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63 Cards in this Set

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1. Cell surface receptors

2.Intracellular receptors
Two main classes of hormone-receptor signaling:
Examples

1. The adenylate cyclase/cAMP system

2. The phosphatidylinositol system

3. The tyrosine kinase system (e.g. the insulin receptor)
Cell surface receptors

Hormones that bind to surface receptors change the activity, not the amount, of a critical enzyme.

Affects the immediate response, not the response later down the pathway.
Examples

1. Steroid hormones

2. Thyroid hormones
Intracellular receptors

Hormones that bind to intraceullular receptors change the amount of a critical enzyme at the level of gene transcription.

Becomes a transcription factor rather than changing the activity.
The amino terminus is outside the cell (part of the extracellular domain).

The carboxy terminus is in the cytosol (part of the cytoplasmic domain).

The extracellular region forms the ligand binding domain.

Usually glycolysated at specific amino acids

The transmembrane domain is comprised of hydrophobic a-helices.

A typical b-adrenergic receptor has seven membrane-spanning helices
a.k.a. heptahelical receptors

The cytoplasmic domain often has a specific function:
- catalytic activity
- a site for phosphorylation

The glucagon and epinephrine receptors are examples of b-adrenergic receptors.
Features of B-adrenergic receptor
The amino terminus is ______ the cell.

Part of the extracellular domain
outside
The carboxy terminus in in the ______

Part of the cytoplasmic domain
cytosol
The _____________ region forms the ligand binding domain.

Usually glycolysated at specific amino acids
Extracellular region
The transmembrane domain is comprised of hydro_____ a-helices.

A typical B-adrenergic receptor has seven membrane-spanning helices (called heptahelical receptors)
hydrophobic a-helices
The __________ domain often has a specific function

- Catalytic activity
- A site for phosphorylation
Cytoplasmic domain
Glucagon and epinephrine receptors are examples of ___-adrenergic receptors
B-adrenergic receptors
Multi-subunit protein complex (a, B, and y) localized within the cell membrane
G-Proteins
The initial _______ of adrenergic receptor activity.
effector
G-proteins bind GTP and GDP to the ___-subunit
alpha
When GDP is bound, the subunits.....
associate
When GTP is bound, the alpha-subunit _______ from the B and y
dissociates
When the alpha-subunit dissociates from the B and y, it results in a _____________ change that affects the interaction of the G-protein with other proteins.

Ex. Adenylate cyclase
Conformational changes
The alpha subunit is an enzyme, what is it called?
GTPase

hydrolyzes GTP to GDP and Pi
1. In the inactive state, the alpha subunit binds GDP and the a, b and g subunits are associated.

2. Hormone binds the receptor, causing a conformational change in the receptor protein.

3. This change in the receptor allows it to bind to the G protein alpha subunit.

4. When the G protein binds the receptor, it has a lower affinity for GDP and a higher affinity for GTP.
a. This causes GTP to replace the GDP bound to the alpha subunit.

5. When GTP is bound to the alpha subunit, the a subunit dissociates from the receptor and the b/g subunit due to a conformational change in the alpha subunit.

6. The free GTP-bound alpha subunit can now bind to adenylate cyclase, activating the enzyme.
a. This causes adenylate cyclase to convert ATP to cAMP and PPi

7. The alpha subunit then hydrolyzes GTP to GDP, which causes the now GDP-bound alpha subunit to dissociate from adenylate cyclase and re-associate with b/g.
Mechanism of G-protein action
Cyclic AMP as a second messenger for hormone receptors
Activates PKA

PKA is degraded by phosphodiesterase

Phosphodiesterases are inhibited by methylzantines, caffeine, theophylline.
a. Prolongs the cAMP response
Hormone binding affects specific metabolic pathways through the _________ of adenylate cyclase activity by the G protein
stimulation.

The G-protein mechanism example is exemplified by the glucagon and epinephrine receptor pathways.
There are cells and specific receptor types in which a G-protein activity ________ adenylate cyclase upon receptor ligand binding.
inhibits
Gs - stimulatory

Gi - inhibitory
The two distinct types of G-proteins
There is signififcant diversity among the different alpha-subunits of the G-proteins

Example: The protein Gi1 in nervous tissue decreases adenylate cyclase activity
The differences in these proteins reside in the alpha subunit
Turning off ligand-mediated signal transduction.

1. The inherent GTPase activity of the alpha subunit of the G-protein stops adenylate cyclase stimulation.

2. Phosphodiesterase activity deactivates cAMP

3. Phosphatase activities in the cell dephosphorylate enzymes that are phosphorylated as a result of protein kinase A.
At the level of downstream enzymatic activities.
Turning off ligand-mediated signal transduction.

1. The receptor is deactivated by phosphorylation of another intracellular protein kinase, b-adrenergic receptor kinase (BARK).

2. Hormone-receptor complex can also be endocytosed (internalized via membrane invagination)

3. The longer the cell is exposed to the hormone, the more likely that one of these inactivation pathways will occur.
Turning off signal transduction at the receptor level.
_____ is phosphorylated by PKA

Activated _____ then phosphorylates the receptor, decreasing its affinity for the hormone.
BARK

B-adrenergic receptor kinase

A feedback kinase that imposes a time limit of the hormone
Hormone-receptor complex can also be endocytosed (internalized via membrane invagination)
The resulting vesicle fuses with the lysosome, where the hormone (and in some cases the receptor) are degraded.

If the receptor is not degraded, it may be recycled back to the membrane
The result is that fewer active receptor molecules are present in the cell membrane.

The cell is therby 'desensitized' to the hormone.
The longer the cell is exposed to the hormone, the more likely that one of these inactivation pathways will occur.
Cholera toxin and cAMP production
Consists of two subunits:
1. One binds a specific ganglioside in the intestinal mucosal cell
2. The other then passes into the intestinal cell
a. This subunit has ADP-ribosylation activity, which modifies the G protein a subunit

b. The modified a subunit has no GTPase activity
Cholera toxin is a protein secreted by the pathogen Vibrio cholerae

No GTPase activity = active adenylate cyclase.

Phosphorylates CFTR receptor
Opens the Cl- channel
Cholera toxin and cAMP production:

The ADP-ribosylated G protein alpha subunit loses its GTPase activity

As a result, cAMP levels remain high in these intestinal cells
1. cAMP is important in these cells for ion balance and consequent water transport.

2. cAMP stimulates transport of chloride ion out of these cells
Infected individuals have an increased secretion of chloride ion from the cell to the intestinal lumen

Results in severe diarrhea
A different signal transduction system for G-protein coupled receptors
The phosphoinositol signaling system
Inositol triphosphate (IP3)

Diacylglycerol (DAG)
The second messengers of the phosphoinositol signaling system
The substrate for forming the second messengers IP3 and DAG is
Phosphoinositol in the cell membrane (PIP2)
Phospholipase C

Activated by the GTP-bound G protein on alpha subunit
The membrane-associated enzyme that catalyzes the reaction in phospholipase C
- Growth Hormone

- Thyrotropin-releasing hormone

- Gonadotropin-releasing hormone

- alpha-1-adrenergic receptor
Examples of hormones that act by the PIP2 system

Phospholipase C is analagous to adenylate cyclase
Hormone binds to the receptor and activates the G-protein system in a manner similar to that of the b-adrenergic system.
In this case, the Ga-GTP subunit activates Phospholipase C
Phospholipase C catalyzes the hydrolysis of ____________ ___________(PIP2) from the cell membrane into the products diacylglycerol (DAG), which stays in the membrane, and inositol triphosphate (IP3), which difuses to the cytosol.
phosphatidylinositol bisphosphate
The IP3 binds to a receptor on the endoplasmic reticulum and activates Ca2+ release from the ER calcium stores into the cytoplasm.

The Ca2+ moves toward the membrane, and together with DAG, activates the membrane-localized Protein kinase C (PKC).
Calcium may have other effects in the cytosol

Calmodulin plays a role in certain cell types
Protein kinase C can then phosphorylate proteins in the cytosol to elicit downstream effects.

e.g. activation of the heterodimeric transcription factors AP1 & AP2 by phosphorylation.
AP1 and AP2 promotes DNA binding and subsequent gene activation analogous to activation of CREB by PKA
The IP3 is recycled back to inositol and can reform phosphatidylinositol in the membrane for additional activation cycles

This process is inhibited by lithium, which is the basis for its use as a treatment for manic-depressive disorders.
Inhibits subsequent reactivation cycles, reducing the effect of phospholipase C-coupled hormone receptor binding by overly abundant ligands
____ activation increses transcription of genes by phosphorylation of CREB

_____ activation stimulates AP1 and AP2, which are also positive transcription factors.
Protein Kinase A

Protein Kinase C
Tyrosine kinase surface hormone receptors

Exists as a preformed dimer of an alpha and beta subunit in the cell membrane
The insulin receptor system.

Growth factor receptors = atypical
- Usually dimerize upon ligand binding
1. Upon insulin binding, the receptor is autophosphorylated on its own tyrosine residues on the cytoplasmic domain (b subunit)

2. The tyrosine kinase activity then acts to phosphorylate other proteins within the cell

3. One of the major effects of insulin action is that glucose transport into the cell is enhanced
Initial steps in the Insulin Receptor pathway.
Name the step:

Phosphorylates itself

Activates the tyrosine kinase activity of the insulin receptor
1. Upon insulin binding, the receptor is autophosphorylated on its own tyrosine residues on the cytoplasmic domain (b subunit)
The tyrosine ______ activity then acts to phosphorylate other proteins within the cell
2

kinase
Name the step:

Mediated by an increase in GLUT 4 transporters in the cell membrane.

There are many intervening steps in the insulin signal transduction pathway between insulin binding and the ultimate effects.
3. One of the major effects of insulin action is that glucose transport into the cell is enhanced

Major effects:
Increases GLUT 4 transporters in the membrane
Mechanism of Insulin Receptor Action

1. Ligand binding to the insulin receptor causes to phosphorylation of the receptor, which activates its cytoplasmic tyrosine kinase activity

2. The primary target for the kinase is Insulin Receptor Substrate (IRS-1), which is phosphorylated by the receptor.

3. IRS-1-P activates phosphatidylinositol-3-kinase (PI3K), an enzyme that then converts phosphatidylinsositol bisphosphate (PIP2) to phosphatidylinositol triphosphate (PIP3).
4. PIP3 activates a PI-dependent kinase (PDK-1), which can then catalyze the phosphorylation of protein kinase B (PKB) and an "atypical" protein kinase C (PKCalternate).

5. Both PKB and PKCa then mediate the increased trafficking of GLUT4-containing vesicles to the cell membrane, which fuse with the membrane, thereby increasing the concentration of GLUT4 molecules in the membrane.
a. This increases the transport of plasma glucose molecules into the cell.

6. IRS-1-P also mediates the mitogenic (growth) effects of insulin signaling.
1. Ligand binding to the insulin receptor causes the ___________ of the receptor, which activates its cytoplasmic tyrosine kinase activity
phosphorylation
2. The primary target for the kinase is ________ _________ _________ (IRS-1), which is phosphorylated by the receptor.
Insulin Receptor Substrate (IRS-1)
3. IRS-1-P activates ______________-__-________(PI3K).

- An enzyme that then converts phosphatidylinsositol bisphosphate (PIP2) to phosphatidylinositol triphosphate (PIP3).
phosphatidylinositol-3-kinase (PI3K)

An enzyme that then converts phosphatidylinsositol bisphosphate (PIP2) to phosphatidylinositol triphosphate (PIP3).
4. PIP3 activates a Phosphatidylinositol-dependent kinase (PDK-1),

PDK-1 can then catalyze the phosphorylation of _________ ______ __ (PKB) and an "atypical" ________ ______ (PKCalternate).
Protein kinase B (PKB) and Protein Kinase C (PKCalternate)
5. Both PKB and PKCa then mediate the increased trafficking of GLUT4-containing vesicles to the cell membrane, which fuse with the membrane, thereby increasing the concentration of _____ molecules in the membrane.

a. This increases the transport of plasma glucose molecules into the cell.
GLUT4
6. IRS-1-P also mediates the _________ (growth) effects of insulin signaling.
mitogenic effects

1. Allow the cell to harness pending energy input

2. General growth promoter
- Protein synthesis
- Mitosis
- DNA replication
Liver & pancreatic b cells

High relative Km for glucose (low affinity)

Only transport glucose at high blood concentrations
GLUT2

Consistent with role of liver in maintaining blood glucose levels, and the pancreatic b cells in sensing high blood [glucose].
Recruited to the plasma membrane in response to insulin

Low relative Km for glucose (high affinity)

Insulin increases glucose transport into adipose cells for synthesis of fatty acids and glycerol by glycolysis for TAG storage.


Insulin increases glucose transport into skeletal muscle cells for glycolysis and glycogen storage.
GLUT4

Skeletal muscle & adipose

Insulin-responsive
This family of receptors encompasses hormones of the steroid, retinoic acid, and thyroid classes.

These hormones can pass through the plasma membrane and interact with receptors inside the cell.

Intracellular receptors are localized to either the cytoplasm or nucleus.
Intracellular Hormone Receptors


In both cases, the hormone-bound receptor moves to the nucleus, where it binds to DNA through a DNA-binding domain in the receptor to either activate or repress target gene transcription.
Where the hormone binds
Ligand (hormone) binding domain
Allows DNA molecules to interact with the structure
DNA binding domain
Disparate between receptors

May contribute to different regulation of the receptor.
Variable domain
The steroid/retinoic acid/thyroid hormone receptors belong to a "___________" and share several conserved features.

Three main functional domains:
1) Ligand (hormone) binding domain
2) DNA binding domain
3) Variable (or antigenic) domain
superfamily
Characterized by an arrangement of cysteine residues that coordinate a zinc ion in a tetrahedral configuration.
a. Forms "fingers" that can interact with specific nucleotides in the major grove of the response element.

The zinc finger domain is utilized by many transcription factors, which can contain as many as 11 fingers.
Zinc fingers

Two zinc fingers comprise the DNA-binding domain of intracellular hormone receptors.

Lie in groove of DNA

Amino acids in fingers make contact in specific DNA sites.

Some factors utilize a combination of Cys and His to coordinate Zn.
1. Hormone is carried in the blood by a carrier protein.

2. The hormone disccociates from the carrier and diffuses across the cell membrane.

3. Hormone binds to an unactivated cytosolic receptor that is "protected" by a heat-shock protein.
4. Hormone binding dissociates the heat-shock protein, exposing a nuclear localization signal and the DNA-binding domain on the receptor.

5. The hormone/receptor complex translocates to the nucleus through a pore complex where it binds to specific DNA elements in the target gene promoter through the zinc finger domain.

6. The receptor-DNA complex nucleates the RNA Pol II holocomplex, which then transcribes the target gene.

7. The resulting mRNA moves to the cytosol, where it is translated into protein.
1. Hormone is carried in the blood by a carrier protein.

2. The hormone dissociates from the carrier, diffuses across the cell membrane and into the nucleus, where it binds to the nuclear receptor.

3. Unliganded nuclear receptors are not bound by HSP, but the DNA-binding domain is masked in the inactive conformation of the receptor.
4. Hormone binding changes the conformation of the receptor, exposing the DNA-binding domain.

5. The activated hormone/receptor complex binds to specific DNA response elements near target genes through the receptor zinc finger domain.

6. The receptor-DNA complex nucleates the RNA Pol II holocomplex, which then transcribes the target gene.

7. The resulting mRNA moves to the cytosol, where it is translated into protein.