Signaling

Question 1

Two types/locations of receptors

Answer 1

Receptors can be located in the
cytoplasm or in the plasma membrane of the cell.

Question 2

Membrane receptors:

Answer 2

Large or polar ligands cannot cross the
lipid bilayer. Insulin, for example, is a protein hormone that cannot diffuse through the plasma membrane; instead, it binds to a transmembrane receptor with an extracellular binding domain.

Question 3

Cytoplasmic receptors:

Answer 3

Small or nonpolar ligands can diffuse
across the nonpolar phospholipid bilayer of the plasma
membrane and enter the cell. Estrogen, for example, is a
lipid-soluble steroid hormone that can easily diffuse across the plasma membrane; it binds to a receptor in the cytoplasm.

Question 4

Signaling method: ligand-gated channels

Answer 4

Ligand binds to receptor on cell surface

⇓

Open a few ligand-gated channels

⇓

A little ion flow

⇓

Hit threashold voltage

⇓

Open many (voltage gated) channels

⇓

big chainge in ion concentrations

⇓

BIG EFFECT

Question 5

Signaling example: ligand-gated channels

Acetyl choline (AcCh)

Answer 5

Ligand binds to receptor on cell surface

AcCh binds

⇓

Open a few ligand-gated (Na+) channels

⇓

A little ion* (Na+) * flow

⇓

Hit threashold voltage

less (-) inside

⇓

Open many (voltage gated Na+) channels

⇓

big chainge in ion (Na+) concentrations

⇓

BIG EFFECT

Muscle contracts

Question 6

Signaling Method: Cascades of Modification

Answer 6

ligand (1st messenger)

⇓

activate receptor in membrane

⇓

activate protein inside cell (usally a chain of activtions = cascade)

⇓

activate a lot of target protein (enzyme or TF…

⇓

lots of product

Examples: Hormones

TSH & epinephrine

• TSH:* stimulates release of thyroid hormone (thyroxine of TH) from thyroid gland.
• Epinepherine* stimulates glycogen breakdown.

*Many water soluble hormones work this way.

Question 7

Signaling Method: by affecting transcription/translation

Answer 7

Ligand binds

⇓

activate a TF

⇓

transcribe a gene

⇓

make lots of mRNA molecules/gene

⇓

mRNA translated

⇓

many new protein molecules/mRNA

Examples:

Thyroid hormone (thyrotropin or TH) & steroid hormones.

Most lipid soluble hormones act in this way. Receptor itself is a TF.

Question 8

Lipid Soluble Ligands

Answer 8

• All use intracellular recptors (steroids, TH, retinoids, vit A, vit D)
• cannot be stored (can pass through membranes, so must be made from soluble precusors as needed

Question 9

Intracellular Receptors

Answer 9

_Types: _

• *lipid soluble ligands *
• hormone binding proteins are needed in blood (are not water soluble, so all lipid soluble ligands travel in blood bound to soluble proteins

All are TFs: activate or repress transcription

• Example: HRE

Have at least 3 domains:

1. Transcription activating (or inhibiting) domain
2. DNA Binding Domain: binds to HRE (specfic to each hormone)
3. Ligand binding domain: binds to a particular steriod (or thyroxine, etc)

• Other domains: receptors also need NLS, and region that allows dimerization, these may be included or separate from the domains listed above.

Question 10

Usual course when TF receptors bind to lipid soluble ligands: Receptor activtion and transcription effect

Answer 10

1. Binding: receptor binds its ligand. Causes conformational change of at least one domain of protein receptor.
2. Disassociation: receptors disassociate from inhibitory proteins.
3. Dimerazation: pairs formed
4. Location:
   •If receptor is in cytoplasm, NLS is uncovered, and receptor moves to nucleus.
5. DNA binding: activted receptor (dimerized & bound to ligand) binds to HRE on DNA.
6. Effect on transcription: activated receptor binds to other proteins associated with the DNA (other TF’s and/or coactivators or inhibitors and stimulates or inhibits transcription)

Question 11

Why do you get different results (patterns of transcription) for different genes in response to the same lipid soluble hormone?

Answer 11

Different genes have different cis-acting regulatory sites. (different genes respond differently to the same combination of TFs)

NOTE: All cells have the same DNA: therefore

• all cells (except immune system have the same cis-acting regulatory sites: same HREs, enhancers, etc
• TRANS-acting factors (ie: horomone receptors and other TFs that vary, between cells, not the cis-acting regulatory sites
• cis-acting regulatory sites do vary beween GENES
• all cells have the same genes for the trans acting factors, receptors, etc. but different genes are used (expressed) to make diffeent regulatory proteins in different cells.

Question 12

A Protein Kinase Receptor

Answer 12

The mammalian hormone insulin binds to a receptor on the outside surface of the cell and initiates a response.

Question 13

A Gated Ion Channel

Answer 13

The acetylcholine receptor (AChR) is a
ligand-gated ion channel for sodium ions. It is made up of five polypeptide subunits. When acetylcholine molecules (ACh) bind to two of the subunits, the gate opens and Na+ flows into the cell. This channel helps regulate membrane polarity

Question 14

GPCR (G Protein Coupled Receptors) Antagonists

Answer 14

block the action of the normal ligand; blocks signaling even in the presence of normal ligand.

Question 15

GPCR (G Protein Coupled Receptors) Agonists

Answer 15

Mimic the action of the normal ligand, causes signaling in the absence of normal ligand.

Question 16

The Structure of G Protein-Linked Receptors

Answer 16

Each G protein-linked receptor has seven transmembrane helices.

A ligand binds to the extracellular portion of the receptor, causing an intracellular portion of the receptor to bind and activate a G protein.

Besides the regions shown, the second cytosolic loop is also involved in G protein interactions in some cases.

Specific amino acids in the cytosolic region are also targets for phosphorylation by G protein-linked receptor kinases (GRKs) and protein kinase A.

Question 17

Compare and contrast G Proteins and GPCRs

Answer 17

G Proteins: activated by receptors

GPCRs: the actual receptor itself

Question 18

Enzyme linked receptors

Answer 18

• Structure: usually single pass proteins that aggregate into dimers when activated
• Function: Many Growth Factors use TK linked receptors or related receptors
• How? Usually generate cascades of modification, but do not usually use 2nd messengers.

Many are linked to protein kinases (intracellular kinase domain in addition to extracellular ligand binding domain), or interact with an intracellular kinase (when activated).

Question 19

What are the active/inactive forms of G protien?

Answer 19

Active: bound to GTP

Inactive: bound to GDP

*Why a switch? G protein does not stay active for long. Turns itself off.

Details:
G protein is activated by dumping GDP and picking up GTP in response to some signal. NOT activated by phosphorylation of hte bound GDP to GTP.

G protein inactivates itself by catalyzing hydrolysis of GTP to GDP.

Question 20

G protein should be able to catalyze:

a) phyosphorylation of GDP –> GTP
b) hydrolysis of GTP –> GDP
c) both
d) either one

Answer 20

b) hydrolysis of GTP –> GDP

When triggered by the appropriate receptor, a G protein binds GTP, which displaces a GDP. The GTP is then quickly hydrolyzed to GDP by the G protein itself, which is a GTPase.

Question 21

Why are ser and thr used by protein kinase to add phosphate.

Answer 21

Ser and Thr are the only AAs with free hydroxyls in their side chains. OH is needed to form an ester bond with a phosphate.

Question 22

Which type of hormone would you find circulating freely in blood? Which would be bound to a protien?

Peptide hormones

or

Steroids

Answer 22

Peptide hormones have hydrophilic regions–> can circulate free in blood.

Steriods are lipids and hydrophobic (lipophilic). To prevent them from forming fat globules in the watery blood, they are carried in association with the hydrophilic proteins.

Question 23

Which is faster acting, epinepherine or a cortisol?

Answer 23

Epinepherine induces a more rapid response, bc simply activates certain proteins that already exist in the cell. Making new proteins from scratch takes longer, wich is the case with steriod hormones like cortisol.

Question 24

What do steroids look like?

Answer 24

Lipid soluble:

greasy/oily phase /dirty

Question 25

What happens to the nucleosomes when chromatin is transcribed?

Answer 25

The transcribed region should retain all or most of its nucleosomes when the RNA polymerase passes through (ie: # of H2B molecules stays the same).

The number of nucleosomes may decrease significantly in the non-transcribed regulatory regions as the nucleosomes are replaced by regulatory proteins.

When transcription occurs, the nucleosome fiber will probably become more loosely coiled, going from a tighter, more closed configuration (such as 30 nm fiber) to a looser, more open config (like beads on a string) –# of nucleosomes does not change but state of packing does. The looser structure will be more sensitive to DNase as the DNA will be moe acessible to nucleases.

Question 26

What are the 3 main sections of a hormone receptor?

Answer 26

1. DNA Binding Domain
2. Transcription Activating Domain
3. Hormone (ie: cortisol, estrogen, etc) binding doinmain **which aslo includes the NLS!!!***

Question 27

How can the addition of one type of hormone (ie: cortisol) turn on transcription of many different genes?

Answer 27

1. All the genes that share the same regulatory sequence can be turned on simultaneously by action of the same TFs. The genes can be located far from each other but each has the same “switch” located in the proximal or distal reg region.
2. When the switch is activated in response to the addition of hormone, the switch (cis-acting sequence) is known as a Hormone Response Element (HRE)
3. All HREs of one kind (ie: corisol response elements) bind the same TF (activated cortisol receptor. All the estrogen response elements bind a different TF = the activated estrogen receptor.)
4. When steroid hormones are added, the hormones ind to their respective receptors. The receptors are then activated, go to the nucleus, bind to their respective HREs and activate (or inhibit) transcription.

Note: Adding hormone, even at low concentration, always means adding many molecules of hormone. Thus adding hormone always activates many copies of the receptor. And there is enough activated receptor to bind to multiple HREs and turn on multiple genes.