21

Cyan gene fused with top half of hybrid protein to be phosphorylated by PKA = consensus sequence in middle

Yellow gene fused with bottom half of same protein

When protein phosphorylated = two halves close together = yellow color = PKA activity

When protein not phosphorylated = far apart = cyan color

Question 22

Epinephrine pathway
In hepatocyte

Answer 22

Epinephrine binds to cell membrane via GPCR = beta adrenergic receptor

G protein is activated

Activates AC to produce more cAMP from ATP

cAMP activates PKA

PKA phosphorylates phosphorylase b kinase

Which phosphorylates glycogen phosphorylase a

Which activates glycogen breakdown to glucose 1 phosphate to glucose

= amplification occurs at each step
= very rapid

Question 23

1 molecule epinephrine is how many molecules of glucose released?

Answer 23

100,000

Question 24

Mechanisms for terminating b adrenergic receptor response

Answer 24

-epinephrine concentration less than Kd = dissociation

-GTP hydrolysis within G alpha
= gtpase

-removal of second messenger
= hydrolysis of cAMp via cyclic nucleotide phosphodiesterase to AMP

-Effects of pathways reversed by phosphoprotein phosphatases
= PP1 for beta adrenergic receptor pathway

Question 25

Desensitization mechanism for beta adrenergic receptor

Answer 25

-beta gamma subunit recruits beta ARK = kinase that phosphorylates ser resides at Carboxyl end of receptor -phosphorylated receptor binds to arrestin = arrests continued activation of incoming G proteins = also recruits clathrin for endocytosis of receptor -arrestin dissociates and receptor loses Ps = returns to surface

Question 26

Desensitization mechanism memory

Answer 26

Beta ARK = bark kinase = fucks with bark of cell = membrane = epinephrine receptor = kinase so phosphorylates = barks like dog listening to sers Barking puts P in ears = barking calls over master = Beta-arrestin = beta boy arresting beta receptor = frat boy doesn’t like things that suck up his drugs = jailed up in clathrin = powerful father helps him = MAPK cascade = same as insulin = cell proliferation = jails are for growing

Question 27

What cascade is triggered by Barr and receptor binding?

Answer 27

MAPK

Question 28

How does G protein interact with atp?

Answer 28

P loop interacts with gamma P of ATP Switching off of G protein relates to hydrogen bonds When Pi cleaved - hydrogen bonds of switches break = Galpha protein inactive

Question 29

Cholera toxin

Answer 29

Secreted by bacteria into intestine Breaks into subunits One subunit interacts with small G protein ARF6 and turns off GTPase activity by pairing NAD+ to P loop = permanently active = super high cAMP and PKA activity = efflux of NaCl is constant = water loss = dehydration

Question 30

Different G proteins

Answer 30

Gs and Gq are stimulatory Gi is inhibitory

Question 31

What does Gq alpha activate?

Answer 31

PLC in Membrane

Question 32

What does PLC stand for? What does it do?

Answer 32

Phospholipase C Converts PIP2 to IP3 + DAG IP3 = ca2+ into Cytosol from ER lumen Ca2+ activates PKC = MAPK/ PI3K pathways = cell proliferation/ glycogen synthesis/ glucose uptake Ca2+ activates insulin release in pancreas = but not beta adrenergic receptors on pancreas

Question 33

What does Gi alpha do?

Answer 33

Inhibits AC = lessens conversion of ATP to cAMP = not so much glycogen breakdown

Question 34

Protein kinases in cell

Answer 34

Receptor thyrosine Kinase ==> PKB = PI3K pathway GPCR (Gs) ==> PKA = beta adrenergic pathway GPCR (Gq) ==> PKC = ca2+ Release = Mapck = PI3K pathway

Question 35

Gs with PKA vs Gq with PKC Memory

Answer 35

Gs = beta adrenergic = cAMP = camp on peak A = PKA Gq = Gq Magazine = men’s magazine = pretty little cumberbatch = PLC activated = IP3 = ingenious person 3 times ==> genius = separate from heart = calcifer = or secret to genius is Bones and Milk = ca2+ Heart is moved by performance of cumberbstch = calcifer moves = kinase C Protein kinase C = peak calcium = calcium up = PKC

Question 36

PIP2 structure reminders

Answer 36

2 esters P group Carbon ring with 3x OH, 2x PO3 groups Two p groups on ring = P^2 Esters = good smells = perfume = P at front

Question 37

PIP3 structure reminders

Answer 37

Phospholipid 2 esters - FA chains P group Carbon ring = 2x OH either end of OC connecting to p group, 3x Po4 groups 3 po4 coming off of ring = p^3 Esters = perfume = P at start

Question 38

IP3 structure reminders

Answer 38

P group Carbon ring = 3x OH, 2x P groups 3 p groups total = IP^3