Receptors and signal transduction

Question 1

How do hydrophobic signaling molecules work?

Answer 1

Includes steroids and related molecules- diffuse through the membrane bind to receptors in the cytosol. The receptor-signal complex moves into the nucleus, where it can bind to transcription control regions in DNA and activate or repress gene expression

Question 2

How do hydrophilic signaling molecules work?

Answer 2

Includes small molecules (Ach, epi), peptides, and proteins (insulin). These molecules can’t diffuse across the cell membrane. They bind to cell surface receptor proteins, triggering a conformational change in the receptor and therefore activating it. Can cause a signaling cascade that leads to short term changes or a long term change in gene expression

Question 3

Ligands

Answer 3

Molecules that can bind to receptors. Can be hydrophobic (membrane soluble) or hydrophilic (membrane insoluble).

Question 4

Types of extracellular signaling (4)

Answer 4

1. Endocrine signaling
2. Paracrine signaling
3. Autocrine signaling
4. Signaling by plasma membrane attached proteins

Question 5

Endocrine signaling

Answer 5

The signaling molecules act on target cells distant from their site of synthesis. The signaling molecules (hormones) are produced and secreted by signaling cells found in the endocrine glands

Question 6

Paracrine signaling

Answer 6

The signaling molecules released by a cell affect only the target cells that are in close proximity. Neurons releasing neurotransmitters are an example

Question 7

Autocrine signaling

Answer 7

Cells respond to substances that they release themselves. This is characteristic of the growth factors released by tumor cells

Question 8

Signaling by plasma membrane attached proteins

Answer 8

Some signaling molecules are integral membrane proteins on the cell surface. The targets of these membrane signals are receptors on the surface of adjacent cells- their proliferation or differentiation are controlled by the signal.

Question 9

What is unique about epinephrine?

Answer 9

Some signaling molecules, including epinephrine, can act at both long and short ranges. Epinephrine functions as a neurotransmitter (paracrine signaling) and as a hormone (endocrine signaling)

Question 10

Human pheromones example

Answer 10

Human pheromones were promoted by the “Athena Institute”- not FDA regulated and similar to vitamins

Question 11

Nicotinic acetylcholine receptor

Answer 11

Expressed in muscle cells at nerve-muscle synapses- it is a ligand gated ion channel that admits K and Na. It is an excitatory receptor that can also be bound by nicotine

Question 12

Receptor-mediated endocytosis

Answer 12

A receptor on the cell surface binds to an extracellular ligand on a macromolecule that it recognizes. The receptor-ligand complex are brought to a plasma membrane region that buds inward to form a coated transport vesicle. The vesicle is usually coated with clathrin

Question 13

Which types of molecules are usually brought into the cell by receptor mediated endocytosis?

Answer 13

LDL, transferrin (iron carrying protein), protein hormones like insulin, and glycoproteins

Question 14

Receptor tyrosine kinases (RTKs)

Answer 14

Bound by extracellular signaling molecules such as growth factors. Implicated in cancer, when mutant forms of growth factors stimulate cell proliferation. Most RTKs are monomeric, but when a ligand binds to the extracellular domain, the two monomers will join together as a dimer

Question 15

RTK components (4)

Answer 15

1. An extracellular domain containing a ligand binding site
2. One hydrophobic transmembrane alpha helix
3. A segment in the cytosol that includes a domain with protein tyrosine kinase activity
4. A C-terminal segment that contains tyrosine residues that will be phosphorylated by the receptor’s own kinase

Question 16

What happens when an RTK forms a dimer?

Answer 16

The two poorly activated kinases are brought together and and phosphorylate each other. Phosphorylation increases the activity of the kinase by making the catalytic site available for ATP or a protein substrate to bind

Question 17

G protein-coupled receptors

Answer 17

These receptors respond to a lot of different signals, but they are found mostly in the CNS and used for neural signaling. Many drugs work by being agonists or antagonists of a GPCR. Protein kinases and second messengers like cAMP often participate in GPCR signal transduction pathways and have short term effects in the cell by changing protein activity

Question 18

Components of a GPCR signal transduction pathway (4)

Answer 18

1. A membrane-embedded receptor that contains 7 transmembrane alpha helices
2. A heterotrimeric G protein that acts as a receptor-activated switch
3. A membrane bound effector protein
4. Proteins that participate in amplification and desensitization of the signaling pathway

Question 19

Steroid receptors

Answer 19

These are intracellular receptors typically located in the cytoplasm or nucleus. These are small hydrophobic molecules that cross the membrane to bind to the receptor. This allows the receptor hormone complex to enter the nucleus and regulate gene activity.

Question 20

Bipartite response to steroid hormones

Answer 20

A primary response protein shuts off primary response genes and turns on secondary response genes, causing the release of secondary response proteins

Question 21

Which vitamin is associated with COVID-19 severity?

Answer 21

People hospitalized with COVID-19 who were deficient in vitamin D experienced worse COVID-19 outcomes than people with normal levels. People with low vitamin D levels are also more likely to test positive for covid. Low vitamin D levels are associated with an increased risk for respiratory tract infections in general. Vitamin D may turn down the “cytokine storm” through immune regulation

Question 22

Functions of vitamin D

Answer 22

Vitamin D is a steroid that helps the body absorb calcium, which is
essential for good bone health. Vitamin D also
helps the immune, muscle, and nervous systems function properly. Most vitamin D is made when an inactive form of the nutrient is activated in your skin when it’s exposed to sunlight. Smaller amounts of vitamin D are in fortified milk and other foods, fatty fish, and eggs.

Question 23

What is the role of vitamin D in cancer prevention?

Answer 23

Research suggests that women with low levels of
vitamin D have a higher risk of breast cancer.
Vitamin D may play a role in controlling normal
breast cell growth and may be able to stop breast
cancer cells from growing.
 There may be a similar relationship with colon
cancer and other non-skin cancers as well.

Question 24

Receptor mediated endocytosis

Answer 24

The ligand and receptor are internalized
into the cell. The cell membrane folds in and particles are transported in a coated vesicle

Question 25

Transferrin receptor

Answer 25

Ferrotransferrin transports iron in the blood. When it binds to a transferrin receptor, RME occurs and the complex is transported in a clathrin coated pit. Inside the cell, low pH causes release of iron from the ligand, but the ligand remains bound to the receptor. The ligand is now considered apotransferrin and is recycled to the cell surface. The pH increases as the endosome nears the cell surface, and apotransferrin is released from the receptor at the cell surface when the pH is neutral (7).

Question 26

Clathrin coated pits

Answer 26

Clathrin coated pits or vesicles are thought to function as intermediates in the endocytosis of most ligands bound to cell surface receptors. Some receptors undergo a conformational change when they bind to a ligand, so they will be retained in a coated pit. Coated pits are used to transport proteins from the plasma membrane and trans golgi network to late endosomes

Question 27

How do viruses use RME to infect cells?

Answer 27

Viruses have a DNA or RNA genome surrounded by a protein shell (capsid). The enveloped virus can enter the cell via RME. It is transported in an endosome, and the capsid comes apart. The virus’ genome is copied and its genes are expressed to make viral proteins. The genes and viral proteins are then assembled to make new viruses. The new viruses are released from the infected cell.

Question 28

LDL packaging

Answer 28

All lipoproteins have an amphipathic shell, a phospholipid monolayer, cholesterol, and a hydrophobic core made of esters or triglycerides. LDL is unique because it only contains one molecule of one type of apolipoprotein , which wraps around the outside of the particle as a band of protein

Question 29

LDL pulse-chase experiment

Answer 29

Human skin fibroblasts were incubated in a medium with radioactive LDL (the pulse). Excess LDL is washed away and the remaining LDL is incubated at 37 degrees C (the chase). The radioactive LDL rapidly disappears from the surface as it is internalized after the cells are warmed (only binding occurs during the pulse, not internalization).

Question 30

Endocytic pathway for internalizing LDL

Answer 30

An LDL receptor on the cell surface binds to an ApoB protein embedded in the phospholipid layer of an LDL particle. The receptor-ligand complex is incorporated in a clathrin coated pit, then an endocytic vesicle. The vesicle fuses with a late endosome, and the resulting pH change in the compartment causes the conformational change in the LDL receptor, and the LDL particle is released. The late endosome fuses with a lysosome and the LDL is broken down. The LDL receptor is recycled to the cell surface. It undergoes a conformational change in response to the pH so it can bind to a new LDL particle

Question 31

Familial hypercholesterolemia

Answer 31

Hereditary disease resulting in extremely high levels of LDL. Some mutations causing this disease prevent the synthesis of the LDL receptor protein, some prevent proper folding of the LDL receptor so it is degraded, others reduce the ability of the LDL receptor to tightly bind LDL. With mutant receptors, LDL can’t be internalized

Question 32

The LDL receptor was identified as a potential therapeutic target in which disease?

Answer 32

Alzheimer’s- it was observed that high concentrations of LDL receptors in the
brain protect mice from Alzheimer’s disease. Crossed a transgenic
Alzheimer’s mouse with a high LDL brain receptor mouse. The progeny still developed Alzheimer’s but much less so due to less “tau” protein generated in the neurons.

Question 33

High cholesterol mouse study

Answer 33

Mouse created at Baylor is not transgenic but rather is a human familial hypercholesterolemia xenograft mouse
model. Mouse chimeric liver is 95% humanized through transplant of LDL diseased human cells (loss of function mutations in LDLR) from liver transplant operation. Mouse was “cured” through single dose transfection of
human wild-type LDLR (gene therapy)

Question 34

Orphan receptors

Answer 34

GPCRs with no known ligand

Question 35

3 classes of GPCRs

Answer 35

Each class binds their ligand differently. Family A binds biological amines, light or odorants, peptides, purines, or lipids. Family B binds peptides. Family C binds biological amines, glutamate, and calcium

Question 36

GPCR general mechanism

Answer 36

When its ligand is not present, a G protein-coupled receptor waits at the plasma membrane in an inactive state. GPCRs all bind the nucleotide guanosine triphosphate (GTP), which they can break down (hydrolyze) to form GDP. A G protein attached to GTP is active, or “on,” while a G protein that’s bound to GDP is inactive, or “off.” With ligand binding, the GPCR is activated and causes the G protein to exchange GDP for GTP. The now-active G protein separates into two pieces (one called the α subunit, the other consisting of the β and γ subunits), which are freed from the GPCR. The subunits can interact with other proteins, triggering a signaling pathway that leads to a response.

Question 37

Robert Lefkowitz and Brian Kolbilka

Answer 37

Awarded a Nobel prize for studying GPCRs

Question 38

How do you Study receptors such as GPCRs? (4)

Answer 38

1. X-ray crystallography can be used that involves analyzing the diffraction patterns coupled to analytical programs to deduce structure
2. Radioligand binding assays can reveal
   competitive agonists or antagonists
3. Measuring the second messenger effect of
   GPCR stimulation
4. Nanodisks put the receptor in the bilayer

Question 39

Competitive binding assays

Answer 39

Used to measure receptor function. The maximum physiological response to a signal occurs when only a fraction of receptors are bound by a ligand. A radioactively labeled ligand is incubated with control cells that don’t express the receptor of interest and cells that do express the receptor. The amount of bound ligand per cell is calculated and plotted

Question 40

Nanodisks

Answer 40

A synthetic model
membrane system that places receptors in their native environment. MSP – Membrane Scaffold Protein serves
as a “belt” that wraps around the membrane
system

Question 41

GTPase switch proteins

Answer 41

These proteins can be turned on and off, activating target proteins. Ras and protein kinases are an example.

Question 42

GTPase switch proteins are regulated by

Answer 42

GEF and GAP. For example, GTPase protein sar1 is activated by ER membrane protein sec12. It acts as a GEF by catalyzing the release of GDP from cytosolic sar1 GDP and the binding of GTP.

Question 43

Muscarinic acetylcholine receptor

Answer 43

A type of GPCR found in cardiac muscle. They are activated when Ach binds, the the rate of contraction of the heart muscle slows down due to an influx of potassium

Question 44

FRET

Answer 44

Changes the wavelength of emitted fluorescence when two fluorescent molecules interact. Used to detect the dissociation of heterotrimeric G proteins after binding to GPCRs in live cells

Question 45

Rod photoreceptor

Answer 45

A phototransducer system using GPCRs. Rhodopsin is the receptor found in rod cells

Question 46

2 roles of retinal pigment epithelium

Answer 46

1. Phagocytose shed outer segment disks
2. Absorb stray photons to avoid back scatter

Question 47

How does the G protein process responsible for vision work?

Answer 47

Activates phosphodiesterase enzyme. In the dark- increased cGMP- channels open at -30 mV and cells release neurotransmitters. Light- decreased cGMP- channels close. cGMP is a second messenger that opens cGMP gated channels to depolarize the membrane

Question 48

Vision and light adaptation

Answer 48

Light acts by lowering the concentration of cGMP and closing of cation channels. The membrane potential becomes more negative (repolarizes) and the release of neurotransmitters is reduced. The more photons are absorbed by rhodopsin, the more cGMP is hydrolyzed, and the more channels are closed

Question 49

Cytokine receptors

Answer 49

Ligand binding causes a conformational change in the receptor and the kinase domains are brought together to phosphorylate each other.

Question 50

Epo

Answer 50

Erythropoietin is a cytokine that triggers the production of RBCs. It induces the proliferation and differentiation of erythroid progenitor cells in the bone marrow. Lance Armstrong was implicated in a doping scandal (injecting epo to increase RBCs)

Question 51

JAK kinase

Answer 51

A tightly bound kinase. In the JAK/STAT pathway, the JAK kinase phosphorylates and activates a STAT transcription factor that moves directly to the nucleus- gene expression is activated

Question 52

Western blots are used to

Answer 52

Distinguish phosphorylated from dephosphorylated proteins activated by epo

Question 53

Ras signal transduction pathway

Answer 53

Activated by receptor tyrosine kinases. The binding of a hormone results in receptor dimerization, kinase activation, and phosphorylation of cytosolic receptor tyrosine residues. Binding of GRB2 and Sos couples receptor to inactive Ras. Sos promotes dissociation of GDP from Ras, GTP binds and the active Ras dissociates from Sos

Question 54

Her 2 receptor

Answer 54

Overexpression of this receptor is not good. It responds to human epidermal growth factor. A normal mammary cell has 20,000 HER receptors, but HER+ cancer cells have 2 million. Herceptin was the first monoclonal antibody generated and FDA approved for breast cancer

Question 55

Kadcyla

Answer 55

Antibody conjugate used to treat late stage metastatic breast cancer. It inhibits HER2 signaling and shedding

Question 56

Insulin receptor (4 steps)

Answer 56

1. When the insulin receptor binds insulin, the activated receptor phosphorylates the IRS-1 protein, which activates the Ras pathway
2. IRS-1 activates PI-3 kinase, membrane lipid PIP3 is created
3. PIP3 binds a protein kinase (Akt) which is activated by other protein kinases
4. Akt catalyzes phosphorylation of key proteins, glucose transporter GLUT4 is recruited to the membrane

Question 57

How were insulin prices modified?

Answer 57

Eli Lilly’s insulin (Humalog) rose drastically in price in a short period of time. After being questioned by Congress, he rebranded his insulin as Insulin Lispro, which will be half the price. Therefore, pressure from Congress can positively address drug prices

Question 58

Stem cell breakthrough treatment in T1D

Answer 58

Researchers (Doug Melton) used human embryonic stem cells to produce beta cells that can produce insulin. Could be used for cell transplantation in T1D patients

Question 59

Tissue engineering in T1D

Answer 59

About 600 different genomic locations that are associated with the risk of diabetes and disease
progression have already been identified. The various genes and pathways that were implicated in the pathogenesis of diabetes converge on a shared output, which is uncontrolled blood sugar that results from pancreatic islet dysfunction. Betalin Therapeutics has created an “Engineered Micro Pancreas” using stem cells seeded onto a decellularized pig lung extracellular matrix. They hope to be in Phase I
clinical trials soon and will
transplant this EMP under the skin

Question 60

Hedgehog

Answer 60

A signaling pathway that uses two membrane proteins to receive and transduce a signal. Hedgehog proteins contain a covalently attached lipid that signals to influence the fate of nearby cells. It plays a role in forming the identities of neurons in the embryonic neural tube

Question 61

Erivedge

Answer 61

Used to treat metastatic BCC. It is a hedgehog pathway inhibitor- disrupts the signal that allows the cancer to grow

Question 62

Second messengers

Answer 62

The first messenger is the extracellular signaling molecule itself. Second messenger molecules bind to an activate/inactivate specific intracellular proteins. This is how the activation of receptors changes cell physiology

Question 63

cAMP

Answer 63

A second messenger that activates protein kinase A. The binding of a hormone causes a conformational change in the receptor, which is activated and binds to a Ga subunit. This triggers dissociated of GDP. GTP binds to Ga and Ga dissociates from the receptor. The hormone dissociates and binds to the effector, activating it. Ga dissociates from the effector

Question 64

Forskolin

Answer 64

This supplement is not FDA regulated. It is supposed to activate adenylate cyclase, which increases levels of cAMP

Question 65

Cholera toxin prevents

Answer 65

GTP hydrolysis

Question 66

Theophylline and caffeine

Answer 66

Act by increasing levels of cAMP

Question 67

Signal amplification

Answer 67

Amplification of a signal is necessary to induce a cellular response. It is possible because receptors and G proteins can diffuse rapidly in the plane of the plasma membrane. The degree of amplification that occurs depends on the number of steps in the cascade and the concentration of its components

Question 68

Signal transduction: Gs vs Gi

Answer 68

GPCRs come in multiple categories- Gs is stimulatory and Gi is inhibitory. Gi inhibits intracellular levels of calcium

Question 69

Chimeric GPCRs

Answer 69

Can identify the Gs and
Gi specificity domains of the GPCRs

Question 70

cAMP dependent protein kinase

Answer 70

Also known as protein kinase A, which is activated by cAMP. These proteins phosphorylate other effector proteins that mediate metabolic effects

Question 71

Regulation of glucose metabolism

Answer 71

An increase in cytosolic cAMP activates PKA. It phosphorylates glycogen synthase, inhibiting its ability to synthesize glycogen. A decrease in cAMP inactivates PKA, leading to the activation of phosphoprotein phosphatase. The activation of PP promotes glycogen synthesis and inhibits glycogen degradation. All of these events occur in the cytoplasm.