GTP binding Proteins-- Ellis
What is one therapeutic strategy to prevent HF?
phosphorylating the B1 adrenergic GPCR receptor (and therefore downregulating its signaling)
What is the original source of energy for the muscle?
glycogen
2 configurations of the GTP binding proteins
1. ON: GTP bound state 2. OFF: GDP bound state
describe epinephrines effects on cardiac and smooth muscle (sympathetic NS)
1. increased cardiac output 2. decreased flow (contraction) to the general circulation 3. increased flow (relaxation) to the skeletal muscle circulation
Gs versus Gi
Gi= inhibitory G protein. It will inhibit adenyl cyclase and will inhibit the production of cAMP--> relaxation Gs= stimulatory G protein. It will stimulate adenylyl cyclase and will increase the production of cAMP--> contraction
list some receptors on platelets (and what each of them interact with)
GpIb: interacts with VWF GpIV: interacts with collagen GpVI: interacts with collagen *Integrin αIIB- β3* (pathologists called it GpIIb-IIIa): interacts with VWF, fibrinogen, fibronectin *GPCR*: interact with TXA2, thrombin, ADP
Role of beta blocker
-*normally, beta agonists will stimulate the heart* -therefore you would think that beta blockers would inhibit the heart (therefore it would be bad to give to someone with heart failure) -but actually, Beta 1 adrenergic receptors play a large role in causing the HF; therefore you want to block beta 1 (without blocking beta 2 or alpha adrenergic receptors)
A 54-year-old man with a history of myocardial infarction (MI) presented with exertional dyspnea. Physical examination was unremarkable. Left ventricular ejection fraction (LVEF) by Doppler echocardiography was 35%, and a stress test was negative for ischemia. The patient was taking aspirin, a statin, and an angiotensin converting enzyme (ACE) inhibitor. He was started on 12.5 mg/d metoprolol controlled-release/extended release (CR/XL) and titrated to a target dose of 200 mg/d over several weeks. describe the meanings behind these clinal findings and describe what each medicine is
-35% LVEF tells you that his heart isn't working well -however, not having ischemia tells you that he did not have a second MI -this leads you to the conclusion that he has heart failure medications: 1. aspirin: is used to stop platelets from clotting and prevent another HA. targets a GPCR (Gq or G13) 2. Statin: is used to lower cholesterol and to prevent HA. does not target GPCR 3. ACE inhibitor: will relax smooth muscle (blood vessels) because the kidney cannot make angiotensin II; therefore the kidney is not signaling for contraction. Therefore his failing heart will not have to do as much work. This will vasodialate his heart. It acts on a GPCR 4. metoprolol: is a *beta blocker*. This is also a GPCR
GRKs
-AKA G protein coupled receptor kinases -phosphorylates GPCR on multiple sites -the arrestin will then come in and bind to those phosphates GRKs plays a role in desensitization of the receptor
GIRK
-AKA G protein regulated inward rectifier potassium channels -used the in the heart muscle cells during parasympathetic response (due to changes in the beta and gamma subunit of Gi) -it works to hyperpolarize the membrane to prevent AP form stimulating contraction--> overall causing relaxation of the cardiac muscle (leading to decreased heart rate)
function of GTP-binding proteins
-GTP-binding proteins bind and hydrolyze the GTP to GDP; or GDP to GTP -this serves as a molecular ON/OFF switch that controls many different processes in the human body
describe the connection between heart failure and HTN
-HTN can lead to HF -HTN will cause chronic stimulation of the sympathetic NS on the heart to help compensate for the increased BP (this may actually be good for the body) -the chronic sympathetic stimulation will cause the heart to always be working harder--> it can lead to HF
PAR mechanism of action
-PAR: protease activator receptor 1. thrombin interacts with PAR 2. this will cause thrombin to cleave the receptor; creating a new receptor. 3. the new amino terminus on the receptor will then serve as a ligand for the receptor
transducin
-a special name for the heterotrimeric GTPases for rhodopsin signaling -has a special name because it was the first one discovered -it has an alpha, beta, and gamma subunit like all the other heterotrimeric GTPases
structure of integrins (located on the platelet)
-all integrins have an alpha and a beta subunit -the integrins span the cell membrane and they can interact with intracellular and/or extracellular proteins
Describe the GPCRs in olfaction
-almost half (339/800) of the GPCRs we have are involved in the sense of smell -however, if an olfactory neuron expressed all of these GPCRs at the same time, we would not be able to distinguish between different smells. To avoid this, *you only have 1 GPCR expressed per olfactory neuron* (other types of neurons will have multiple GPCRs so this is specific to olfactory neurons)
seven membrane spanning helices (include the structure)
-another name for G protein coupled receptors (GPCR) -this is because they span the membrane 7 times -they are made up of alpha helixes (this is the perfect structure because it allows the hydrophilic AA to be sequestered on the inside; therefore the hydrophobic AA can interact with the hydrophobic, lipid plasma membrane)
receptor recycling
-arrestin promotes the internalization of the receptor -The receptor can then go to the lysosome and get degraded--> an overall reduction in the amount of receptors (but this isn't necessary for desensitization to occur) -it can also strip the GPCR clear of the phosphates and recycle it back (therefore it can now bind and be activated--> downstream signaling effects)
Why does epinephrine not bind to the beta 2 adrenergic receptor in the fasted state like it does in skeletal muscle?
-because the glucagon is undergoing its mechanism of action by binding to the beta adrenergic receptors already -you do not want them to act on the same receptor; therefore the epinephrine binds to the alpha 1 adrenergic receptor
glucagon
-breaks down glycogen in the liver during the fed state -it acts through the GPCR (called the glucagon receptor)
compare and contrast cis and trans bonds. What is the importance of this in rhodopsin signaling
-cis: double bond is on the same side -trans double bond is on opposite sides cis bonds have a lower melting point (it is easier for them to melt) because the stereochemistry prevents them from packing together as tightly -therefore, *the inactive retinal ligand has a lower melting point than the active retinal ligand of rhodopsin*
mechanism of action for drugs used for depression and the side effects
-depression medications target serotonin receptors (agonists because they enhance the serotonin receptor) -however, serotonin also plays a role in the GI tract (it increases water uptake in the GI tract) -side effects: therefore a medication that increases serotonin will cause increased water uptake in the GI tract--> diarrhea
GPCR desensitization (include the 2 types)
-desenitization is prolonged -2 types: 1. homologous: loss of activity of a single type of receptor in response to agonist binding. There is desensitization of the target receptor itself 2. heterologous: simultaneous loss in activity (desensitization) of multiple subtypes-- sometimes even in the absence of agonists. You are desensitizing the downstream signaling pathways
Briefly describe how we ensure we only have 1 GPCR per olfactory neuron
-during development the olfactory neuron will just randomly pick 1 of the epigenetically silenced genes (it is methylated, therefore it is inactive) -it then shifts from the receptor being epigenetically silenced (methylated) to being in the activated state -however, it is possible that during the development the neuron will randomly pick up a pseudogene instead of a gene for a functional olfactory GPCR. This would cause the olfaction to not be functional in the neuron
epinephrine's role in regulation of glycolysis of the skeletal muscle
-epinephrine is the fight or flight hormone -it can further promote glycogen breakdown, due to its interaction with *adrenergic receptors* -adrenergic receptors are GPCRs
compare and contrast the sense of smell between humans and mice
-humans do not use their sense of smell nearly as much. We have 339 GPCRs dedicated to smell -mice have over 1,000 GPCRs involved in olfaction
Why is pharmacology stupid?
-in pharmacodynamics the drug target is often referred to as a receptor. This terminology probably began because a ton (40% of medications) target GPCR (which are actual receptors) -however, this word is misleading because not all drug targets are receptors; drug targets can also be things like enzymes, RNA, and DNA -so pharmacologists use the word receptor (R) to describe what the drug acts on; but biochemists use the word target (T) (because not all targets of drugs are receptors)
Plavix (clopidoogrel)-- mechanism of action and what it is used for
-inhibits ADP from binding to P2Y12; therefore it inhibits Gi 1. Gi inhibits adenyl cyclase; therefore inhibiting Gi will increase adenylyl cyclase activity. 2. Increased adenyl cyclase will cause an increase in cAMP 3. *cAMP inhibits platelet activation*; therefore increased levels of cAMP will decrease the stickiness and cause anti-aggregation of platelets -used to reduced the risk for HA and stroke
classical view of desensitization
-is INCORRECT -said that phosphorylation was responsible for the desensitization carried out by downstream kinases in the 2nd messenger pathways -basically they thought that downstream kinases (like PKC and PKA) were causing the phosphorylation of the receptor
heterotrimeric GTPases (structure)
-is a large ATPase -has 3 subunits: alpha, beta, and gamma -the alpha subunit binds GTP (the beta and gamma subunits do not) -there is a tremendous degree of complexity with the hereotrimeric GTPases because there is a lot of different alpha, beta, and gamma subunits
Dynamin family (role and pathway)
-is a large GTPase that is involved in creating vesicles 1. the vesicles will bud off of the membranes 2. the vesicles will continue to bud until they have reached a defined size (they are still attached to the plasma membrane though) 3. The dyamin (in the ON state) will come in and aggregate around the neck of the budding vesicle 4. *GTP hydrolysis then occurs* (converts GTP to GDP) 5. This will allow the vesicle to be popped off from the membrane, and the vesicle will be released from the membrane
ACE inhibitor mechanism of action
-it blocks the production of angiotensin II -therefore it will stop contraction signal of the smooth muscle arterioles of the general circulation -this promotes relaxation -it is often used in HF patients because it allows the heart to relax and not have to pump as hard
IP receptor
-it is a GPCR 1. PGI2 (an inhibitory prostaglandin) binds to the GPCR IP receptor 2. it is associated with a *Gs* (therefore it activates adenylate cyclase) 3. increased cAMP will cause platelet activation to decrease (you are less likely to aggregate platelets and form a clot) role: *shuts down coagulation*
Gαt (definition and pathway)
-it is a special name for the G alpha subunit for the rhodopsin signaling 1. it works by *activating a phosphodiesterase* which cleaves and inactivates cGMP 2. this shuts down the cGMP regulated channel 3. the membrane becomes *hyperpolarized* 4. this transmits a signal to the brain (note: normally in other tissues depolarization will cause signaling to the brain)
Describe the switch between ON to OFF
-it is turned off by *GTP hydrolysis*: GTP is replaced by GDP -this causes the OFF state to be re-instated (until you get a new input)
Describe Ras role in cancer
-it works by connecting the tyrosine kinase with the cell proliferation cascade -some mutations will activate Ras and cause cancer; this is because the *GTPase activity gets reduced* (therefore it is staying in the ON conformation)
What is the major receptor in the heart? What are some other minor ones?
-major: beta 1 adrenergic receptor -minor: beta 2 adrenergic receptor alpha adrenergic receptors
Describe how vesicles are recycled. what GTPase is involved in this?
-new vesicles are formed by the recycling of phospholipids (this is kind of like you are recycling the vesicles) 1. vesicles are endocytosized 2. the vesicles are recycled and re-clustered 3. the vesicles to get re-loaded with neurotransmitter 4. the vesicles are then ready to bud off the membrane again -*dynamin* (a large GTPase) plays an important role in this recycling (it is involved in the endocytosis of the vesicle back into the membrane)
Describe the mechanism of action for opioids and the side effects
-opioid medications target the *OPR receptors* (a GPCR for opioid drugs) -the opioids will raise the threshold for the pain sensations to reach the brain (therefore it will diminish pain) -the opioids will also inhibit the negative inhibition (AKA stimulate) the pleasure centers i the brain-- this is why taking opioids causes euphoria -side effects: opioids can cause respiratory depression and euphoria
Ras
-part of the small GTPase superfamily -will *regulate cell proliferation* (and other diverse behaviors) -there are around 30 Ras proteins in the GTPase superfamily
Rab
-part of the small GTPase superfamily -is involved in *vesicle trafficking* -the vesicles ultimately fuse with the plasma membrane. This allows the vesicles to dump their contents
Arf
-part of the small GTPase superfamily -it *regulates vesicles with the cytoskeleton* (like actin); therefore it is involved in vesicle transport-- the cytoskeleton is what actually moves the vesicles throughout the body
Ran
-part of the small GTPase superfamily -it is important in *transporting* cargo into and out of the *nucleus*
Rho
-part of the small GTPase superfamily -it is involved in *cytoskeletal dynamics*
arrestin (include its 2 roles)
-prevents signaling from happening -arrestin binds to the G alpha subunit -if arrestin is bound, the receptor cannot bind its G alpha subunit and therefore cannot cause downstream signaling (the receptor is desensitized) -arrestin can also promote the internalization of the receptor -all you need to have desensitization is to have arrestin bound to the G protein (you don't need it to be internalized)
cAMP
-produced from adenyl cyclase -it *inhibits platelet activation* (aka means you are less likely to form a clot) -it also causes contraction -it is a target for preventing HA and stroke
example of a heterologous GPCR desensitization
-prolonged epinephrine can desensitize ATR1 (even though it isn't stimulated) because of effects on the common downstream signaling pathways -this shows how it affects downstream signaling pathways, rather than the receptor itself
example of homologous GPCR desensitization
-prolonged epinephrine stimulation of the smooth muscle can cause desensitization to the alpha adrenergic receptor -therefore, in order to get the same effect on contraction of the general smooth muscle you need more epinephrine -this shows the desensitization of the alpha adrenergic receptor
What specially causes the desensitization to epinephrine? How does this change the GPCR?
-prolonged stimulation will cause the receptor to be phosphorylated (beta 1 adrenergic receptor) -the 3rd loop (5th and 6th helix) of the receptor gets phosphorylated during desensitization
P2Y1
-receptor for ADP on the platelet -interacts with Gq -it is not critical for the anti-platelet action of clopidogril
Describe 2 reasons behind side effects of drugs that target GPCRs
-remember: different GPCR have similarities (they are the same family of molecule) -because of these similarities, the drugs that target GPCR will often cause similar side effects because: 1. they affect the same GPCR in an off-target organ in the body OR 2. because it affects a different GPCR that has a similar enough structure to the GPCR the drug means to target
Describe the ligand in rhodopsin signaling
-remember: rhodopsin signaling is NOT ligand mediated 1. light enters 2. the ligand (retinal) undergoes a conformational change: it isomerizes the cis double bond to a trans double bond 3. this alters the conformation of rhodopsin 4. this alters the relationship with Gαt -in the inactive state the retinal is kinked (it has a cis double bound at position 11 of the hydrocarbon chain) -in the active state, the retinal is unkinked (the hydrocarbon tail now has a trans double bond at position 11)
describe the ligand binding domain of rhodopsin receptors
-rhodopsin is stimulated by light -it is weird because its binding pocket doesn't need to be exposed to the external environment (this is different from other receptors)
Describe rhodopsin signaling
-rhodopsin signaling is achieved through a GPCR -the GPCR senses and is activated by photons of light (instead of a ligand like other GPCRs)-- it is not ligand mediated -because the GPCR does not have a ligand bind to it in order to be activated, the binding site is closed (all other GPCR binding sites are open in a binding pocket so that the ligand can bind) -the ligand (*retinol*) is ALWAYS bound to the rhodopsin (even in the inactive state); this is different from other GPCRs that have to be activated by having the ligand bind to them
Gi (include the 2 pathways)
-stands for inhibitory G protein: it inhibits adenylyl cyclase (and therefore decrease cAMP levels) -P2Y12 binds here (therefore this is the target for clopidogrel) 2 pathways (side pathway): probably not important for us to know 1. P2Y12 binds to Gi 2. this affect the *beta-gamma subunit* (other G protein activation affects the alpha subunit) 3. PI-3-K is activated 4. PIP3 5. increases Rap1 main pathway: 1. P2Y12 binds to Gi 2. Gi inhibits the adenylyl cyclase 3. this causes cAMP levels to decrease--> *less contraction*
Describe changes in the receptors the body makes when it has HF. What are the good and bad players involved in HF?
-the beta 2 (with chronic sympathetic stimulation) will actually switch over and start interacting with Gi, instead of the Gs it normally interacts with. This may be a part of physiological compensation -a failing heart will switch from increasing cAMP to decreasing cAMP (tells you that cAMP may be the bad player that causes HF). The increased cAMP causes heart contraction; the *decreased cAMP causes heart relaxation* -a patient with HF will increase the amount of alpha adrenergic receptors (alpha 1-A and alpha 1-B). The alpha receptors are pro-survival (they are a good thing). They will be protective to cardiomyocytes/try to save heart cells -beta 1 is the bad guy. it will induce apoptosis of cardiomyocytes and promote proliferation of cardiac fibroblasts--> fibrosis of heart--> HF
What is the problem with GPCR signaling in the smooth muscle cells of the general arteriole circulation?
-the contraction of the smooth muscle will cause an increase in your BP -you want the blood to flow to skeletal muscles by dilating the blood vessels that supply the legs -you want to cut off blood supply to the places you do not need blood (like the stomach to digest)
Describe how the GPCR are similar/different
-the different GPCR will have different AA in the pocket that binds ligand. *the size of the pocket and the AA side chains in the pocket determine what ligands will bind in the pocket* -the GPCR have similar AA (because they all have the 7 helices that span the membrane); but they will have divergent AA sequences in the pocket that will allow them to bind different things
mechanism of drugs for BP and heart failure and the reason behind the side effects
-the drugs are *antagonists that block the adrenergic receptors*; therefore the adrenergic receptors don't function -but you get side effects because adrenergic receptors are located in other areas besides just the heart -therefore these drugs will block adrenergic receptors that are also located in other tissues (like the liver and muscle)
describe the association the heterotrimeric GTPases have
-the heterotrimeric GTPases are *associated with the GPCR* -the heterotrimeric GTPase is important in the formation of the GPCR -The GPCR will be inactive when GDP is bound to the alpha subunit -The GPCR will be activated when GTP is bound to the alpha subunit of the heterotrimeric GTPase
describe the difference between the active and the inactive platelet. What causes this difference?
-the inactive platelet all be discoid in shape -the active platelet will have projections coming off of it. the projections are due to the actin cytoskeleton -inactive platelets have the actin cytoskeleton underneath the plasma membrane. when platelet activation occurs, the signaling that occurs will begin to chop up the original actin cytoskeleton and you will start building the actin cytoskeleton back up in different patterns and shapes -this change in actin configurations is due to the *GTPases*
Describe the role of the kidney in smooth muscle contraction. What drugs inhibit this?
-the kidney produces angiotensin II to alter the blood pressure (by causing contraction of the general smooth muscle arterial system) -*ACE inhibitors*: blocks angiotensin II and therefore it blocks contraction of the general arterial smooth muscle (and causes relaxation). It is prescribed to patients with heart failure
generally describe the GPCR family
-there are ~800 different GPCR in the human body -the families are very different; but have similar roles and certain structures that are similar (hence why they are considered to be in the same family)
Why are GPCR a good target for drugs?
-they are exposed to the external environment; therefore they are easily available for drugs to bind to them -the drugs do not have to cross the cell membrane in order to assert its effect
Describe the overlap between G13 and Gq
-they overlap at MLCK -G13 pathway: MPase will increase MLCK -Gq pathway: increase Ca2+ levels will increase MLCK remember: MLCK will activate myosin to interact with the actin--> smooth muscle contraction and a change in the shape
GTP-gamma S
-this is a state where *GTP can't be hydrolyzed to GDP* -this will cause the dynamin to accumulate around the head of the vesicle that is still fused to the membrane -however, the *vesicle will not be able to detach from the membrane* (because the GTP can't be converted to GDP) -overall you will get elongated helices on the budding vesicle
compare and contrast TXA2 and PGI2
-thromboxane A2: causes platelet coagulation through the Gq signaling -PGI2: inhibits platelet coagulation through IP receptor and Gs signaling
aspirin
-used to inhibit production of TXA2 -TXA2 binds to the G13 and Gq pathway on the platelet; therefore the G13 and Gq pathways are blocked -this will cause platelets to be less prone to change in shape and will cause them to be less sticky; therefore aspirin is *used to prevent thrombi/coagulation*
describe the drug used to treat movement disorders
-uses dopamine -dopamine is an agonist (it will enhance the receptor)
What would be the perfect beta blocker for someone with HF?
-want to block beta 1 -do not want to block beta 2 (because beta 2 can switch over to start decreasing cAMP--> decreasing calcium--> decreasing contraction)
GTPase Activator Proteins (GAP)
-will cause *GTP hydrolysis* -therefore it converts the ON confirmation to the OFF confirmation (GTP is replaced by GDP)
Guanine Exchange Factors (GEF)
-will cause the *nucleotide exchange* (cause GDP to leave and GTP to bind to the active site) -therefore it converts the GTP binding protein from the OFF to the ON conformation
Describe why you get side effects with GPCR blockers (like beta blockers, etc)
-you have the same GPCR receptors in many different areas of the body. this will cause side effects in off-target tissues -you have to weigh the costs and rewards of GPCR blockers before you prescribe it (it does you no good to treat bladder incontinence if you will cause GI problems in the process)-- just an example I made up
How do you get maximal induction of glycogenolysis in skeletal muscle?
-you need the fight or flight/epinephrine response PLUS the increased calcium response -these are 2 different pathways to induce glycogenolysis; when both of these pathways are working you get maximal activation of glycogenolysis
describe the switch between OFF to ON
-you will switch between the ON and OFF signals 1. typically the switch is on the OFF state (GDP bound) 2. but in response to some sort of input (there a ton of vastly different input signals), the GDP will leave 3. *nucleotide exchange*: GTP now binds to the spot where GDP was (the binding site) 4. This causes the switch to be turned ON 5. the ON switch will cause an output signal that will change conformation in the cell
Describe the sympathetic GPCR signaling in smooth muscle cells of the skeletal muscle circulation
-your goal is to get the arteries of the skeletal muscles to relax so that you can deliver more blood to the skeletal muscles. You do not want these to be contracted -*in smooth muscle cAMP stimulates muscle contraction*; therefore during sympathetic stimulation you would want to decrease contraction to the muscle by ??? 1. epinephrine binds to the *beta 2 adrenergic* receptor 2. this stimulates *Gs* 3. this will stimulate adenyl cyclase and therefore increase cAMP 4. *in smooth muscle, cAMP promotes relaxation* (in the cardiac muscle cAMP causes contraction) 5. activated cAMP inhibits the MLCK--> relaxation 6. cAMP also activates protein kinase C; therefore you inhibit the regulatory system needed for contraction (ultimately causing relaxation)
describe the parasympathetic nerve signaling in the heart muscle cells (myocytes)
1. *Ach* binds to the *M2 Muscarinic receptor* 2. this will cause GTP to bind to the Gi protein 3. *Gi* will inhibit adenylyl cyclase--> reduced cAMP levels--> inactivates protein kinase and causes a decrease in calcium 4. the *decreased calcium will cause decreased contraction* in the heart rate (AKA causes relaxation of the heart muscle)--> slower heart rate
describe the mechanism behind activating platelets
1. *Gq* protein will lead to an increased in calcium (through the normal signaling of *IP3*) 2. the *increased calcium* in the platelet will influence MLCK 3. The *MLCK* will increase the number of contractile elements (actin and myosin) 3. the actin and myosin contraction and relaxation will drive the *platelet shape* and changes in the vesicle trafficking 4. the actin and myosin will also facilitate *projections and vesicle movement* throughout the cell
How can Ach have an effect on the basal heart rate? (parasympathetic NS)
1. Ach binds to M2 muscarinic receptor 2. this activates Gi and *changes the beta and gamma subunits* 3. beta-gamma subunit then induces *GIRK* 4. GIRK hyperpolarizes the membrane of myosin; therefore the AP is harder to of through and stimulate the contraction 5. this will cause the heart rate to slow down (slows down the contraction of the heart)--> relaxation remember: basal HR is affected through the beta and gamma subunits; NOT through the cAMP pathway (that just affects the heart rate in the rest and digest)
What are the 2 arms of the different pathways that will regulate glycolysis in the muscle?
1. Calcium 2. epinephrine
Overall describe how phosphorylation of the GPCR receptor happens
1. GRKs phosphorylates the GPCR 2. arrestin then comes in and binds to the phosphorylation (it will specially bind to the G alpha subunit) 3. this means the G alpha cannot activate the downstream signaling pathway (even if a ligand is bound to GPCR and activating it)
list the regulators of the GTP-binding proteins
1. Guanine Exchange Factors (*GEF*): causes nucleotide exchange. Converts GDP to GTP (goes from OFF to ON) 2. GTPase Activator Proteins (*GAP*): causes GTP hydrolysis. Converts GTP to GDP (goes from ON to OFF)
describe the pathway of how the GPCR works and how the signaling stops
1. Ligand binds to the GPCR and activates it 2. This causes a conformational change in helix 5 and 6 (they are elongated) 3. This causes the switch from GDP to GTP (GDP leaves and a GTP binds to the same spot) 4. The extra phosphate changes the conformation of the G alpha subunit (so the alpha subunit can interact with the GPCR in a new way) 5. This changes the conformation/relationship with the beta-gamma complex (like Gs). 6. these changes allow the conformation of alpha subunit to change and makes it available for downstream signaling 7. The beta-gamma complex is now available for interaction for downstream interactions (but the main player of downstream signaling pathways is the G alpha subunit) 8. the GTPase activity of the alpha subunit (GTP converted to GDP) will return the system back to the inactive state This is an example of GPCR acting like a GEF
describe the mechanism of G13
1. TXA2 or thrombin bind to TP or PAR 2. TP and PAR interact with G13 3. G13 activates *Rho-GEF* 4. Rho-GEF acts on *RhoA* 5. RhoA activates *ROCK* (a protein kinase) 6. ROCK *inactivates myosin phosphatase (MPase)* 7. this *stimulates the myosin-light chain kinase* (MLCK) 8. MLCK will *activate myosin* to interact with the actin--> smooth muscle *contraction* and a change in the shape
pathway of Gq
1. TXA2, ADP, or thrombin bind to their receptors; which activates Gq 2. Gq stimulates phospholipase C (*PLC*) 3. PLC releases IP3 OR DAG a. IP3 will cause an *increase in Ca2+* b. DAG will activate protein kinase C (PKC) 4. both Ca2+ and PKC form CalDAG-GEF 5. this causes an increase in Rap1
What interacts with Gq
1. TXA2---> TP--> Gq 2. thrombin--> PAR--> Gq 3. ADP--> P2Y1--> Gq
What binds to G13?
1. TXA2--> TP--> G13 2. thrombin--> PAR--> G13
list the different clinical GPCRs we have talked about and their clinical significance
1. adrenergic: target of antagonists to treat BP and heart failure 2. serotonin: target to treat depression 3. dopamine: target of an agonist to treat movement disorders 4. *HRH1*: target to treat hypersensitivity 5. OPR: target of opioids 6. *CXCR4 (chemokines)*: used as drug targets to treat HIV and cancer 7. *Wnt*: involved in cancer
calciums role (2) in regulation of glycolysis of skeletal muscle
1. an increased amount of calcium in the cytoplasm will *stimulate phosphorylase kinase activity* -this will activate the phosphorylase and will *promote glycogen breakdown* (glycolysis) 2. the increased calcium will also cause *muscle contraction* -the muscle contraction will consume energy (therefore it will cause glycolysis to occur). therefore, calcium is involved in the regulation of glycolysis in the skeletal muscle
what may actually be compensatory (good) for the body to fight against HTN (and therefore try to prevent HF)?
1. chronic stimulation of the SNS 2. desensitization of GPCR (through phosphorylation)
describe how GTPases are involved in translation
1. eIF2 is bound to GTP and the initiator tRNA (ON) 2. this brings the tRNA to the 40s subunit 3. translation is initiated at the ribosome 4. However, you want to be able to stop translation after the materials are delivered and the factor has done its job 5. in order to stop translation you need to turn the GTPase OFF using *eIF2b* 6. eIF2b works by hydrolyzing the GTP back to GDP (OFF) overall: -the eIF2 is bound to GTP--> translation is ON and occurring -the eIF2b hydrolyzes GTP to GDP--> translation is OFF and not occurring need to rewatch this slide
describe the regulation of glycogen metabolism in the liver due to epinephrine (when you are in the fed state)
1. epinephrine acts on the beta 2 adrenergic receptor (like it does in skeletal muscle) 2. the receptor interacts with HTM-G protein (Gs in this case) 3. Gs is a G stimulatory protein (it stimulates the G protein) 4. When the G protein is stimulated, *adenylate cyclase* will be activated 5. adenylate cyclase promotes the production of cAMP 6. cAMP will convert inactive protein kinase A to active protein kinase A 7. protein kinase A will activate phosphorylase kinase 8. phosphorylase kinase will convert inactive phosphorylase to active phosphorylase 9. active phosphorylase will cause glycogenolysis *this is the exact same pathway as epinephrine in the skeletal muscle*
describe sympathetic GPCR signaling in smooth muscle cells of the general arteriole circulation (AKA blood vessels)
1. epinephrine binds to an *alpha 1 adrenergic receptor*. The epinephrine will not augment the calcium that is already in the smooth muscle, it needs to generate calcium in order to stimulate smooth muscle contraction 2. This stimulates *Gq/G11* 3. this will increase IP3 and DAG 4. IP3 will mobilize calcium from the calcium stores 5. the calcium will then bind to calmodulin and activate the MLCK 6. this will cause contraction of the smooth muscle the Gq/G11 will also have calcium effects by *inhibiting myosin phosphatase* through the RhoA/ROCK pathway. When myosin phosphatase is inhibited you get greater phosphorylation of the myosin head group; therefore you get more contraction
describe the regulation of glycogen metabolism due to epinephrine in skeletal muscle
1. epinephrine binds to the *beta 2 adrenergic receptor* 2. the receptor interacts with *Gs* 3. Gs is a G stimulatory protein (it stimulates the G protein) 4. When the G protein is stimulated, *adenylate cyclase* will be activated 5. adenylate cyclase promotes the production of cAMP 6. cAMP will convert inactive protein kinase A to active protein kinase A 7. protein kinase A will stimulate phosphorylase 8. phosphorylase will cause glycogenolysis
How does your body know it picked a functional olfactory GPCR gene rather than a pseudogene? How do you only pick 1 functional gene?
1. if you picked a functional olfactory GPCR gene there will be *ER stress* 2. ER stress occurs because you are forming a lot of proteins at one time; plus the seven spanning membrane proteins (GPCRs) take a lot of work to process in the ER 4. the *ER stress will cause eIF2 to be phosphorylated* 5. this causes protein synthesis to be inhibited; therefore the ER can buy time to resolve the stress 6. However, some things are activated by the ER stress. 7. these activated things will go to the nucleus and shut down transcription, and the epigenetic regulator *LDS1 is turned off* 8. this will prevent you from picking up another olfactory gene (because your cell knows it already has a functional one); therefore more olfactory genes are not turned on
outside-in activation of integrins
1. integrins recognize a tripeptide (RGD) extracellularly 2. this causes a change in the integrins shape 3. this shape change alters the binding characteristics of the intracellular and extracellular domain of the integrin 4. this opens up the information and allows the integrin to interact with *talin* on the intracellular domain
describe the mechanism of action for angiotensin II
1. it is produced by the kidney 2. angiotensin is a ligand that binds to *ATR1- angiotensin receptor 1* (which is a GPCR) 3. ATR1 interacts with *Gq/G11* 4. this causes *contraction* of the general circulatory smooth muscle (arterioles) angiotensin works in response to the kidney; not in response to the SNS (Aka you don't have to be in the fight or flight mode to get increased angiotensin II)
describe the sympathetic nerve signaling in cardiac muscle cells (myocytes)
1. norepinephrine will bind to *Beta 1 adrenergic receptor* 2. this will stimulate *Gs* 3. Gs will activate stimulate adenylyl cyclase 4. this causes cAMP levels to increase 5. cAMP will phosphorylate one of the plasma membrane calcium channels 6. this allows more calcium to come in and signal the ryanodine receptor 7. this burst of calcium will stimulate *contraction*
describe the difference between normal and pathogenic hypertrophy of the heart
1. normal hypertrophy: -due to continued exercise (because the heart is working harder to pump and it adapts) -all parts of the heart will enlarge evenly, but the muscle will not get thicker 2. pathological hypertrophy: -is often due to HTN or valve stenosis-- the heart is having to work harder to pump the blood; therefore it will begin to hypertrophy. This will eventually cause *enhanced sympathetic stimulation of the heart* in order to compensate -the heart does not expand in exact proportions-- the muscle wall of the heart is thickening -normal heart tissue (cardiomyocytes) gets replaced by fibrosis (scar tissue). This causes the heart to lose elasticity--> decreased force and less filling of the heart--> death
compare and contrast epinephrine in the skeletal muscle and in the liver
1. skeletal muscle: epinephrine acts on the beta 2 adregneric receptor; through the use of Gs 2. fasted state in liver: epinephrine acts on the alpha 1 adrenergic receptor; through the use of Gq. then works by increasing calcium levels 3. fed state in the liver: epinephrine acts on the beta 2 adrenergic receptor (like in the skeletal muscle); through the use of Gs
2 types of GTP binding proteins superfamily
1. small GTPases: monomeric enzymes of approximately 20-30 kD 2. large GTPases
generalities of the sympathetic and parasympathetic NS (role, neurotransmitter, receptor)
1. sympathetic NS -role: fight or flight. Increases the heart rate in order to deliver ore blood to the skeletal muscles -NT: norepinephrine and epinephrine -receptor for NT: *adrenergic receptors* 2. parasympathetic NS -role: rest and digest. decreases the heart rate -NT: Ach (acetylcholine) -receptor for NT: *muscarinic receptors*
inside-out activation of integrins
1. talin binds to the intracellular domain of the integrin 2. this causes a change in the domain of the extracellular and intracellular integrin domain 3. the integrin can bind things extracellularly
describe the desensitization of opioids and its effect
1. the opioid receptors can become desensitized 2. this means you need to take more medication in order to have the same effect on dulling the pain (or causing euphoria if you are taking recreationally) 3. however, *opioid receptors do NOT downregulate (become desensitized) at the same rate in different parts of your body* example: you start to have to take more morphine to decrease your pain (because your opioid receptors have become desensitized). however, the opioid receptors in the lung is still fully functional; therefore this increased amount of opioid can cause respiratory distress
describe the regulation of glycogen metabolism due to calcium in skeletal muscle
1. there is an increase in calcium released into the extracellular pace 2. the calcium binds with calmodulin to form the *Ca2+-calmodulin complex* 3. this complex will regulate the glycogen breakdown by stimulating the activity of *phosphorylase kinase* -role of phosphorylase kinase: converts phosphorylase b (inative form) to phosphorylase a (active form)
List some of the large GTPases
1. translation factors 2. dynamin family 3. heterotrimeric GTPases
describe the regulation of glycogen metabolism in the liver due to epinephrine (when you are in the fasted state)
1. you all already using the glucagon to induce glycogenolysis (because you are in the fasted state), but to get maximal induction you also need to elevate the calcium levels. This is down using epinephrine 2. epinephrine binds to the *alpha 1 adrenergic receptor* (not Beta 2 adrenergic receptor like in skeletal muscle) 3. this receptor activates HTM-G protein (specifically *Gq*) 4. *Gq will regulate calcium* levels through inisotol triphosphate (IP3) and diacylyglycerol (DAG) 4. IP3 and DAG release calcium from the claim stores--> and increase in calcium levels 5. the calcium can then interact with calmodulin and form the Ca2+-calmodulin complex 6. this complex stimulates phosphorylase kinase and therefore regulates glycogen breakdown 7. this allows for maximal induction (along with the glucagon that is acting on the beta 2 adrenergic receptors)
How can you re-sensitize GPCR activity?
GPCR activity can be restored after removing the agonist
Which is a better way of thinking: Integrin αIIB- β3 or GpIIb-IIIa? why?
Integrin αIIB- β3 this is because of its mechanism
Do we rely on the sympathetic NS for our heart to beat? why or why not?
NO; we do not rely on the SNS for our heart to beat -our heart will beat even if we are not stimulating the SNS (fight or flight response)--DUH -the SNS only modulates our heartbeat (it will increase our heartbeat) -the SNS just takes our normal heart beat and makes it faster
what receptors and G proteins does ADP interact with? Which one of these pathways does clopidorgel affect?
P2Y12 --> Gi (this is inhibited by clopidogril) P2Y1--> Gq (this is not inhibited by clopidogril)
list some of the small GTPase superfamily molecules
Ras Rab Ran Rho Arf there are 153 members of this family (that were discovered from the human genome project)
TXA2, thrombin, ADP in platelet (what receptor does it bind and what GPCR does it interact with)
TXA2: interacts with TP receptor. the TP receptor interacts with G13 or Gq thrombin: interacts with the PAR receptor. PAR receptor interacts with G13 or Gq ADP: interacts with P2Y1 and P2Y12. P2Y1 interacts with Gq. P2Y12 interacts with Gi
describe the biochemical mechanism of thrombin on clotting
This is an example of outside-in signaling 1. thrombin interacts PAR and cleaves it 2. this activates Gq 3. this causes Gq to release IP3 (which will increase calcium) and DAG (which will increase protein kinase C) 4. increased Calcium and PKC will stimulate GEF 5. rap1 exchanges GDP for GTP; therefore it gets turned ON 6. this recruits talin and RIAM 7. the inactive talin gets rolled out; this exposes binding sites that were not initially available 8. this now allows Integrin αIIB- β3 to bind things extracellularly 9. Integrin αIIB- β3 can now bind fibrin, fibrinogen, or VWF at the site of a clot; this allows the platelet to jump in a be a part of the clot
desensitization (what causes it and what is the effect)
a loss of response due to prolonged or repeated administration of an *agonist*. This causes a *receptor to become unresponsive*
List some GPCRs
adenosine adrenergic chemokine dopamine histamine rhodopsin
describe the ligand binding domain of adenosine receptors
adenosine purines can perform signaling roles in the heart
Describe the difference between beta 1 and beta 2 adrenergic receptors
beta 1 is subject to desensitization and down regulation due to the chronic response of the sympathetic stimulation beta 2 is not subject to desensitization. beta 1 is bad; beta 2 is good beta 2 can switch to decrease cAMP; beta 1 increases cAMP only
describe the ligand binding domain of adrenergic receptors
bind relatively small molecules (like epinephrine)
What causes the desensitization linked to receptor phosphorylation?
due to arrestin and GRKs
Why is the use of alpha blockers in patients with HF controversial?
in the heart: -you want alpha function to be preserved because it sends out pro-survival signals in the general circulation: -you want to block alpha receptors because they are *causing contraction in the general vasculature* they need to have clinical trials done to help decide if you should put HF pts on alpha blockers
describe the regulation of glycogen metabolism in the liver due to glucagon (when you are fasted)
it has the same signaling pathway as epinephrine in the skeletal muscle 1. glucagon binds to the glucagon receptor (which is a GPCR) (note: it was the beta 2 adrenergic receptor in the skeletal muscle) 2. the receptor interacts with HTM-G protein (Gs in this case) 3. Gs is a G stimulatory protein (it stimulates the G protein) 4. When the G protein is stimulated, *adenylate cyclase* will be activated 5. adenylate cyclase promotes the production of cAMP 6. cAMP will convert inactive protein kinase A to *active protein kinase A* 7. protein kinase A will stimulate *phosphorylase kinase* 8. phosphorylase kinase will convert inactive phosphorylase to *active phosphorylase* 9. active phosphorylase will cause *glycogenolysis*
Why are GCPR important clinically?
over 40% of approved drugs target the GPCR family in 1 way or another
describe the ligand binding domain of chemokine receptors
they have a much larger binding site (because chemokines are bigger molecules)
Why do you use a beta blocker on patients with heart failure?
you want to block beta 1 in people with HF; because the Beta 1 is causing the fibrosis in the heart that makes the heart pump harder and causes HF
What happens metabolically if you are undergoing a fight or flight response?
your skeletal muscle will undergo an increased amount of glycolysis (break down of glycogen stores in the muscle) in order to have enough available energy to run or fight
