Hormone Signalling Pathways

Question 1

what are hormones?

where are they synthesized and secreted by?

where are they released?

where do they bind? (important)

what happens after binding?

Answer 1

Messenger molecules

synthesized and secreted by “endocrine cells”

released into ECF or blood stream!

Bind to specific receptors on TARGET TISSUES (target cells are far away from where it was secreted… hence messenger)

activation of signal transduction (through ligand binding) and/or alternating gene expression –> leads to specific response

Question 2

what’s good to know about the amount of hormone secreted?

what about downstream?

Answer 2

little secreted, but leads to a huge amplification signal!

Question 3

steps of hormone signaling? (9 steps)

Answer 3

biosynthesis

storage

secretion

transport to tissue/cells

recognition and binding to receptors

activation of signal 
transduction pathway (ON SWITCH)

amplification and relay of signal

cellular response

degradation (OFF SWITCH)

Question 4

at which step of hormone signaling does the problem occur for type 1 diabetes?

type 2?

hypothyroidism?

Answer 4

biosynthesis… you don’t form enough insulin

binding to receptors! we have enough insulin but it’s not binding

biosynthesis..

Question 5

Signal termination can occurs by 3 ways… how?

Answer 5

ligand dissociation of the receptor

receptor is internalized / removed from the membrane

enzyme in the downstream signaling that prevents the formation of the second messenger.

Question 6

Cell is stressed.. what happens?

what are the two possible things that can be generated?

where are the receptors of the ligands in each case?

any subtypes of each?

Answer 6

generates signaling molecule (hydrophobic / hydrophilic) from the signaling cell

BOTH it is released and makes it to the target cell via the blood

if it’s hydrophobic it can pass through the plasma membrane (it doesn’t need a gate)… receptors are INSIDE THE CELL (either cytoplasmic and it moves to the nucleus once ligand is bound affect gene expression.. or in the nucleus already and when the hormone binds it turns on of off gene expression

if it’s hydrophilic it can’t pass the layer so it needs a receptor… receptors are OUTSIDE the cell

Question 7

What are the 4 types of signaling (with example of each)

and how do they work?

Answer 7

Endocrine signaling –> classic hormone signaling. (ex. epinephrine made from adrenal medulla –> to heart and affects HR) –> goes through blood and gets to its target cells

Paracrine signaling (Testosterone secreted by Leydig cells in the testes –> released into ECF –> neighboring cell (Sartoli cell) is the target cell –> stimulate sperm formation

Autocrine signaling (T lymphocytes –> Interleukin is released by the signaling cell, but it binds to receptors that are present on the same cell –> turns on gene expression.

Juxtacrine signaling (Fibroblasts –> secreting Heparin-binding EGF to the cell adjacent to it. they are Juxtacrine to each

Question 8

What are the water soluble hormones (hydrophilic)

lipid soluble (hydrophobic)

Answer 8

epi, insulin, glucagon, GH

estrogen, testosterone, cortisol, 1,25-dihydroxy cholecalciferol

Question 9

what are the two receptors that are involved in hydrophilic hormone signaling that we care about?

Answer 9

G protein- coupled receptors (GPCRs)

Receptor tyrosine Kinases (RTKs)

Question 10

How do lipophilic hormones get to their target receptor?

what happens once they get there? (include specifics)

Answer 10

can pass through the plasma membrane. receptors are located in the cytoplasm or the nucleus

if in the cytoplasm –> the molecule/receptor complex is going to the nucleus.. it binds to the specific DNA sequence (in the promoter region!) called the “hormone response element HRE”… this turns or off the expression of multiple genes.

if in the nucleus, it’ll just bind there

both of these alter gene expression through regulation of transcription.

Question 11

Hydrophilic medications.. what’s two examples? what’s its half life?

Answer 11

SUPER SHORT HALF-LIFE.. it is as needed. (seconds to minutes)

Epinephrine –> contained in auto injectors, used to treat severe acute allergic reaction

Insulin –> shot given right before eating.

Question 12

Lipophilic medications… what’s an example? what’s its half life?

Answer 12

longer half-life.. hours to days

oral contraceptives –> contains ethinyl estradiol that you need to take daily.

Question 13

How do GPCR’s work?

what kind of protein is it?

what kind of ligand binds to it?

how does it become active?

how does it become inactive again?

Answer 13

present on the surface of the plasma membrane. they’re 7 transmembrane domain sitting there.

the ligand is the hydrophilic hormone that binds to the receptor.

the receptor is connected to heterotrimeric G proteins (which has an alpha, beta, gamma subunit)

in the inactive form the alpha subunit is bound to a GDP..

once a signal comes in it tells the G protein to activate… GEF (Guanine Exchange Factor) turns the GDP to GTP, activating the G protein. The alpha and GTP together leave, and that alpha subunit is connected to some kind of enzyme to do its job.

once the alpha subunit has done its thing, the alpha subunit has intrinsic GTPase activity but it’s not super fast.. it can be accelerated through
GTPase Activating Protein (GAP)

back to inactive form.

Question 14

what are the 4 variations of GPCR signaling?

what does it stimulate/inhibit?

what is elevated or lowered?

Answer 14

Gs…

stimulates adenylate cyclase… messenger molecule is cAMP that is elevated

Gi…

inhibits adenylate cyclase so outcome is lower cAMP

Gt…

stimulates cGMP phosphodiesterase.. so it breaks cGMP down so that goes down

Gq…

activates phospholipase C, increases intracellular calcium

Question 15

What does epinephrine bind to on G proteins?

what’s the physiologic response of each?

Know the receptors!

what’s strange about epinephrine to note?

Answer 15

when binding to beta adrenergic receptor it uses the Gs pathway… so increasing cAMP levels.

the effect is relaxation of bronchial and intestinal smooth muscle, contraction of heart muscle

when binding to alpha adrenergic receptor it uses Gi… constriction of smooth muscle!

so epinephrine can bind to two different things that have the opposite effect. so it has the power to do different and opposite stuff

Question 16

What does Histamine bind to on the GPCR?

Physiologic response?

Answer 16

activates Gs –> bronchoconstriction and symptoms of allergic reactions.

(H2 receptor)

Question 17

What does Dopamine bind to on the GPCR?

Physiologic Response?

Answer 17

Gi –> increase HR

Question 18

what does acetylcholine bind to on the GPCR?

Physiologic Response?

Answer 18

Gq –> Bronchoconstriction and stimulation of salivary glands

Question 19

What does light bind to on the GPCR?

Answer 19

Gt –> Vision

Question 20

What does RTK do?

structure?

what is the general idea of what happens once something binds to the extracellular receptor?

what are the two signaling pathways from there?

Answer 20

3 different domains..

1) one is the ligand binding domain which is the Extracellular domain.
2) another membrane spanning domain and a third intracellular domain
3) the intracellular has a tyrosine residue.

when you have a binding of a ligand (on the extracellular side) causes dimerization.. which phosphorylates the tyrosine residues on the internal side, which recruits a bunch of stuff to it, activating it.

it can go through a RAS dependent and RAS independent signaling.

Question 21

What is the structure of insulin?

how are these connected to each other?

Answer 21

A chain and B chain. There’s a C chain that connects A and B but it is removed.

they remain close together by the disulfide bridges (2 bridges) and also there’s a disulfide bond on the A chain.

Question 22

When insulin is formed, what is its shape in the inactive form?

what about when it’s active?

Answer 22

a hexamer with a Zn in the middle of it.

the active form is a monomer (hexameter dissociates) which then goes to bind to the RTK

Question 23

What is insulin initially generated as?

what happens to it?

where is this happening?

what happens once insulin is formed?

Answer 23

Preproinsulin mRNA (by ribosomal transcription)

this then goes to the lumen of the ER –> proteolysis forms proinsulin –> transported to the golgi apparatus –> lose the pro so you have insulin

all this is happening in the pancreatic beta cells.

secreted into blood stream, bind to target and exert its effect

Question 24

what are the two phases of secretion for insulin?

Answer 24

first comes from a limited pool of insulin granules “readily releasable pool” (RRP) – 5%.

the second comes from the reserve pool. Granules must undergo mobilization until there is a steady release

Question 25

What is the signal for insulin release? (pathway)

1st step that needs to happen for insulin to be secreted?

what happens once its in the beta cells?

what’s the signal that helps inhibit the potassium ATP channel?

what then causes the insulin granules to be secreted?

Answer 25

glucose is taken up into the cell by GLUT2 of the beta cells via Facilitated Diffusion

it’ll enter the glycolytic pathway and converted to G6P through glucokinase.

then glycolysis, TCA, and the glucose has generated ATP!

so you have elevated ATP and that inhibits a potassium ATP channel. this causes depolarization of the plasma membrane that spreads across until it reaches a Ca2+ channel!

lots of calcium enters the cell, extra calcium comes in and causes insulin granules to be mobilized and released into the blood stream!

Question 26

What is sulfonylurea drugs?

Answer 26

they use the potassium channel as their target

in diabetes type 2, glucose isn’t up taking as well, so medicine targets at the level of the potassium channel to increase ca2+ –> secrete more insulin to uptake glucose!

Question 27

What is the Insulin signaling through RTK?

what happens once the tyrosines are phosphorylated?

One arm of the pathway? What does this one increase the transcription of?

What happens in the other arm of the pathway? What is ultimately recruited and what is increased in activation?

Answer 27

insulin from the pancreatic beta cells gets to the target cell RTK.

insulin binds to the RTK (doesn’t really dimerize since insulin is a dimer already). in response to binding, there’s a phosphorylation of the tyrosine in the intracellular domains.

recruitment of IRS-1 (insulin receptor substrate)

recruits GRB-2 after IRS-1 comes.

once recruited, it turns on the RAS or MAPK pathway.

the RAS phosphorylates several proteins which increase glucokinase transcription (which increases glucose utilization)

in the other way IRS-1 increases PI-3-kinase which activates PKB which phosphorylates stuff increases messages to GLUT4 and more gets inserted to the plasma membrane of the target cells and glucose comes in, and it activates glycogen synthase.

Question 28

Net effect of insulin on RTK? (3 things)

Answer 28

increase glucose uptake, increase glycogen synthesis

increase glucose utilization (through glucokinase)!

Question 29

How is termination of insulin signaling happening? (2 ways)

Answer 29

the receptor becomes internalized by the target cell.

it can be degraded by proteases OR recycled back to the plasma membrane and do it again..

Insulin down regulates its own receptor by decreasing the rate of synthesis or degradation.

Question 30

What is insulin resistance?

Answer 30

failure of normal amounts of insulin to elicit expected response.

you’ve given the person enough insulin and glucose but the glucose isn’t cleared from the body.

Question 31

What are reasons for insulin resistance (4 reasons)

what about the receptor?

what about signaling?

what enzyme is responsible for signaling problems?

what activates that enzyme?

what does this defect lead to?

Answer 31

down regulation of the insulin receptor (RTK)

defects in the insulin receptor (75 mutations found thus far)

Defects in Signaling
Defects in IRS1 and IRS2 (insulin signaling) –> phosphorylating serine instead of tyrosine (via Ser/Thr kinase) inhibits downstream stuff… this inactivates IRS1 and 2.

this same process also actually inhibits PKB so you’re not going to have all the stuff that we have on that right arm path.

ser/thr kinase is the enzyme doing all this… this is activated by cytokines, free fatty acids, DAG, ceramics, inflammatory molecules

this is a precursor for type 2 diabetes because the body compensates by trying to make more insulin, the receptor isn’t working, so things go really bad.

REMEMBER: insulin downregulates its own receptor.

Question 32

What are nuclear receptors?

orphan receptors?

importance of Nuclear Receptors?

Answer 32

work as transcription factors for lipophilic signaling.

they’re named based on the ligand that binds to them.

we don’t know what the ligands are… unknown ligands. (discovered by DNA sequencing)

VERY IMPORTANT DRUG TARGETS

Question 33

molecular structure of NRs?

what are the three domains of NRs?

what happens when it’s inactive?

what happens when the ligand binds to the domain?

what happens because of this conformational change?

final outcome?

Answer 33

all NRs have the basic structure with 3 major domains!

Ligand Binding domain
DNA binding Domain
“Activation function domain or Transcription Activating domain”

when it’s inactive, it’s surrounded by inhibitory proteins. –> keep it inactive

when the ligand binds to the ligand binding domain, it undergoes massive conformational changes… then all of the inhibitory proteins are dissociated and coactivators are bound to the ligand binding domain and the transcription activating domain.

this change allows it to bind to the DNA and serve as a transcription factor –> undergoes transcription and increase or decrease mRNA production.

Question 34

what is conserved on the nuclear receptors?

Answer 34

DNA-binding domain.

found on pretty much all nuclear domain

(HRE regions)

Question 35

What is the response (early) to steroid hormone? (primary)

late? (secondary)

example of both?

Answer 35

hormone binding to its receptor and a set of proteins are generated initially… or the primary-response proteins.

primary-response proteins that are generated shut off the primary response and turn on the secondary response!

testosterone increases spermatogenesis (primary) but builds secondary sex characteristics (body hair, puberty). the secondary response takes years to response (hence the delayed)

Question 36

Estrogen receptors?

types?

where is each type found?

how do these become active?

what are the different forms of active estrogen?

Answer 36

(works through the nuclear receptor)

alpha and beta.. both of which are made by product of different genes.

alpha is present abundantly in uterus, vagina, ovaries

beta in ovaries, prostate but also lung brain, bone, vasculature.

some cells have both!

for the estrogen receptor to work they need to dimerize (a + a), (b + b), or (a + b)

Question 37

mech of action of ERs?

what does the enzyme that is activated do?

what is the overall action of ER then?

Answer 37

nuclear receptor so 3 domains

estrogen binds to estrogen receptor –> dimerizes the receptor ==> binds to estrogen response element on the DNA –> recruits coactivators –> promotes the action of HISTONE ACETYL TRANSFERASE (HAT)!

Hat loosens up the chromatin and allows transcription to occur!

Question 38

What does tomoxafin do?

how does it work?

what happens to tomoxafin and then how does it participate in the estrogen reaction mechanism?

what’s the end goal?

Answer 38

treats breast cancer!

antagonizes estrogen. it binds to the same receptor but activates a different pathway that results in inhibiting transcription of estrogen.

so it is metabolized by the liver by cytochrome P450 to 4-hydroxy-tamoxifen –> binds to same receptor, dimerizes it –> recruits HISTONE DEACETYLASE instead.. this makes the chromatin super dense and prevents transcription

Question 39

what’s some data saying about ER signaling?

Answer 39

we think it’s in the nucleus but there are some studies that show it’s in the cytoplasm… some even say there’s evidence on the cytoplasm.

so some say it’s “non-genomic”.. but primary evidence is it’s a nuclear receptor.

Question 40

which cells secrete insulin?

which cells are targets of insulin?

Answer 40

Beta cells of the pancreas

Liver, skeletal muscle, adipocytes, brain