Lecture 5: Hormone Signaling Pathways

Question 1

Why is only a small amount of hormone necessary to generate a signal?

Answer 1

The effect of hormone binding to its receptor is magnified via amplification.

Question 2

Once the ligand binds to its receptor - the receptor complex can do what to cellular pathways?

Answer 2

• Activate or inhibit cellular pathway (s) that elicit a particular cellular response (enzyme activity, gene expression)

Question 3

Differentiate endocrine, paracrine, autocrine, and juxtacrine signaling; give an example of a molecule that participates in each one?

Answer 3

Endocrine: signaling molecule released by a cell distant to target and transported via bloodstream to target. i.e., epinephrine

Paracrine: signaling molecule released by one cell type and diffused to a neighboring target cells of different cell type i.e. testosterone

Autocrine: signaling molecule acts on the same cell type as the secreting cells themselves i.e. IL-1

Juxtacrine: signaling molecule stays attached to the secreting cell and binds to a receptor on an adjacent target cell i.e. heparin-binding epidermal growth factor

Question 4

Can a signaling molecule be used in more than one type of signaling?

Answer 4

Yes, some molecules may participate in more than one type of signaling

Question 5

What are the receptor involved in hydrophilic hormone signaling; what does the signaling molecule-receptor complex initiate?

Answer 5

• GPCRs
• RTKs
• Initiates production of second messenger molecules inside cell
• Both found on the surface of target cells, since the signaling molecule won’t be able to diffuse throught the membrane

Question 6

What are the receptor types for lipophillic hormone signaling (differentiate between the two); binding to the receptor does what?

Answer 6

• Cytoplasmic receptors: exist as inactive complex. Upon binding, the hormone-receptor complex translocates to nucleus where it binds to a specific DNA sequence called the hormone response element (HRE)

- Nuclear receptors: already present in nucleus bound to DNA. The hormone allows for interaction w/ additional proteins and activates the complex

• Signaling molecule-receptor complex acts as a transcription factor

Question 7

Differentiate between hydrophilic and lipophilic medications?

Answer 7

Hydrophilic: have short 1/2-lives (seconds to mins.). Given a the time of need, like epinephrine.

Lipophilic: have long 1/2-lives (hours to days). Need to take daily, like oral contrapceptives.

Question 8

What are lipophilic signaling molecules?

Answer 8

• Steroid hormones: progesterone, estradiol, testosterone, cortisol
• Thyroid hormone: thyroxine (T4)
• Retinoids: retinol, retinoic acid

Question 9

What are some hydrophilic signaling molecules?

Answer 9

• AA derivatives: histamine, serotonin, melatonin, dopamine, NE, epi
• Acetylcholine
• Polypeptides: insulin, glucagon, cytokines, TSH

Question 10

Explain the general mechanism for GPCR signaling

Answer 10

• Trimeric G-proteins contain three subunits (α, β, γ)
• Inactive G protein has GDP bound to α subunit, and to become active G protein must exchange GDP for GTP using GEF, and α-subunit separates from beta and gamma subunits
• To return to inactive state, intrinsic GTPase activity of G protein hydrolyzes bound GTP into GDP + Pi w/ help from GTPase-activating protein (GAP)

Question 11

What occurs when a signaling molecule binds to a GPCR using Gs pathway?

Answer 11

• Stimulates adenyly cyclase, which increases cAMP that can activate PKA.
• PKA will phosphorylate target proteins to alter their activity

Question 12

What occurs when a signaling molecule binds to a GPCR using Gt pathway?

Answer 12

• Light hits a GPCR leading to the stimulation of cGMP phosphodiesterase, which converts cGMP –> 5-GMP, halting any action being promoted by cGMP

Question 13

What occurs when a signaling molecule binds to a GPCR using Gi pathway?

Answer 13

• Signaling molecule binds causing the inhibition of adenylyl cyclase so NO cAMP is produced, and PKA is NOT activated

Question 14

What occurs when a signaling molecule binds to a GPCR using Gq pathway?

Answer 14

• Activates PLC, which produces the second messengers DAG and IP3
• IP3 leads to increased Ca2+
• DAG leads to the activation of PKC, which phosphorylates target proteins to alter their activities

Question 15

Epinephrine, histamine, and NE bind to what type of receptors, activating which GPCR pathways?

Answer 15

• Epinephrine binds β-adrenergic receptor activating Gs
• Histamine binds H2-receptor activating Gs
• Epinephrine/NE bind α-adrenergic receptor activating Gi

Question 16

Dopamine, ACh, and light bind to what receptors utilizing which GPCR pathway?

Answer 16

• Dopamine binds D2-receptor utilizing the Gi pathway
• ACh binds the M3-receptor utilizing the Gq pathway
• Light activates rhodopsin receptor utilizing Gt pathway

Question 17

What is the structure of insulin?

Answer 17

• Two polypeptide chains referred to as the A chain (21 AA’s) and B chain (30 AA’s)
• Linked together by two disulfide bridges (S-S)

Question 18

What is the secondary and tertiary structure of insulin; active vs inactive structure?

Answer 18

• 3-fold symmetry w/ Zinc in the center connected via histidines
• Inactive stored in body as a Hexamer

- Active form is a monomer

Question 19

Describe the basic process underlying insulin synthesis and secretion

Answer 19

• Glucose upregulates preproinsulin mRNA
• Translated into preproinsulin protein in the ribosome
• Cleaved by a protease to form proinsulin
• Folded and transported to Golgi to be packaged into granules
• C-chain cleaved to form A-chain + B-chain insulin structure

Question 20

What are the 2 phases of insulin release after glucose stimulation?

Answer 20

1) First (rapid but transient) release comes from a limited pool of granules referred to as the readily releasabel pool (RRP)
2) Second (sustained) phase comes from a larger pool referred to as the reserve pool.

Question 21

How does glucose cause the release of insulin granules from the pancreatic β-cell?

Answer 21

• Glucose enters the cell via GLUT 2 and will undergo glycolysis –> PDH –> TCA producing ATP/ADP
• ATP/ADP caused the closing of ATP-sensitive K+ channels, which lead to the opening of Ca2+ channels
• Opening of the Ca2+ channels stimulate the release of insulin granules into the blood

Question 22

Describe the mechanism upon insulin binding the RTK that involves the RAS pathway.

Answer 22

• Insulin binds RTK, receptor becomes dimerized and autophosphorylates tyrosine residues.
• IRS-1 is recruited and becomes phosphorylated, attracting the scaffolding protein called GRB-2, which activates the RAS pathway
• RAS pathway phosphorylates nuclear proteins causing an increase in the transcription of Glucokinase and increased glucose uptake

Question 23

Describe the mechanism upon insulin binding the RTK that involves the RAS-independent pathway.

Answer 23

• Insulin binds RTK, receptors dimerizes, autophosphorylates tyrosine residues.
• IRS-1 is recruited and phosphorylated, then recruits PI 3-kinase, which activates PKB via phosphorylation
• PKB phosphorylates many nuclear proteins and trigger the translocation of GLUT4 to plasma membrane; activation of glycogen synthase
• Leads to increased glucose uptake and glycogen synthesis

Question 24

How can insulin be measured in a Quantifiable manner?

Answer 24

Measured as amount of glucose cleared from the blood in response to a fixed dose of insulin

Question 25

What are some of the defects that can lead to insulin resistance?

Answer 25

- Defect in insulin signaling - Defect in the insulin receptor due to **mutations** - Defects in the insulin binding domain extracellularly and/or intracellularly (cause severe insulin resistance) - Defects in IRS1 and IRS2 - Phosphorylation of serine instead of tyrosine disrupts downstream signal

Question 26

What molecules can lead to the phosphorylation of Ser/Thr and contribute to insulin resistance; what is the bodies response?

Answer 26

- Cytokines, FFA's DAG, ceramide, and inflammatory molecules - Lead to degradation of the protein because body recognized it as foreign

Question 27

How do epinephrine and cortisol affect glucose metabolism?

Answer 27

**Epinephrine:** stmulates glycogen **breakdown** by promoting **glucagon secretion** **Cortisol:** stimulates **gluconeogenesis** upon the depletio of glycogen stores, by inducing transcription of enzymes involved in the pathway

Question 28

What are the 2 types of Nuclear Receptors; examples of the hormones that bind?

Answer 28

**Classic NR**: ligands are lipophilic hormones. Glucocorticoids, mineralcorticoids, estrogen, progesterone, and androgens **Orphan Receptors**: other receptos discovered by DNA sequencing (ligands unknown)

Question 29

What does the term "adopted" orphan receptor mean; examples of these ligands?

Answer 29

- When a ligand has been discovered for an orphan receptor it becomes known as "adopted" - Retinoids, thyroid hormones, Vit D, xenobiotics, androstane, etc..

Question 30

Where are Nuclear Receptors located and why are they important?

Answer 30

- Located in **nucleus** or in **cytosol** - Important **drug targets**

Question 31

What are the 3 major domains of nuclear receptors and function of each?

Answer 31

1) **Activation Function 1 Domain (AF1)** - modify the conformation of the entire receptor 2) **DNA binding domain (DBD)** - binds to regulatory sequences on DNA called hormone response element (HRE) 3) **Ligand binding domain (LBD) -** binds to various molecules (agonist or antagonist) which regulates ligand-dependent activation of receptor

Question 32

What is the primary (early) response to steroid hormone vs. secondary (delayed) response?

Answer 32

- Ligand binds, conformational change occurs, and will either come to nucleus to interact w/ DNA (cytoplasmic receptor) or form active complex to the HRE (nuclear recptor) - Activating gene expression will induce synthesis of primary-response proteins, which can go on to block primary-response genes and turn on secondary-response genes, mediating the secondary (delayed) response.

Question 33

What are the 2 types of estrogen receptors and both acts as estrogen-dependent what; which cells express them?

Answer 33

- ERα and ERβ - Both are estrogen-dependent **transcription factors** - Many cells express **BOTH ERα** and **ERβ**

Question 34

Where is ERα expressed most abundantly?

Answer 34

- Female reproductive tract (**uterus, vagina, and ovaries)** - Mammary gland, hypothalamus, endothelial cells, and vascular smooth muscle

Question 35

Where is ERβ expressed most abundantly?

Answer 35

- Prostate, ovaries, w/ lower expression in lung, brain, bone, and vasculature

Question 36

Explain the binding of estrogen to the estrogen receptors (ERα,ERβ)

Answer 36

- Estrogen receptor located **in the nucleus**, upon binding of estrogen undergoes a conformaional change, which involve the binding of many nuclear proteins - Results in activation of **HAT**, an enzyme that **modifies** **chromatin structure.** - **Recruits** proteins that comprise the **general transcription apparatus,** leading to enhances transcription to form mRNA

Question 37

How does Tamoxifen affect the mechanism of estrogen signaling and what is it commonly used for?

Answer 37

- Breast cancer drug similar in structure to that of estrogen. - Tamoxifen, **an antagonist**, is metabolized by CYP450 in liver to **4-hydroxy-tamoxifen**, which has slightly different structure than estrogen so binds receptor in different place - Different downstream proteins are activated and bind the complex and the end result is the activation of **Histone Deacetylase (HDAC),** which keeps DNA in chromatin form and **inhibits transcription** - Effective cancer drug because it stops the proliferation of cancer cells

Question 38

What are Non-genomic estrogen signaling pathways?

Answer 38

- Some ER's located in the **plasma membrane** - Belong to the **GPCR and RTK** type of receptors - Effects mediated through metabolic changes as well as changes in gene expression