G Proteins Flashcards
G Protein Definition
Guanosine nucleotide binding proteins
GPCR details
G Protein coupled receptors
- Plasma membrane receptor = 7 transmembrane helical segments
- G protein active with GTP
- Effector enzyme in plasma membrane activated by G protein
Generic pathway for G proteins
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
Two big functions of GPCRs
Detect hormones/growth factors
Aid in smell/ taste
Beta adrenergic receptor
Base form for GPCRs
Mediates epinephrine effects = adrenaline
Where are beta adrenergic receptors located?
Muscle, liver, adipose tissue
On membrane
G Protein structure
3 subunits:
alpha = binding site for GTP or gdp = GTPase functionality
GPCR is transmembrane component not G protein
Beta
Gamma
When gdp bound = heterotrimeric
When is Gs accurate?
Stimulated G protein = activated = GTP
AC Enzyme function
Adenylyl Cyclase = integral plasma membrane protein
Synthesizes cAMP from ATP = removes PPi
Stimulated by Galpha protein
ATP structure reminders
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 !!!
cAMP structure reminders
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
AMP structure reminders
Like cAMP but p is not interacting with glucose ring
Nitrogen, glucose, phosphorus
cAMP minus the c = comrades
Cyclic nucleotide phosphodiesterase
cAMP -> AMP via hydrolysis
What does cAMP activate?
Protein kinase A
= PKA
Memory: the camp at peak A = best mountain camp around = fight or flight
Why is PKA important?
Key to phosphorylation of important ser or thr residues of proteins for fight or flight response
How many cAMPs to activate PKA?
4
2 per regulatory subunit
PKA structure details
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
PKA consensus sequence
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
FRET
Fluorescence resonance energy transfer
Fret proportional to (1/d^6) between fluorescent proteins
Lower fluorescence if close enough
FRET for detecting cAMP in cells
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
FRET for PKA activity in cells
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
Epinephrine pathway
In hepatocyte
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
1 molecule epinephrine is how many molecules of glucose released?
100,000
Mechanisms for terminating b adrenergic receptor response
-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
Desensitization mechanism for beta adrenergic receptor
-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
Desensitization mechanism memory
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
What cascade is triggered by Barr and receptor binding?
MAPK
How does G protein interact with atp?
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
Cholera toxin
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
Different G proteins
Gs and Gq are stimulatory
Gi is inhibitory
What does Gq alpha activate?
PLC in Membrane
What does PLC stand for? What does it do?
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
What does Gi alpha do?
Inhibits AC
= lessens conversion of ATP to cAMP = not so much glycogen breakdown
Protein kinases in cell
Receptor thyrosine Kinase ==> PKB = PI3K pathway
GPCR (Gs) ==> PKA = beta adrenergic pathway
GPCR (Gq) ==> PKC = ca2+ Release = Mapck = PI3K pathway
Gs with PKA vs Gq with PKC Memory
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
PIP2 structure reminders
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
PIP3 structure reminders
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
IP3 structure reminders
P group
Carbon ring = 3x OH, 2x P groups
3 p groups total = IP^3
