STUDY GUIDE_COLOGY

GEF

(causes GDP to dissociate from the G-protein and GTP to bind) results in ACTIVATION of GDP → GTP. It's a type of RGS

GAP

- proteins stimulate GTP hydrolysis to GDP and result INACTIVATION of g-protein. Most common and best understood GTP→GDP. It's a type of RGS

DISSOCIATION not association

-Activation of G-Protein is due to ___________ of the subunits

cAMP isn't being turned off

-If you had epinephrine creating camp, and also caffeine or theophylline/methyl xanthine(all which block phosphodiesterase), then the effect of the epinephrine is exaggerated because the ____________.

The agonist binds the GPCR Gq is activated and activates IP3 IP3 results in increased Ca2+ levels in the cell 4 Ca2+ binds calmodulin to activate it Ca2+/calmodulin binds PP2B (aka calcineurin) to activate it PP2B (calcineurin) removes the phosphate from Inhibitor 1 Inhibitor 1 is not longer active and cannot inhibit PP1 Therefore, PP1 is active and can dephosphorylate it's target enzymes and turn the

19. How can an agonist binding to a GPCR/Gq receptor indirectly regulate the activity of a PP1 phosphatase as described in slide 8 in the Signal Transduction III section?

• ATP pumps it out • Stores in Endoplasmic Reticulum (important reservoir) • Mitochondria acts like sponge that keeps excess Ca+2 in ITS

21. How does a cell maintain the 10,000-fold calcium gradient difference between the outside and inside of the cell?

Calmodulin - by undergoing a conformational change it is enabled to bind to specific proteins for a specific response. Can undergo post-translational modifications such as phosphyorylation, acetylation, methylation, and proteolytic cleavage which can potenentially modulate its actions • Ex: Myosin light chain kinase smooth muscle, adenylate cyclase, phosodiesterase (decrease cAMP), Ca/CaM dependent protein Kinase, phospholipase A2 (prostaglandin etc)

23. Explain how calmodulin plays a "pivotal role" in signal transduction.

Calmodium undergoes a conformational change upon binding to calcium; up to 4 calciums

Describe how calmodulin is activated?

no, Some are soluble and others are membrane-associated

Are all PDE type are soluble ?

Calcium causes PKC translocation to plasma where membrane where it binds phosphatidylserine (PS) DAG in presence of Ca+2 activates PKC (acts like cataylist) 1. Agonist binds receptor 2. G-protein (Gq) dissociates to Gq and BetaGamma 3. Activation of Phospholipase C 4. Phosphatdyl inositol 4,5-phosphate (PIP2) converted to DAG (Diacylglycerol) and IP3 5. IP3 binds receptors on ER and leads to release of Ca+2 6. DAG stays in membrane and activates PKC (Protein Kinase C) DAG = increase sensitivity

Describe how PS, DAG, and calcium act together to activate protein kinase C (PKC).

Receptor affinity is high when g-protein is bound-GDP is bound and inactive (no agonist) when agonist binds, the receptor changes shape to have low affinity for gprotein.

Describe how receptor affinity changes during the G-protein cycle.

Calcium causes PKC translocation to plasma where membrane where it binds phosphatidylserine (PS) DAG in presence of Ca+2 activates PKC (acts like cataylist) 1. Agonist binds receptor 2. G-protein (Gq) dissociates to Gq and BetaGamma 3. Activation of Phospholipase C 4. Phosphatdyl inositol 4,5-phosphate (PIP2) converted to DAG (Diacylglycerol) and IP3 5. IP3 binds receptors on ER and leads to release of Ca+2 6. DAG stays in membrane and activates PKC (Protein Kinase C) DAG = increase sensitivity

Describe how the activation of phospholipase C leads to IP3 and DAG and what these second messengers each do in the cell.

ACh ??? Increase intracellular Ca+2 via voltage dependent and/or acivationof GPCR/Gq and increase IP3 Calcium binds to calmodulin (CaM) forming "active" Ca+2/CaM complex Ca+2 /CaM binds to Myosin light chain kinase (MLCK) activating it MLCK phosphorylates myosin light chains Phosphorylation causes Actin binding site to be exposed = contraction

Describe the biochemical steps from binding of acetylcholine to its receptor on a smooth muscle cell and the contraction of the muscle.

Pathway of the bottom left area: Epi → β adrenergic receptor → Gs → AC → cAMP → activation of cAMP-PK and phosphorylation of MLK → decreases affinity of Ca+2/CaM for MLCK causing it to be inactive; LC20 (myosin light chain tail) is not phosphorylated→ contraction can NOT occur

Describe the biochemical steps from binding of epinephrine to the β adrenergic receptor on a smooth muscle cell and the relaxation of the muscle.

Voltage Gated Channels Coupled Gq g-protein generate IP3 inside

Describe two mechanisms for increasing intracellular calcium levels.

yes

Does adding fats keeps the proteins /subunits in the membrane?

B-arrestin prevents the receptor from interaction with Gas

For the mechanism for rapid homologous desensitization of a GPCR, what happen after BARK phosphorylates the Beta receptor which then attracts the binding of the protein B-arrestin

BARK phosphorylates the Beta receptor which then attracts the binding of the protein B-arrestin

For the mechanism for rapid homologous desensitization of a GPCR, what happen after The βγ subunit binds to BARK (beta adrenergic receptor kinase)

The βγ subunit binds to BARK (beta adrenergic receptor kinase)

For the mechanism for rapid homologous desensitization of a GPCR, what happen after agonist binds to beta receptor and causes activation of G-protein (dissociation of Gas from βγ subunit?

agonist binds to beta receptor and causes activation of G-protein (dissociation of Gas from βγ subunit

For the mechanism for rapid homologous desensitization of a GPCR, what happen first?

Guanine nucleotide binding protein

G-Protein AKA

3, heterotrimetric alpha beta gamma subunit

G-Proteins contain how many of subunits and what types

Guanine-nucleotide exchange factors

GEF is aka

Protein kinase A phosphorylates the β subunit of phosphorylase kinase, which subsequently activates glycogen phosphorylase.

IN all of the biochemical steps starting from the binding of epinephrine to the β - adrenergic receptor on a hepatocyte to the breakdown of stored glycogen and the release of glucose into the blood, WHAT HAPPEN AFTER The GTP-bound form of the α-subunit of Gs activates adenylate cyclase, a transmembrane protein that catalyzes the formation of the secondary messenger cAMP from ATP.

By inhibiting proteins - they keep things active longer; therapeutic examples - opiate extender and parkinsons

How an RGS inhibitor could have therapeutic potential?

(Slide 31 - SignalTransductionIII.ppt - "Smooth muscle relaxation via Nitric oxide (NO) and cyclic GMP")

How does activation of a muscarinic cholinergic receptor on vascular endothelial cells result in relaxation of the overlying smooth muscle?

Phosducin is a cytosolic protein which binds with high affinity to the βγ subunits; prevention of βγ subunit reassociation with Alpha subunit prolonging biological activity

How does the cytosolic protein phosducin "prolong the biological activity" of the Gα subunits of the G - protein?

The fatty acid group on alpha will help to keep the alpha in place in the membrane, otherwise it will be swimming around, and into the cytosol.

How does the squiggle keep the squiggle keeps the alpha subunit of G protein in the membrane?

11

How many phosphosdiesterase (PDE) has been found?

The GTP-bound form of the α-subunit of Gs activates adenylate cyclase, a transmembrane protein that catalyzes the formation of the secondary messenger cAMP from ATP.

IN all of the biochemical steps starting from the binding of epinephrine to the β - adrenergic receptor on a hepatocyte to the breakdown of stored glycogen and the release of glucose into the blood, WHAT HAPPEN AFTER Epinephrine and glucagon bind to specific 7TM receptors in the plasma membranes of muscle and liver cells. Epinephrine binds to the β-adrenergic receptor in muscle, whereas glucagon binds to glucagon receptor. Binding events activate the α subunit of the Gs protein. A specific external signal has been transmitted into the cell through structural changes, first in the receptor and then in the G protein.

Glycogen phosphorylase cleaves the bond at the 1 position by substitution of a phosphoryl group. It breaks down glucose polymer. (Glycogen is left with one less glucose molecule and the free glucose is in the form of glucose-1-phosphate.

IN all of the biochemical steps starting from the binding of epinephrine to the β - adrenergic receptor on a hepatocyte to the breakdown of stored glycogen and the release of glucose into the blood, WHAT HAPPEN AFTER Protein kinase A phosphorylates the β subunit of phosphorylase kinase, which subsequently activates glycogen phosphorylase.

The elevated cytosolic level of cyclic AMP activates protein kinase A through the binding of cAMP to the regulatory subunits, which then dissociate from the catalytic subunits. The free catalytic subunits are now active.

IN all of the biochemical steps starting from the binding of epinephrine to the β - adrenergic receptor on a hepatocyte to the breakdown of stored glycogen and the release of glucose into the blood, WHAT HAPPEN AFTER The GTP-bound form of the α-subunit of Gs activates adenylate cyclase, a transmembrane protein that catalyzes the formation of the secondary messenger cAMP from ATP.

Epinephrine and glucagon bind to specific 7TM receptors in the plasma membranes of muscle and liver cells. Epinephrine binds to the β-adrenergic receptor in muscle, whereas glucagon binds to glucagon receptor. Binding events activate the α subunit of the Gs protein. A specific external signal has been transmitted into the cell through structural changes, first in the receptor and then in the G protein.

IN all of the biochemical steps starting from the binding of epinephrine to the β - adrenergic receptor on a hepatocyte to the breakdown of stored glycogen and the release of glucose into the blood, WHAT HAPPEN FIRST?

PHOSPHODIESTERASE ENZYME

IN cAMP, The cyclic bond is a very strong bond, that can ONLY BE BROKEN BY A

analgesic potentiators

INhibitors of RGS could be possibly effective specific _________

Gs is sexy which like to stimulate Adenylate. Gi is idiot and hateful which does opposite of Gs. Gq is the one from GQ magazine which like to promote coolness like phospholipase C G12 is an outcast which like to promote goofy stuff like GEF

Imagine and relate G proteins general family and their function

cAMP-PK is like 2 hollow suPerK camp (holoenzyme) with 4 empty space for (all these describes whole regulator subunits). These 2 hollow suPerK camp is connect to 2 ROCKET C ( active catalytic subunits) on top of each structure. As a whole, everything is called an inactive holoenzyme. When 4 camp of people come in to fill up the 4 empty space of SuPerK camp, it transforms and shoot off the 2 ROCKET C. the ROCKET C scare any protoss that has money (ATP + protein) and catalyze it to take all its money and hid in their body (phosphoprotein)

Imagine and relate the structure of cAMP-dependent protein kinase and how is it activated?

Gi - sound like a girl that hate ion so since K+ and Ca+ channel are ion channel, they're being inhibit. Gi also hate Gs so it does the opposite by inhibit adenylate cyclase

Imagine and relate why Gi inhibit K+ and Ca+ channel and Adenylate cyclase

on 1 side of the camp, it stop store glucogen storage by phosphorylate glycogen synthase machine (phosphate glycogen synthase is INACTIVE). On other side of camp, it encourage break down of glucose by add adding phosphate to phosphorylase kinase. Eliminate storage and encourage making food to eat AT THE SAME TIME!

Imagine the "dual regulation" of cAMP-PK in the control of glycogen breakdown and synthesis

When agonist bind to GPCR/Gq receptor, Ip man (IP3) encourage his bone to thicken so increase calcium. He use his calcium to hit 1 camera (CAM/ calmodulin) four time and activate it. The CAM was connect to a wire look like neuron (Calcineurin code name PP2B) and activate it. PP2B sends signal to remove phosphate from inhibitor switch 1 (inhibitor 1). Since The inhibitor switch also cannot inhibit the TERMINATOR (PP1). Since this terminator is active, it can REMOVE phosphate from target enzyme and eventually turn it off.

Imagine the GPCR/Gq receptor binding process

After ACh bind muscarinic receptor, add in the story of Gq and stop at the activation of camera (cam). Instead of The camera activate Calcineurin, it can activate signal to Muscle control click (MLCK myosin ligh chain kinase) to turn on. The control click can phosphoryte myosin light chain which expose myosin head for actin to bind for contraction to occur.

Imagine the biochemical steps from binding of acetylcholine to its receptor on a smooth muscle cell and the contraction of the muscle.

Since Epi sounds feminine it use the Gs protein to active (alpha male story) AC - cAMP. 2. PK people move into CAMP - cAMP-PK. Instead use energy to break down food to eat, they use phospho money to wrap up the Muscle control click (MLCK-PO4). Since it's wrap up, the camera (CAM) can't send signal to turn music on (muscle). Now everyone can relax (muscle relaxation).

Imagine the biochemical steps from binding of epinephrine to the β adrenergic receptor on a smooth muscle cell and the relaxation of the muscle.

Alpha male WITH GDP (no money) was original attach to Girl (Beta gamma on Gprotein) but he got signal to get more money (turn GDP to GTP). He decide to leave the Girl since he has more money now. With more money, he shows off and impress (activate) a dude on cycle (adenylate cyclase) to build him a CAMP bachelor's pad (turn ATP to CAMP). However he did pay the guy so the government (GTPase - GAP) found out about the fraud and take his money away (dephosphatase GTP to GDP). Now Alpha male with no money decide to reattach his girl beta gamma.

Imagine what G protein cycle like a real like situation

After agonist bind to G protein receptor, G s go out to stimulate Adenylate cyclase leave Beta gamma open. The dog attack and attract the cop B-arrestin protein to attach and Beta subunit to jail. This turn protein into a desensitize criminal and Prevent it from rebind to Gs.

Imagine why For the mechanism for rapid homologous desensitization of a GPCR, what happen after The βγ subunit binds to BARK (beta adrenergic receptor kinase)

Mother protein GTPase like the GAP because it's easy and fast to shop there (accelerate)

Imagine why GAPs stands for GTPase accelerating proteins

GTPase desphorylates GTP on alpha subunit

In steps of G protein Cycle, what happen after Alpha subunit dissociates and associates with adenylate cyclase, AC take ATP and makes cAMP

Alpha subunit with GTP dissociates and associates with adenylate cyclase, AC take ATP and makes cAMP

In steps of G protein Cycle, what happen after Alpha subunit loses GDP and binds GTP

Alpha subunit reassociate with the beta/gamma subunit and GPCR

In steps of G protein Cycle, what happen after GTPase desphorylates GTP from alpha subunit

Alpha subunit with GDP gain a phosphate and turn into GTP (GEF add that phosphate)

In steps of G protein Cycle, what happen after hormone or drug binds its membrane receptor?

hormone or drug binds GPCR membrane receptor

In steps of G protein Cycle, what happen first?

Protein Kinase A phosphorylates the β subunit of phosphorylase kinase, which subsequently activates glycogen phosphorylase. (Glycogen → glucose-6-phosphate)

In the "dual regulation" of cAMP-PK in the control of glycogen breakdown and synthesis, WHAT HAPPEN AFTER Activated by cAMP - the activated catalytic subunits of cAMP-PK catalyze the transfer of phosphate (PO4) from ATP to serine or threonine residues in a variety of proteins.

Protein Kinase A sets the stage for the shutdown of glycogen degradation by adding a phosphoryl group to the α subunit of phosphorylase kinase after first phosphorylating the β subunit. This addition of a phosphoryl group renders the enzyme a better substrate for dephosphorylation and consequent inactivation by the enzyme protein phosphatase (PP1). Protein phosphatase 1 also removes the phosphoryl group from glycogen phosphorylase, converting the enzyme into the usually inactive b form.

In the "dual regulation" of cAMP-PK in the control of glycogen breakdown and synthesis, WHAT HAPPEN AFTER Protein Kinase A phosphorylates the β subunit of phosphorylase kinase, which subsequently activates glycogen phosphorylase. (Glycogen → glucose-6-phosphate)

cAMP-PK phosphorylates Glycogen Synthase, which is inactive when phosphorylated, which stops the conversion of UDPG to Glycogen

In the "dual regulation" of cAMP-PK in the control of glycogen breakdown and synthesis, WHAT HAPPEN AFTER Protein Kinase A sets the stage for the shutdown of glycogen degradation by adding a phosphoryl group to the α subunit of phosphorylase kinase after first phosphorylating the β subunit. This addition of a phosphoryl group renders the enzyme a better substrate for dephosphorylation and consequent inactivation by the enzyme protein phosphatase (PP1). Protein phosphatase 1 also removes the phosphoryl group from glycogen phosphorylase, converting the enzyme into the usually inactive b form.

Inactivates glycogen synthase to prevent glyconeogenesis -Glycogen synthase grows adds glucose to a UDP-glucose to grow glycogen chains Activates phosphorylase kinase -Phosphoryalse kinases breaks glycogen bonds using phosphate to remove G-1-P (glucose)

In the "dual regulation" of cAMP-PK in the control of glycogen breakdown and synthesis, WHAT HAPPEN FIRST?

cAMP-PK inactivates Glycogen Synthase AND it activates Phosphorylase Kinase by adding a phosphate. When Glycogen Synthase is inactivated, glycogen synthesis ceases so that glycogen breakdown can occur. When Phosphorylase Kinase is activated, it goes on to active phosphorylase, which goes on to break glycogen to glucose.

In the "dual regulation" of cAMP-PK in the control of glycogen breakdown and synthesis, WHAT IS THE SUMMARY?

cAMP-PK phosphorylates Inhibitor 1 and activates it. Inhibitor 1 will inhibit PP1. If PP1 is inactivated, it cannot dephosphorylate the enzyme ensuring that the phosphate will stay on the enzyme.

In the above regulatory pathway (question 19) how does cAMP-PK insure that the phosphorylated enzyme substrate stays phosphorylated?

GTPase Accelerating Proteins

Referred to as (GAPs)

Gs

This G Protein increase adenylate cyclase = increase cAMP (52-45k)

Gi

This G protein has variety of functions - first one was inhibitory (41-39k a. Adenylate cyclase inhibition; K+ channel activation; Ca+2 channel inhibition

hormone receptors (Rec), a stimulatory G protein (Gs), catalytic adenylyl cyclase (AC), phosphodiesterases (PDE) that hydrolyze cAMP, cAMP-dependent kinases, with regulatory (R) and catalytic (C) subunits, protein substrates (S) of the kinases, and phosphatases (P'ase), which remove phosphates from substrate proteins. Open arrows denote regulatory effects.

The cAMP second messenger pathway. Key proteins include

Gq

This G Protein activate phospholipase C

G12

This G Protein activates GEF

Serine-threonin phosphatases

This enzyme can catalyze the dephosphorylation of proteins that have been phosphorylated on serine or threonine residues. (Hydrolyze to remove phosphate)

Gs, Gi, Gq, G12

What are the 4 general families of heterotrimeric G-proteins

They differ in their preference for either cAMP (ex. PDE4, PDE7, PDE8) or cGMP

What are the difference in different types of PDE

Activation, Inhibition, Stimulation

What are the functions of the released βγ subunits of the G-protein? Describe at least 3.

PP1 and PP2B

What are the main 2 classes or serine-threonin phosphoprotein phosphatases?

PP2B are activated by calcium/calmodulin. Calcineurin is regulated by a series of inhibitors called immunophillins.

What are the unique features of PP2B of serine-threonine phosphoprotein phosphatases?

PP1 contain 4 subtypes (α, β, γ1,γ2). Each subtype exhibit very distinct patterns of tissue distribution. PP1 enzymes dephosphorylate a broad range of phosphoproteins. PP1 are inhibited by the binding several know inhibitors (Inhibitor 1, 2, DARPP-32, and NIPPI) <-- a part of the structure can inhibit phsophatase)

What are the uniques feature about PP1 (Protein phosphatase) of serine-threonin phosphoprotein phosphatases?

All the membrane-bound enzymes; (1) All are activated by Gαs. They have tissue specific patterns of expression ie: AC1 is neural specific.

What are the various ways we have discussed that the various isoforms of adenylate cyclase can be activated? 1st one

(2) All are activated except for AC9 by forskiolin.

What are the various ways we have discussed that the various isoforms of adenylate cyclase can be activated? 2nd one

(3) AC1 and AC9 are activated by Ca 2+ calmodulin.

What are the various ways we have discussed that the various isoforms of adenylate cyclase can be activated? 3rd one

(4) AC2, AC4, AC7 are stimulated by free BetaGamma subunits of the activated G-protein

What are the various ways we have discussed that the various isoforms of adenylate cyclase can be activated? 4th one

xanthine derivatives like caffeine & theophylline

What can inhibit certain PDEs resulting in ↑cAMP

Activate a K+ channel in the brain via Gi by opioid alpha2 adrenergic, muscarinic cholinergic, and GABAb receptors

What does βγ subunits of the G-protein do to K+ channel?

Activation of MAP-kinase (mitogen-activated protein kinase) these kinases are activated by growth factors ... thus GPCR via releasing

What does βγ subunits of the G-protein do to MAP-kinase?

In presence of Gas BetaGamma subunits inhibit AC1 and AC8

What does βγ subunits of the G-protein do to adenylate cyclase (AC)_ types AC1 and AC8 in presence of gas betagamma?

Stimulates adenylate cyclase (AC)_ types AC2, AC4 and AC7

What does βγ subunits of the G-protein do to adenylate cyclase (AC)_ types AC2, AC4 and AC7?

Inhibit of voltage-active L-type Ca+2 channels

What does βγ subunits of the G-protein do to voltage-active L-type Ca+2 channels?

ACh attaches to receptor that increases Ca+2 into endothelial cell that pumps Nitric Oxide to the smooth muscle cell. Note: it continues beyond PKG (also cGMP-PK = same as cAMP-PK)

What is the biochemical mechanism of action of nitric oxide (NO) and cGMP in smooth muscle relaxation?

BARK (beta adrenergic receptor kinase)

What is the main enzyme involve in rapid homologous desensitization of a GPCR?

cAMP-PK is a holoenzyme (dissociates to be activated) Two regulatory subunits, each having a catalytic part 2 cAMP bind each regulatory subunit and changes the conformation The conformational change causes the release of the catalytic unit The active catalytic subunits catalyse the transfer of phosphate from ATP to serine or threonine residues on proteins (it is a kinase)

What is the structure of cAMP-dependent protein kinase and how is it activated?

95% of phosphorylation occurs on the serine residues. 3-4% on theorine residues. 1% on tyrosine residues

What parts of the protein substrate is phosphorylated by cAMP-PK?

GPCR

What type of G protein is a GEF and also a receptor

cytosolic protein

What type of protein is phosducin?

G-Protein

What type of receptor is a 7 transmembrane receptor that is a large protein family of transmembrane receptors that sense molecules outside the cell and activate inside signal transduction pathways and ultimately cellular response)

prevent of βγ subunit reassociation with Alpha subunit prolonging biological activity

When Phosducin binds with high affinity to the βγ subunits, what would happen?

Type II cAMP-PK

Which type of cAMP-PK can autophosphorylation- prevents the reassociation between R and C. This means that the catalytic subunits phosphorylate the RII of the same enzyme. This makes it easier for cAMP to dissociate the holoenzyme (activation) as well as prevents the reassociation (inactivation) of RII and C once activated by cAMP

Type 2 cAMP-PK

Which type of cAMP-PK is more more likely to be on the membran?

Type 1 cAMP-PK

Which type of cAMP-PK is more soluble?

Type 2 cAMP-PK

Which type of cAMP-PK tends to bound to membrane via a group of proteins called "A kinase anchor proteins" or AKAPs?

b/c the receptor changes shape to have low affinity for GDP and get rid of it so it can bind to GTP

Why receptor affinity has low affinity for Gprotein when activated?

5'-AMP

c. Cyclic AMP is subsequently broken down by cAMP phosphodiesterase to ______ which does not have the cyclic phosphate group and does not regulate cellular processes to any great extent.

RGS's

discovered in 1996 composed of diverse groups of proteins each with specific tissue localization) best function is to inhibit G-protein signaling by increasing GTP hydrolysis.

the squiggle keeps the alpha in the membrane

in G protein, the alpha subunit as the "squiggle", what is its function?

Long chain fatty acids, 20 carbons long, 16 carbons, etc -Attached to cysteine residues in proteins to attach to the membrane

in G protein, what is the "squiggle" attach alpha and gamma subunits

none

in cAMP, 5'amp has effect or none in producing a biological response

cyclic nature of 3'5' cyclic amp

in cAMP, The ______ is required for biological response

The phosphodiesterase

in cAMP, what enzyme inactivates the molecule of cyclic amp and also cyclic gmp

phosphodiesterase

what is an enzyme that degrades the phosphodiester bond in cAMP