G protein - Kucera

Question 1

Ionotropic are ___meric proteins with __ subunits. They are __-gated receptors and affect rapid and short duration responses (quickly on and quickly off).

Answer 1

Ionotropic are multimeric proteins with 5 subunits. They are ligand-gated receptors and affect rapid and short duration responses (quickly on and quickly off).

Question 2

Metabotropics:

–Do not have ___ channel as part of the receptor

–Receptor is a single protein with __ membrane-spanning domains

–__-proteins are activated intermediates

–Onset and duration of responses is longer/shorter than ionotropic receptors

Answer 2

–Do not have ion channel as part of the receptor

–Receptor is a single protein with 7 membrane-spanning domains

G-proteins are activated intermediates

–Onset and duration of responses is longer than ionotropic receptors

Question 3

Explain G-protein coupled receptors

Answer 3

GPCR:

GPCR is attached to G-alpha, G-beta and G-gamma. The G-alpha, when bound to GDP is inactive. When GTP replaces this GDP, the G-alpha subunit is activated, released and binds to adenylate cyclase. Once GTP-G-alpha subunit binds to adenylate cyclase (effector enzyme), AC creates cAMP and ppi. cAMP binds to the regulation subunit of PK which releases the catalytic subunit of PK. The catalytic subunit of PKA is now active and will add a phosphate group to a target protein, either inhibiting it or activating it. This generates a cell response.

Question 4

Explain how Phosphoinositides are under the control of GPCRs:

Answer 4

When a hormone binds to a GPCR, it activates the G-alpha subunit which then binds to PLC (phospholipase C). The PLC cleaves PIP2 into DAG and IP3. IP3 then goes to the ER membrane and sits/activates a Ca2+ channel that is embedded in the ER membrane. This causes a release of Ca2+ into the intracellular space. The Ca2+ along with DAG will activate PKC (protein kinase C). PKC then binds to target proteins, adds a phosphate to them ad generates a cellular response.

Question 5

1. IP3 is released from ___ embedded in the ____ _____
2. IP3 binds to the IP3 receptor in the __ ___
3. The binding induces opening of the ____ channel which is part of the IP3 receptor

One major route for the calcium ion is to bind to calcium binding proteins. These proteins associate with and regulate the activity of an assortment of cell proteins which have important cellular functions.

_____ is a prominent calcium binding protein

Answer 5

1.IP3 is released from PIP2 embedded in the plasma membrane

2. IP3 binds to the IP3 receptor in the endoplasmic reticulum
3. The binding induces opening of the calcium channel which is part of the IP3 receptor

One major route for the calcium ion is to bind to calcium binding proteins. These proteins associate with and regulate the activity of an assortment of cell proteins which have important cellular functions.

Calmodulin is a prominent calcium binding protein

Question 6

List from largest to smallest:

G-alpha, G-beta, G-gamma

G-____ subunits:

1. bind to receptors
2. determine identity of effector
3. directly activate (or inhibit) ____

G-beta, and G-gamma subunits: directly activate (or inhibit) effectors

Answer 6

List from largest to smallest:

G-alpha > G-beta > G-gamma

G-alpha subunits:

1. bind to receptors
2. determine identity of effector
3. directly activate (or inhibit) effectors

G-beta, and G-gamma subunits: directly activate (or inhibit) effectors

Question 7

GPCR:

• Examples: ____ (relating to or denoting nerve cells in which epinephrine (adrenaline), norepinephrine (noradrenaline), or a similar substance acts as a neurotransmitter); ACh-muscarinic
• Membrane-bound; forms ternary complex with GTP-binding protein and effector
• Mechanism: activate ____ messengers (cyclic AMP, phosphoinositol, ion channels)

Answer 7

GCPR:

• Examples: adrenergic; ACh muscarinic
• Membrane-bound; forms ternary complex with GTP-binding protein and effector
• Mechanism: activate second messengers (cyclic AMP, phosphoinositol, ion channels)

Question 8

GPCR:

• Made up of _,__,__ subunits
• Four Subclasses and fuction?

–G-alpha-__

–G-alpha-__/___

–G-alpha-__/___

–G-alpha-__/___

•The __ and __ subunits do not vary from one to another

Answer 8

GPCR:

• Made up of alpha, beta, gamma subunits
• Four Subclasses

–G-alpha-s (stimulatory G protein)

–G-alpha-i/o (inhibitory G protein)

–G-alpha-q/11 (activates phospholipase C- PLC)

–G-alpha-12/13 (interact with rho-guanine exchange factors, which are important for growth and proliferation of cells).

•The beta and gamma subunits do not vary from one to another

Question 9

G-alpha

• Stimulates __ __generating cAMP
• cAMP stimulates ___
• PKA phosphorylates ion channel and opens the channel
• The neuron is made more excitable

Answer 9

• Stimulates adenylyl cyclase generating cAMP
• cAMP stimulates protein kinase A
• PKA phosphorylates ion channel and opens the channel
• The neuron is made more excitable

Question 10

G-alpha i/o

•____adenylyl cyclase

____ level of cAMP

•Cellular response is ____ to the response elicited through G-alpha-s

Answer 10

G-alpha-i/o

inhibits adenylyl cyclase

•Decreases level of cAMP

•Cellular response is opposite to the response elicited through Gas

Question 11

G-alpha-q/11

•Stimulates____ causing the cleavage of __ to __ and __

___ causes the release of ___ from the endoplasmic reticulum

___ stimulates protein kinase C that activates other downstream targets

Answer 11

•Stimulates phospholipase C causing the cleavage of PIP2 to DAG and IP3

•IP3 causes the release of calcium from the endoplasmic reticulum

•DAG stimulates protein kinase C that activates other downstream targets

Question 12

G-alpha-12/13

• Activates ____ nucleotide exchange factors (___)
• Some of the _____ are ____-associated and promote cellular proliferation
• Rho family of small GTPases are important ___ of multiple cellular activities

Answer 12

• Activates 3 Rho guanine nucleotide exchange factors (RhoGEFs)
• Some of the RhoGEFs are leukemia-associated and promote cellular proliferation
• Rho family of small GTPases are important regulators of multiple cellular activities

Question 13

G-beta/G-gamma complex

•Effectors of these subunits are different ion channels.

G-beta/G-gamma complex effects:

–______ channels (___). A channel that is “inwardly-rectifying” is one that passes current (positive charge) more easily in the inward direction (into the cell) than in the outward direction (out of the cell). It is thought that this current may play an important role in regulating neuronal activity, by helping to stabilize the resting membrane potential of the cell. The G protein-coupled inwardly-rectifying potassium channels (GIRKs) are a family of inward-rectifier potassium ion channels which are activated (opened) via a signal transduction cascade starting with ligand-stimulated G protein-coupled receptors (GPCRs)

–____ channels. The N-type calcium channel is a type of voltage-dependent calcium channel

Answer 13

G-beta/G-gamma complex

•Effectors of these subunits are different ion channels.

G-beta/G-gamma complex effects:

–G-protein-regulated inwardly rectifying K+ channels (GIRK). A channel that is “inwardly-rectifying” is one that passes current (positive charge) more easily in the inward direction (into the cell) than in the outward direction (out of the cell). It is thought that this current may play an important role in regulating neuronal activity, by helping to stabilize the resting membrane potential of the cell. The G protein-coupled inwardly-rectifying potassium channels (GIRKs) are a family of inward-rectifier potassium ion channels which are activated (opened) via a signal transduction cascade starting with ligand-stimulated G protein-coupled receptors (GPCRs)

–N-type voltage-gated Ca2+ channels. The N-type calcium channel is a type of voltage-dependent calcium channel

Question 14

Autonomic/Peripheral Receptors:

•Adrenergic (____/_____)

–Sympathetic Receptors (All are ____’s)

On the GPCR, if the receptor is an:

a1-adrenergic receptor, then it will signal to the G-alpha-q (it will be ____ to the effector molecule)

a2-adrenergic receptor, then it will signal to the G-alpha i/o (____ to the effector molecule)

If the receptor on the GPCR is a:

b-adrenergic receptor, then it will signal to the G-alpha (____ to the effectors)

β1, β2, and β3-adrenergic receptors on the G-alpha, will _____ the effector molecule.

β2-adrenergic receptors can also be on the G-alpha inhibitory proteins and ____ the effector.

Answer 14

Autonomic/Peripheral Receptors:

•Adrenergic (epinephrine/norepinephrine)

–Sympathetic Receptors (All are GPCR’s)

On the GPCR, if the receptor is an:

a1-adrenergic receptor, then it will signal to the G-alpha-q (it will be stimulatory to the effector molecule)

a2-adrenergic receptor, then it will signal to the G-alpha i/o (inhibitory to the effector molecule)

If the receptor on the GPCR is a:

b-adrenergic receptor, then it will signal to the G-alpha (stimulatory to the effectors)

β1, β2, and β3-adrenergic receptors on the G-alpha, will stimulate the effector molecule.

β2-adrenergic receptors can also be on the G-alpha inhibitory proteins and inhibit the effector.

Question 15

•Cholinergic (acetylcholine)

–Parasympathetic Receptors (muscle; ionotropic and GPCR’s)

•Muscarinic GPCR’s

–M1 Family (M1, M3, M5): Gaq (_____-odd ones)

–M2 Family (M2, M4) : Gai/o (_____- even ones)

Answer 15

•Cholinergic (acetylcholine)

–Parasympathetic Receptors (muscle; ionotropic and GPCR’s)

•Muscarinic GPCR’s

–M1 Family (M1, M3, M5): Gaq (stimulatory)

–M2 Family (M2, M4) : Gai/o (inhibitory)

i - “ee”nhibitory

Question 16

The identity of G-alpha determines the effector:

Stimulatory functions:

G-a-s: stimulate __ ___

G-a-q: stimulate ___ turnover

Inhibitory functions:

Gai: inhibit __ ___; open inward K+ channels (GIRK); inhibit Ca++ channels

Gao: same as ___

Answer 16

Stimulatory functions:

G-a-s: stimulate adenylyl cyclase

G-a-q: stimulate phosphoinositol turnover

Inhibitory functions:

Gai: inhibit adenylyl cyclase; open inward K+ channels (GIRK); inhibit Ca++ channels

Gao: same as Gai

Question 17

Identity of Ga Determines Receptor Family

Which receptors signal through through the following G-proteins?

_____ - Gat

_____ - Gas

____ - Gai/o

____ (serotonin) - Gaq

Answer 17

Identity of Ga Determines Receptor Family

Rhodopsin receptor through - Gat

beta-adrenergic receptor - Gas

opioid receptor - Gai/o

5-HT-2 (serotonin) receptor - Gaq

Question 18

Answer 18

Question 19

G protein activation:

1. In the basal (inactive) state, receptor is bound to G-protein heterotrimer, and ___ binds to GDP
2. When agonist binds to receptor, conformational changes dissociate GDP from Ga and is replaced by ___
3. Binding of GTP causes the G-protein to __ from the receptor, while Ga and Gbg dissociate from each other
4. ____-bound Ga, and dissociated Gbg, activate (or inhibit) their effectors
5. GTPase converts __ to __, regenerating GDP-bound inactive G-protein heterotrimer
6. G-protein heterotrimer binds to receptor, basal (inactive) state

Answer 19

1. In the basal (inactive) state, receptor is bound to G-protein heterotrimer, and Ga binds to GDP
2. When agonist binds to receptor, conformational changes dissociate GDP from Ga and is replaced by GTP
3. Binding of GTP causes the G-protein to dissociate from the receptor, while Ga and Gbg dissociate from each other
4. GTP-bound Ga, and dissociated Gbg, activate (or inhibit) their effectors
5. GTPase converts GTP to GDP, regenerating GDP-bound inactive G-protein heterotrimer
6. G-protein heterotrimer binds to receptor, basal (inactive) state

Question 20

When an individual gets cholera, they get massive diarrhea. Why does this happen?

Answer 20

Cholera toxin blocks GTPase on Gas which irreversibly stimulates cAMP. GTPase converts GTP back to GDP, regenerating GDP-bound inactive G-protein heterotrimer. If you inhibit GTPase, cAMP will be forever stimulated and will never go back to being inactivated.

Chloride channels will be stimulated and opened and brought into the lumen of the intestines, brining in water. This will cause massive diarrhea.

Question 21

How does pertussis toxin work?

Answer 21

PT blocks activation of Gai/o and irreversibly prevents agonist activation of inhibitory receptors.

The pertussis toxin prevents agonist activation of inhibitory receptors, so you see an increase in cAMP in those particular cell types. This causes the disease process.

Question 22

How are gluconeogenesis enzymes synthesized?

1.Glucagon binds to its receptor.

This activates a ___ receptor!

2. ____ levels go up
3. This activates ___
4. PKA catalytic subunit enters the nucleus
5. C subunit phosphorylates ___

6. ____ interacts with several transcription factors one of which is ____, together they activate transcription of genes that encode gluconeogenic enzymes

Answer 22

1.Glucagon binds to its receptor.

This activates a G-protein coupled receptor!

2.cAMP levels go up

3.cAMP activates PKA

4. PKA catalytic subunit enters the nucleus
5. C subunit phosphorylates CREB (cAMP Response Element Binding Protein)

6.CREB interacts with several transcription factors one of which is FOXO, together they activate transcription of genes that encode gluconeogenic enzymes

Question 23

cAMP is also involved in neuroscience. cAMP will activate ___, which activates ___, which up-regulated gene synthesis of endocannabinoids by binding to the ___ gene.

From Wiki: The endocannabinoid system (ECS) is a group of endogenous cannabinoid receptors located in the mammalian brain and throughout the central and peripheral nervous systems, consisting of neuromodulatory lipids and their receptors. Known as “the body’s own cannabinoid system”,[1] the ECS is involved in a variety of physiological processes including appetite, pain-sensation, mood, and memory, and in mediating the psychoactive effects of cannabis.

Answer 23

cAMP is also involved in neuroscience. cAMP will activate protein kinase A, which activates CREB, which up-regulated gene synthesis of endocannabinoids by binding to the CRE gene.

From Wiki: The endocannabinoid system (ECS) is a group of endogenous cannabinoid receptors located in the mammalian brain and throughout the central and peripheral nervous systems, consisting of neuromodulatory lipids and their receptors. Known as “the body’s own cannabinoid system”,[1] the ECS is involved in a variety of physiological processes including appetite, pain-sensation, mood, and memory, and in mediating the psychoactive effects of cannabis.

Question 24

G-protein coupled receptors:

Desensitization occurs in two steps:

1. Reversible: ___ of receptor uncouples the receptor from G-protein so that binding of agonist no longer activates effector. Regeneration of the active receptor occurs by ____.
2. Irreversible: if receptor remains _____ for a long period of time, it binds to __-____ and is sequestered into the cell where it is degraded (downregulation).

Answer 24

G-protein coupled receptors: Desensitization occurs in two steps:

1. Reversible: Phosphorylation of receptor uncouples the receptor from G-protein so that binding of agonist no longer activates effector. Regeneration of the active receptor occurs by de-phosphorylation.
2. Irreversible: if receptor remains phosphorylated for a long period of time, it binds to beta-arrestin and is sequestered into the cell where it is degraded (downregulation).