Molecular Cell Biology II (BIO-235)

Role of the βγ subunit (4)

1. always remain associated with each other

2. targets scaffolding proteins in yeast mating response

3. can activate phospholipase C pathway

4. can be considered a secondary messenger

What makes steroid hormones unique in terms of receptor location? (3)

1. amphipathic: polar end dissolves in blood, hydrophobic end crosses plasma membrane without transporters

2. receptors found directly inside the cell, closer to the nucleus

3. directly affect gene expression

What therapeutic is given for opioid addiction?

Methadone

What determines whether a ligand binds a plasma membrane vs. intracellular receptor?

• hydrophilic ligand → CAN’T cross membrane → bind plasma membrane receptors

• hydrophobic ligands → cross membrane → bind intracellular/nuclear receptors

examples of hydrophobic ligands

steroid hormones and retinoids

What are receptor serine kinases?

phosphorylates serine residues

What are threonine kinases?

phosphorylates threonine residues

What is naloxone (narcan) and how does it work?

antidote for opioid overdose that acts as a competitive antagonist at mu-receptors

what is signal amplification?

a single signaling molecule outside a cell can generate a large, rapid intracellular response

what is an example of signal amplification?

1 molecule of epinephrine → release of 10^8 molecules of glucose from glycogen

what is an example of a threonine kinase?

• TGF-beta (transforming growth factor beta)

what are secondary messengers?

molecules made inside the cell in response to a signal

what are the major secondary messengers? (5)

1. cAMP

2. IP3

3. DAG

4. Ca2+

5. PIP3

autocrine signaling

the source and target are the same cell - the cell signals itself

what is thebaine?

highly addictive compound from opium that is used to synthesize OxyCotin

why is thebaine important historically?

Purdue Pharma mass-marked OxyCotin based on thebaine

What family is at the center of the resulting opioid crisis?

Sackler

What is hydrolysis?

water is used to break down complex molecular bonds

In the adnylyl cyclase pathway, what could the ligand be and what is the outcome?

1. could be epinephrine OR glucagon

2. glycogen → glucose → raised blood sugar

juxtacrine signaling

requires direct contact between source and signal cell because the signal is on the surface of the signaling cell

Gs (4)

• stimulatory

• activates adenylyl cylase

• increases cAMP

• sensitive to cholera toxin

Gi (4)

• inhibitory

• inhibits adenylyl cyclase

• lowers cAMP

• sensitive to pertussis toxin

Go

inhibitory type

Gq (2)

• activates phospholipase C

• used by alpha adrenergic receptors

endorphins

body’s endogenous opioid-like peptides

phosphodiesterase inhibitors

inhibits phophodiesterase from breaking down cAMP which leads to heightened levels and prolonged signaling

what is are two examples of a phosphodiesterase inhibitor?

1. caffeine

2. theophylline

which ligand is a protein hormone?

insulin

what is cAMP?

second messenger made from ATP by adylyl cyclase

what is the target of cAMP?

protein kinase A (PKA)

what are the effects of cAMP?

glycogen → glucose = increased blood glucose

key difference between kinase and phosphorylase in terms of phosphate source

kinase: phosphate comes from ATP

phosphorylase: phosphate comes from inorganic phosphate

rank opioid ligands by potency

sufetanil → fetanyl → heroin → morphine → codeine

structural mimicry in opioids

mu receptor is easily fooled by structures that mimic endorphin

cholera toxic: mechanism

blocks GTP hydrolysis so Gs is locked on

cholera toxic: target

Gs alpha subunit

cholera toxin: disease

severe diarrhea and dehydration

pertussis toxin: target

Gi alpha subunit

pertussis toxin: mechanism

inhibits Gi → can’t inhibit adenylyl cyclase

pertussis toxin: disease

whooping cough

what are receptor tyrosine kinases (RTKs)?

cell surface receptors that translate extracellular signals into intracellular action

how do RTKs work?

dimerize and autophosphorylate → phosphorylate tyrosine residues

what are examples of RTK? (2)

1. insulin receptor

2. epidermal growth factor receptor (EGFR)

G-alphas and crosstalk: key rules (5)

• Some G-alpha subunits are specific and others can cross talk

• Gs is shared between glucagon and beta-adrenergic receptor → both raise blood sugar

• Gq is used by alpha-adrenergic → phospholipse C pathway

• Gi inhibits adenylyl clase → used to turn off cAMP production

• the g-alpha subunit determines the downstream outcome

insulin receptor structure

• unusual RTK: has 2 alpha subunits and 2 beta subunits

what is the alpha subunit in the insulin receptor structure?

membrane-spanning receptor protein

what is the beta subunit in the insulin receptor structure?

kinase domain

what is the kd value? what does it measure?

dissociation constant; binding affinity

what does the kd value tell you?

concentration at which half of receptors are occupied

lower kd means…

higher affinity

higher kd means…

lower affinity

what is affinity?

the strength of the binding interaction between a single molecule and its receptor/target

what is a g protein? (2)

• functions as the on/off switch for the receptor

• binds GDP (inactive) or GTP (active)

what nucleotides does a g protein bind?

guanine

what determines activity in g-proteins?

whether guanine is GTP or GDP

what does GTP stand for?

guanosine triphosphate

what does GDP stand for?

guanosine diphosphate

4 classes of hormones

1. amino-acid based

2. peptide

3. protein

4. lipid/steroid

amino-acid based hormones (2)

epinephrine and epinephrine

peptide hormone

antidiuretic

protein hormone (2)

1. insulin

2. glucagon

lipid/steroid hormones (4)

1. testosterone

2. estrogen

3. progesterone

4. cortisol

what are lipid/steroid hormones derived from?

cholesterol

what activates PKA?

cAMP

what activates PKB?

PIP3

what activates PKC?

DAG and phorbol esters

examples of GPCRs (6)

1. opioid receptors

2. rhodopsin

3. taste and smell receptors

4. thyroid stimulating hormone receptor

5. glucagon receptor

6. alpha-adrenergic

what three amino acids are commonly phophorylated by kinases and why?

• tyrosine, serine, threonine

• all have exposed hydroxyl group which is the phosphate attachment point

what are the two main classes of plasma membrane receptors?

1. seven membrane-spanning large heterotrimeric G-protein coupled receptors (GPCRs)

2. receptor kinases

signaling crosstalk

when two different signaling pathways intersect, sharing molecules or outcomes

example of signaling crosstalk

glucagon and epinephrine: different ligands, different receptors, both activate the same Gs protein → same downstream effect = increase in blood glucose

opiate

naturally produced compounds found from the opium poppy; contains morphine, codeine, thebaine

opioid

used to be synthetic molecules but now a general term for anything that binds opioid receptors

coreceptor

additional protein that stabilizes ligand-receptor interaction to increase the probability that the receptor will be activated

endocrine signaling

signals travel long distances through circulatory or lymphatic system

what signaling class is insulin?

protein

why does insulin’s signaling class matter?

because it can’t cross the plasma membrane so it binds to a receptor on the cell surface

what is insulin’s receptor?

RTK (receptor tyrosine kinase)

what are the three mechanisms of receptor downregulation?

1. receptor-mediated endocytosis

2. phosphorylation/desensitization

3. beta-arrestin

receptor-mediated endocytosis

receptor pulled into cell → internalized/degraded

phosphorylation/desensitization

GRK adds phosphate → receptor unresponsive; reduces affinity for ligand

beta-arrestin

binds phosphorylated receptor → blocks G protein activates; promotes endocytosis

three main pathways of signal transduction

1. phospholipase C PWY

2. adnynyl cyclase PWY

3. insulin receptor PWY

exocrine signaling

secretes substance through a duct

what two things activates PKC?

1. phorbol esters

2. DAG

glucagon: receptor

GPCR

insulin: receptor

RTK (receptor tyrosine kinase)

glucagon: G-protein

Gs

insulin: G-protein

none (RTK)

glucagon: impact on blood glucose

raises

insulin: impact on blood glucose

lowers

glucagon: glycogen

breaks down

insulin: glycogen

synthesizes

where are adrenergic proteins?

made in adrenal glands on the kidneys

what are adrenergic proteins?

epinephrine and norepinephrine

calcium

secondary messenger released from ER that rapidly increases its low cytosolic concentration in response to external signals

two classes of g-proteins

1. large heterotrimeric G-proteins

2. small monomeric G-proteins

large heterotrimeric G-proteins

alpha, beta, gamma subunits

small monomeric G-proteins

single subunit

most important example of a small monomeric G-protein

RAS