G-proteins & cAMP regulation

oliceridine

g protein-biased ligand of MOR, shows lower rates of gastrointestinal dysfunction and respiratory depression compared to morphine. its analgesic effect is equivalent to that of morphine

what happens with drugs

dopamine receptor affected by cocaine and amphetamines, opiate receptor by heroin, serotonin 5-HT1A receptor by MDMA (ecstasy) -- these are all GPCRs 1. you get high, then you become addicted 2. become addicted because the receptors listed above bind these drugs much tighter than they do their natural ligands. The cell therefore overproduces more and more receptors to try and come back to homeostatic state, so the receptors don't reset properly and the whole signaling cascade downstream is messed up. after exposure, more receptor is needed to get the "normal" physiological response. the body responds by altering gene expression and receptor levels in the brain

The graph shown reflects both ligand binding and response data obtained for a new hormone-receptor. the following can be said about this hormone receptor and the signaling pathway it stimulates a. the receptor is a "direct" receptor, in which hormone binding mediates a direct response b. the receptor is an "indirect" receptor, in which a second messenger mediates a faster response than could be mediated by a direct receptor c. the hormone (ligand) binding correlates well with the response d. the binding of all receptors by the hormone (ligand) is needed to mediate a maximal response e. none of the above

e - none of the above a is incorrect because the graph shows that it is not a 1:1 ratio b is incorrect because the response would be slower c. incorrect because it does not correlate well d. incorrect because only a few receptors need to be bound for maximal response

partial agonist

gives you part of the response

full agonist

gives you same result that normal chem in body does

cAMP binds to

regulatory subunit of Protein kinase A (PKA) & activates it. it changes the shape of the regulatory subunit and releases the Catalytic subunit

mutations in rhodopsin GRK lead to

retinitis pigmentosa

GTPase-accelerating/activating proteins (GAPs)

stimulate the rate of GTP hydrolysis to GDP and inactivate GTPases/G-proteins. they are catalysts for increasing the enzymatic ability of the timer and help hydrolyzing ability of alpha subunit

m-opioid receptor

target of prototypical m-opioid receptor agonist, morphine. morphine has adverse effects such as pain tolerance, constipation and respiratory suppression, thereby limiting its clinical utility. the b-arrestin pathway was found to signal these adverse effects.

GTPase switches

- GTPase switch proteins play important roles in intracellular signal transduction - GTPases are active when bound to GTP and inactive when bound to GTP - the timeframe of activation depends on inherent GTPase activity (the timer function) - GEFs & GAPs

validation of purified Gs

- cholera toxin was already known to covalently link NAD to substrate and also to cause a massive increase in cAMP so it somehow activated AC - had 3 main G proteins in purified membranes, Ga1, Ga2, Gb - sticking covalent-linked NAD onto something in AC-responsive cells leads to massive activation in AC and large increase in cAMP, then added cholera toxin with radioactive NAD to 3 protein columns and now will only stain things that cholera sticks on to. LOOK AT SLIDES

biased activation: both G-protein and arrestin can actually send signals

- the G-protein pathway and b-arrestin pathways are often spatially and temporally distinct, and mediate unique physiological or pathophysiological effects : - initiation of biased GPCR signaling is imparted by GPCR conformational changes - different ligands stabilize distinct active GPCR conformations, which is considered to result in varying degrees of biased signaling towards G proteins or b-arrestins. - current theory = phospho-barcode on the GPCR has an important role in GPCR-biased signaling & the way that GRKs and other molecules phosphorylate sites on the receptor based on the conformational changes and availability of those sites to be phsophorylated. this leads to a stronger receptor signaling/arrestin signaling path

Gs-protein pathway: B-adrenergic receptor

1. epinephrine binds to its specific receptor 2. hormone-receptor complex causes the GDP bound to Gs-alpha (Gsa) to be replaced by GTP, activating Gsa 3. activated Gs-alpha separates from Gs-beta gamma (Gs-By), moves to adenylyl cyclase and activates it. many Gsa subunits may be activated by one occupied receptor 4. adenylyl cyclase catalyzes formation of cAMP 5. cAMP activats PKA 6. phosphorylation of cellular proteins by PKA causes the cellular response to epinephrine 7. cAMP is degraded by phosphodiesterase into AMP, reversing the activation of PKA

inhibitory G-proteins also regulate

AC and cAMP

GEFs vs GAPs

GEFs: help a GDP-bound GTPase (Gprotein) to replace GDP with GTP GAPs: help a GTP-bound GTPase to accelerate its own INHERENT GTPase enzymatic activity to cleave the terminal phosphate group from the GTP to turn it into GDP, thus inactivating the GTPase

G-protein activation/inactivation process

GTP binds to alpha subunit of trimeric G protein -> activates it and dissociates into Ga unit and a Gby complex --> Ga unit activates next part of the sequence, then hydrolyses GTP which inactivates this subunit and causes it to dissociate from the target protein --> inactive alpha subunit reassembles with By complex to re-form inactive G-protein

alpha subunit of G-protein itself is a

GTPase - this reflects how the system is a timer system that shuts itself off

G proteins are also called

GTPases or guanine nucleotide-binding proteins

G-protein coupled receptor

a plasma membrane receptor that works with the help of a G protein, embedded in the plasma membrane

retinitis pigmentosa

a progressive degeneration of the retina and can be caused by mutations in rhodopsin's ability to activate a G protein.

3 broad subclasses of trimeric-GPCR linked effector proteins

a. adenylyl cyclase b. phospholipase C c. ion channels these are effectors, and can have different effectors/different second messengers downstream which each target different second messengers

G-protein gated ion channels

a. effector can interact with ion channel to open b. Ga subunit can interact with ion channel c. GBy subunit (s) can interact with ion channel

Binding of signal to GPCR

activates the GPCR --> stimulates GDP/GTP exchange on the G protein

G protein

acts as an off/on switch: if GDP is bound to the G protein, it is inactive. If GTP is bound, it becomes active.

caffeine acts to mimic a mild "adrenaline rush" in the following ways: a. it is an analogue of the neurotransmitter, adenosine, which acts in the brain to prevent sleep b. it is a blocker of cAMP phosphodiesterase, resulting in the accumulation of cAMP in a variety of cells/tissues c. it stimulates adenylyl cyclase, resulting in increased cAMP production d. all of the above

b a is incorrect because it adenosine PROMOTES sleep c is incorrect because caffeine doesn't stimulate adenylyl cyclase

the following is true of the first crude reconstitution of the adrenaline receptor response using red blood cells and adrenal cortical cells performed in rodbells lab: a. they mixed rbc membrane fractions containing AR with adrenal cortical cell cytoplasmic extracts containing AC b. they fused adrenal cortical cells that lacked adrenaline receptors to RBCs, which as a result of NEM treatment, lacked AC activity c. the reconstitution demonstrated that sutherland's model was correct in that the adrenaline receptor and AC behaved as a single entity d. none of the above

b is correct a is incorrect because the rbcs had both AR and AC c is incorrect because it showed that they were separate entities

carvedilol

b-arrestin-biased agonist for b-AR, has been demonstrated to inhibit the G protein-mediated toxic effect of catecholamines (dopamine, norepinephrine, adrenalin) and stimulate b-arrestin-mediated cell survival signaling pathways. large scale clinical trials indicated that carvedilol may significantly improve survival in patients with heart failure compared with other nonbiased b-blockers. lowers heart rate, blood pressure and strain on the heart

biased signaling can be encoded through 3 general mechanisms

biased ligand biased receptor biased system

inverse agonist

binds and shuts down the system

desensitization

blocking active receptors from turning on additional G proteins homologous - blocking one receptor lowers the response for that pathway heterogenous - blocks both receptors' pathways even though only one receptor is actually blocked. depends on receptor phosphorylation

phosphodiesterase enzymes catalyze

cAMP + H20 --> AMP

regulatory subunit is bound to

catalytic subunit and inhibits its enzymatic activity when reg unit is not bound to cAMP

vibrio cholera

causes cholera. in a normal gut: H2O, NaCl, NaHCO3 secretion controlled by hormones via Gs/cAMP signal pathways V. cholera secretes enterotoxin, chemically modifies alpha-Gs which results in no GTPase activity so it stays on and doesn't respond to GAPs or itself. this results in a massive signal cascade of cAMP which leads to severe watery diarrhea because cholera opens ion channels in the gut ==> dehydration/death

biased system

cell itself can give more priority toward a specific path & doesn't really matter which ligand or receptor binds

arrestins

compete with G proteins to bind phosphorylated GPCRs. upon binding, the GPCRs become desensitized, even though ligands are bound extracellularly. Brings the receptor into the cell as an endosome, which then potentially acts as its own platform for signaling, either independently or in the same initial pathway.

biased ligand

creates conformational shift that pushes more towards arrestin or GPCR pathway

ways to terminate the response completely

desensitization & turnover - GTPase autotimer - G-protein reassociates - G protein-coupled receptor kinase (GRK) phosphorylates a GPCR - arrestins - accelerated by GAPs or regulators of G protein signaling (RGSs)

Alfred gilman's experiment to try and falsify rodbell's new model for adrenaline response

didn't believe in Rodbell's transducer. - used S49 lymphoma cells that have persistent division: -> increasing cAMP in media = cells stop dividing -> treatment of cells with adrenaline = cells stop dividing. adrenaline signaling through cAMP tells cancer cells to stop dividing - created S49 cyc-mutants ("cyclase-") and observed effects on response to adrenaline and response to forskolin. If the mutation is in the AC, then you would expect cells to continue to grow even when you add adrenaline problem: can you assume its just the cyclase that is mutated? no - could be receptor instead, tested for that with radioactive adrenaline binding FOUND: - some of these cells that DO have adrenaline receptor (binding) but no adrenaline response (don't stop dividing) still respond to forskolin which directly activates AC (stop dividing) - this means they must have a functional AC - therefore there must be another mutation in another part of the pathway (transducer) ==> proved rodbell was right - the cyc- cells didn't lack AC, they lacked the G protein

biased receptor

different isoforms of receptor bind to same ligand, but isoform 1 is more towards receptor signaling. isoform 2 towards arrestin

different second messengers have

different target proteins

resensitized

if receptors are recycled and returned to cell surface after being internalized by arrestin, the cell remains sensitive to the ligand

what provided the first evidence suggesting a transducer of receptor action. explain the steps

in vitro reconstitution using purified membranes. purified membranes + hormone (adrenaline) + ATP --> cAMP - some purchased lots of ATP worked, some didn't - pure ATP that rodbell made in the lab did not work. They did not get an active AC to produce cAMP - adding GTP worked - during the reaction, GTP decreased and GDP increased, showing that the source leading to activation was GTP not ATP - GTPase (breaks down GTP) terminated AC activation - non-hydrolysable GTP analogue causes prolonged activation - they found out the original ATP lots purchased had some that were contaminated with GTP, which is why some lots worked and some didn't.

class B receptors

interact strongly with b-arrestin and the complexes are targeted to endosomes for subsequent G protein-coupled receptor (GPCR) degradation and ERK1/2 activation. leads to sustained signaling because its more stable

Class A receptors

interact with b-arresting and the complexes are targeted to clathrin-coated pits for subsequent ERK1/2 activation near the memrbane. process is transient (happens near membrane) and is followed by rapid recycling (or degradation) of the receptors back to the plasma membrane

Internalization of b-arrestin

internalized without forming a complex with the b1-adrenergic (b1-AR), b-arrestin briefly kisses b1-AR, locates to clathrin-coated structures at the plasma membrane, and then activates ERK1/2

antagonist

keeps you at baseline, stops additional signaling by your own chemicals

disorders associated with G protein-coupled receptors

loss of function mutation that results in a nonfunctional signaling pathway. gain of function mutations may create a constitutively activated G protein. certain polymorphisms in G protein-related genes may result in an increased susceptibility to asthma or High blood pressure & decreased susceptibility to HIV retinitis pigmentosa, some benign thyroid tumors, certain polymorphisms in G protein-related genes

different types of G protein

monomeric: only has 1 subunit trimeric: 3 subunits (alpha, beta, gamma)

is it enough to just turn off cAMP?

no - have to actually turn off the receptor or else G proteins will just keep activating AC and this will keep making more cAMP

GPCR that is activated stimulates GRK to

phosphorylate the GPCR on multiple sites, then arrestin binds to the phosphorylated GPCR

G protein-coupled receptor kinase (GRK)

phosphorylates a GPCR

cAMP-phosphodiesterase is activated by

phosphorylation catalyzed by PKA. It is also activated by binding to cAMP/PKA-R (regulatory) subunit. therefore cAMP stimulates its OWN degradation, leading to rapid shutoff of cAMP signal

Guanine nucleotide-exchange factors: GEFs

proteins that promote the replacement of bound GDP molecule for a NEW molecule of GTP, thereby activating GTPases (aka G proteins) . This is NOT activating the inherent GTPase activity, it actually activates the Ga subunit

what did rodbell theorize about the adrenaline receptor pathway

that there must be a "transducer" that acts between the receptors and the adenylyl cyclase. predicted that the mechanism was: adrenaline --> binds to adrenaline receptor --> activates transducer ---> interacts with adenylyl cyclase ----> converts ATP to cAMP -----> mediates adrenaline response ----> cAMP phosphodiesterase breaks down cAMP to AMP

all activation/associations based on affinity so that means

they are based on structure/morphological changes and changes in amino acid side chains exposed

inactive PKA made of

two regulatory subunits and 2 catalytic subunits

whooping cough

very severe coughing, highly contagious. -some G-protein coupled receptors are inhibitory, for instance alpha-Gi of GiPCR inhibits adenylyl cyclase -pertussis toxin binds to immune T cells, inhibits alpha-Gi which leads to prolonged activation of AC - blocking of alpha-Gi results in prolonged activation of adenylyl cyclase ==> increase in cAMP levels ==> impairment of immune cells ==> can't clear out infection - bordetella pertussis produces the pertussis toxin, which ADP-ribosylates Gi-alpha and inhibits nucleotide exchange, so it can't go from inactive state to active inhibitory state