MCB 135A Unit 1

general steroid hormone signaling pathway

steroid diffuses through plasma membrane --> binds to inactive receptor --> active SR complex can bind DNA and can be imported into the nucleus --> can 1) dimerize, 2) form a composite or 3) tether

dissociation constant (Kd)

measure of affinity of the receptor for its ligand

the higher the dissociation constant, the lower the affinity

HRE

hormone regulated element

GRE

glucocorticoid regulated element

TGTTCT

ERE

estrogen regulated element

TGACCT

nuclear receptor gene

from 5' to 3' end:

AF-1: co-regulator binding

- regulation and protein-protein binding

- least conserved region because it's different for each receptor

DBD: DNA-binding domain

- zinc finger

- DNA binding

- transcription factor binding

- defines the family of genes

LBD: ligand binding domain

- S (steroid) binding

- protein binding

- used to classify if the receptor is known, orphan or adopted

orphan receptors

has no ligands

~50% of receptors have no ligand

lipid molecules have low affinity binding

- function without signaling molecule

- acts as transcription factor without ligand

- constitutively active because they bind to DNA without ligands

adopted receptors

used to be orphan receptors but found ligand

usually are lipid metabolites

metabolite

substance formed in or necessary for metabolism

whole cell binding assay

- used to determine if there is binding between a ligand and its receptor

cells (Rec + vs Rec-) --> *Steroid --> low speed spin/centrifugation 100xg

*S is found in the pellet

Rec+ has both S and SR in the cell

Rec- has just *S in the cell

homogenization of cells

"blowing open" cells

separate by size at a molecular level

spin cells at 100,000xg --> collect supernatant cytosol that holds the receptors --> add S (radioactive) --> use biochemistry to analyze and compare relative sizes of *S and S(radioactive)R

gel filtration

chromatographic technique used to separate proteins of different sizes

quantifies *SR and determines size

native conformation

functionally active conformation of a protein

*S/DNA/protein can all bind

denatured confirmation

denatured receptor cannot bind *S/DNA/Protein

3 Cell type cytosol extraction experiment

differences in sizes of *SR in different cell types

- reasons for this include:

1) receptor is bigger in one cell

2) other proteins attach to receptor x *S complex

sucrose gradient centrifugation

looks at density differences between different proteins and can differentiate between smaller density differences

cytoplasm --> low vs high salt --> +*S --> size fractionation --> sucrose gradient

high salt lowers amount of *SR because it unbinds receptor from overall protein

antibody pulldown assay

uses antibody, which has specificity due to epitopes that recognize particular antigens on a protein

epitope binds to antigen on protein of interest --> antibody is covalently attached to a bead --> spin mixture --> in pellet is the bead x Ab x protein of interest x *S

negative control: add a bead without antibody

dimerize

two SR complexes come together on DNA

composite

SR complex forms with transcription factor on DNA

tethering

SR is attached to transcription factor on DNA

antibody pull-down assays

quantifying receptor using specificity of antibody

- antibodies recognize specific antigenic sites on proteins

- need to have positive and negative control in experiments

- negative control: take out component of assay and it should not work

ex: in bead-Ab pulldown assay, insert bead without antibody and it should not elicit a signal response

- positive control: the experiment should always give the desired response in this case

ex: when testing a steroid that is supposed to emulate the effects of estrogen, the positive control would use estrogen to see the normal response

experiment example:

tissue --> cross-sections of cells --> Ab-estrogen receptor --> secondary Ab with fluorescent tag --> use to determine which cells have a particular receptor

glass fiber filter assay

quantify the amount of *S on a filter

cytosol with S and receptor added to a filter, add wash to wash out excess S

looks at how much *S is used?

Western Blot

- look at inherent size of protein backbone

- denature the protein

procedure:

cells --> extract (grind cells up) --> denature proteins --> use SDS-ionic detergent to separate different proteins --> use polyacrylamide gel electrophoresis to separate proteins based on size

positive control

conditions you know will work

negative control

elimination of experimental component that renders an ensured absence of signal

experiment to determine whether a protein is bound to the protein of interest in native conditions

1) use co-immunoprecipitation to collect protein of interest

2) use HPLC to determine size of ligand-receptor compound

or

3) use western blot to determine the presence of a particular protein

- need to use western blot to denature to find the presence of protein of interest

Kd

dissociation constant

- measure of affinity of a protein for its ligand

lower the kd, higher the affinity

saturation binding

experimental approach used to determine how much of the substrate (steroid) is required for the sample to be concentrated

the smaller the Kd, the greater the affinity of a receptor

example experiment:

take cell extract and add S at varying concentrations --> measure SR using any binding assay (such as glass fiber filter, Ab pulldown assay)

results of this experiment: the curve will not plateau when adding only S because you are actually measuring the specific and non-specific binding of S (meaning that S could be binding to anything)

total binding = specific and non-specific binding

specific binding

amount of receptors are limited, we know for sure that the ligand/steroid is binding to the receptor/protein that we want

non-specific binding

protein indiscriminately binds multiple ligands, steroid binds indiscriminately to different receptors

experiment to determine specific and non-specific binding

cell extract + [S] various concentrations +/- S100x excess --> binding assay SR --> for each concentration of *S, subtract nonspecific binding from total binding to get specific binding

non-specific vs. specific binding

total binding: *S binds to both the high affinity limited receptor and non-specific unlimited low affinity "junk" sites

non-specific binding: which is why we add unlabeled S100x (in excess). S(excess) will compete with *S for specific sites, but there will be no competition for the non-specific junk sites because both can bind

non-specific binding: S(excess) + S --> S(excess) takes up all the open receptor sites on the protein of interest, meaning that S only attaches to non-specific sites

total binding - non-specific binding = specific binding (all the times that *S actually binds to receptor/protein of interest)

Scatchard Plot

A plot of bound ligand concentration divided by free ligand concentration, against bound ligand concentration, i.e. [RL] / [L] versus [RL] where [L] and [RL] are the concentrations of respectively the free ligand and the receptor-ligand complex. The slope of the straight-line plot equals −1/KD and the intercept on the y-axis is [R]tot / KD, where [R]tot is the concentration of the receptor protein and KD is the equilibrium dissociation constant.

Bound/Free = (-1/Kd)B + Rtotal/Kd

slope = function of affinity = (-1/Kd); greater the absolute value of Kd, the higher the affinity

y-axis = bound/free (bound ligand/free ligand)

x-axis = bound ligand and receptor

why does the slope go downward for a scatchard plot

as you get closer to saturation, you need more steroid

increase the bound form to saturate, but now you have much more free than bound as you reach saturation

negative cooperativity

binding of the initial ligand decreases the binding affinity of the hormone to other sites

constant change in Kd to less affinity as more hormone ligand is added

in a scatchard plot, the graph is not linear but negative exponential (1/lnx)

positive cooperativity

binding of an initial ligand increases the binding affinity of other sites on the protein

constant change in Kd to more affinity as more hormone ligand is added

in scatchard plot, the graph is like curve facing inward

competition binding

measures relative affinity of different ligands for a specific receptor

compares an unknown to a known

determines relative affinity

better competition would mean that the competition has higher affinity than the original

never goes down to zero because of non-specific binding

standard curve

competition of known unlabeled ligand with known labeled ligand

x-axis: unlabeled steroid

y-axis: *SR

test of unknowns

competition of unlabeled unknown with labeled known ligand

each runs its own binding assay experiment with varying concentrations

agonist

a molecule that binds to a hormone's receptor site and elicits the same response

with nuclear receptors, the agonist binds to the receptor site, enters the nuclear membrane, binds to the promoter sequence on the DNA + TF + co-regulators and facilitates gene transcription

binding sites may differ from antagonist binding sites

antagonist

binds to the receptor site and elicits no response

when interacting with the ligand binding site, the antagonist can change the conformation of the receptor

binding sites may differ from agonist binding sites

no response and prevents agonists from working

ligand triggered conformational change

change in receptor shape due to the presence of an antagonist (or agonist) that prevents (or facilitates) the function of the protein

law of mass action

more ligand, more likely to bind

can outcompete a ligand that may have more affinity if there is enough of the ligand

dextamethasone

cortisol agonist that binds to the binding site of glucocorticoid receptors, has a higher affinity than regular cortisol

RU486

antagonist of cortisol and progesterone

commonly found in the abortion pill; progesterone maintains placenta attachment during pregnancy

- stops placenta attachment which can lead to miscarriage/abortion

tamoxifen

stops the growth of breast cancer cells and was used as a breast cancer treatment

found to be an agonist of uterine cell and bone cell growth, meaning that it could cause uterine / bone cancer

lower affinity than estrogen

helix 12

receptor of steroid that changes conformation based on what binds to its active site

tamoxifen: does not form a clamp and allows co-repressor binding

estrogen: forms clamp and allows co-regulator binding

SERM

selective estrogen receptor modulator

ex: such as tamoxifen, which affects the structure of the protein

raloxifene

SERM, antagonist of mammary cells and uterus cells, agonist of bone cells

higher affinity than estrogen

EM-652 (acolbifene)

antagonist of mammary, uterine and bone cells

greater affinity than raloxifene and estrogen

testosterone

steroid hormone for androgen receptor that facilitates growth of various sex specific cells and functions

binds to androgen receptor, enters nucleus, binds with TF and co-activator to transcribe gene

in excess in prostate cancer, leads to proliferation of these cancerous cells

bicalutamide

antagonist of androgen receptor

enters nuclear membrane, may bind to TF and recruits co-repressor to prevent transcription of RNA

problem with antagonists and prostate cancer

in late stage prostate cancer, the androgen receptor works without the ligand and becomes completely hormone/ligand independent

enzalutamide

anti-androgen drug used for prostate cancer

prevents constitutive androgen receptor from entering the nucleus and acting on the target gene

activation of steroid receptor

activation allows for competence:

nuclear import

DNA binding

TF binding

dimerization

experiment to determine activation:

1) collect cytosol from any cell

2) add radiolabeled steroid + isolated nuclei from wherever you want

3) incubate at 4º, 25º, low salt and high salt/physiological salt

4) spin at 10,000xg

5) determine radioactivity of pellet

results:

1) found that there were high levels of *SR in high salt and high-temp conditions (basically physiological conditions)

this experiment shows activation, but the body does this normally

question: why isn't every receptor activated in the body if the experiments are activated at physiological conditions

answer: missing the ligands --> ligands help to activate binding

how does activation occur?

activation creates an increase in positive charge on the protein receptor surface (positive charge because DNA is negatively charged)

two possible mechanisms for activation:

1) conformational change that puts more positive charge on the receptor surface

or

2) additional repressor protein bound to the receptor protein is separated and creates a positive charge on surface of receptor protein

most often it is #2, can use gel filtration to determine difference and what x is

cytosol --add *S--> -/+ activation conditions --> gel filtration

in low salt, have more SR than S

in high salt, have equal amounts of SR and S

hsp90

heat shock protein 90 , common repressor protein that upon activation detaches from the receptor protein

keeps receptor in the inactive state

addition of a ligand triggers activation and triggers detachment of the protein / degradation of HSP90

can increase the affinity of ligands that activate the receptor protein

DNA binding assay

cytosol + *S --> activate --> DNA fragments, 200bp --> gel filtration column

(negative control / make sure that the receptor is specifically binding DNA = add RNA)

we see different peaks at different sizes that show SR-DNA binding, SR, and S

*SR needs DNA to stay in the nucleus

is it the DNA that keeps the receptor in the nucleus?

cytosol + *S --> activate --> nuclei w/ DNase and w/ no DNase --> spin --> pellet and measure radioactivity

we see that with DNase, there is little *SR in the nucleus, but without DNase there is a lot of receptor steroid complex in the nucleus

*SR needs other proteins to stay in the nucleus, not just DNA

cytosol + *Dex --> temperature activation --> various concentrations of salt --> DNA vs. nuclear import

- found that SR binds to DNA best at lower concentrations of salt

- even at high concentrations of salt, SR can be found in the nucleus

- at physiological concentration of salt, SR does not bind that well to DNA

this tells us that the receptor interacts with components of the nucleus in addition to DNA

while DNA is necessary for *SR to stay in the nucleus, other proteins are necessary to keep it in the nucleus

ex: circannual rhythm - progesterone receptor nuclear import in hen oviducts

- they found that receptor enters the nucleus better at different times of the year

- experiment where they just added DNA --> no PR in nucleus

- added DNA + proteins --> PR in the nucleus

defective response from receptor

defect can be anywhere in the pathway, meaning that a mutation can affect anywhere in the pathway (from receptor protein production to defect in binding)

S49 lymphoma cells

add glucocorticoid to these cells --> cells die

cells that survive the dexamethosone --> Dex receptor variants/mutants --> have a defect somewhere in the pathway

isolate mutant cell clone, grow and characterize

cell viability assay

cells + dex --> see if cells survive or not

WT cells die, mutant cells survive because they are unable to bind dex

can cells bind glucocorticoid?

cells (w/ GR) + radioactive Dex --spin--> pellet with cells (quantify how much radioactive SR is present)

categories of cells that you may find in the pellet

1) cells that have no binding of *Dex due to lack of receptor expression or an S binding defect (mutation in the ligand binding domain)

2) cells that retain and bind *Dex

Nt-

nuclear translocation mutant (does not retain a lot of *Dex)

binds less DNA than WT, low specific sequence binding, same size as WT

point mutation in critical amino acid needed for DNA binding

NTi

mutant that retains more *Dex than wild type

higher affinity when binding to random DNA sequences, but lower affinity when binding to specific sequences

found that it has a mutation in the N-terminus region of the glucocorticoid receptor --> deletion of N-terminus (evidenced by smaller band in western blot)

N-terminus Ab does not bind

d-

deathless cell, meaning that it does not die but binds an equal amount when compared to wildtype

same receptor as WT, mutation is in the target gene (endonuclease in target gene that leads to post-receptor effects)

S-

no steroid binding because has point mutation in the section coding for the LBD

ability to get into/remain in nucleus depends on the receptor in the cytosol

cytosol with (WT, NT-, NTi, etc.) + *Dex + nuclei from any source --spin--> nuclear pellet

use salt gradient fractionation to determine how well the receptors bind to the steroid

cytosol (WT, NT-, NTi, d-) + *dex --> temperature activation --> apply to DNA column --> elute by salt gradient

salt gradient

used as an assay procedure to determine affinity of receptors (salt interferes with protein-protein/protein-DNA interactions)

the higher the concentration of salt, the greater the affinity of the protein and whatever it is binding to

found that NT- had the least affinity, smaller amount of *SR because it is not that efficient in binding DNA in the first place

NTi had higher affinity for binding to DNA

what is up with NTi?

tried to figure out what was happening with this mutant and why affinity was so different

cyto (WT vs. NTi) + *Dex --> activate temp to get rid of HSP90 --> gel filtration

- found that the *SR complex for NTi was smaller than that of WT

wanted to find if this was because of the intrinsic protein backbone of the receptor --> put in western blot

found differences in size between the two (NTi is smaller than WT)

identification of functional domains

pure GR protein --protease (limited amount) --> biochemically separate various GR protein into fragments by size --> assay fragments for *Dex binding, DNA binding and Ab binding

found that

- full functional protein: WT @ 94 kDa

- missing N-terminus: NTi @ 39 kDa

- smallest fragment, bound to steroid: 25 kDa

3 parts of receptor protein

1) modulator (AF-1 binds to co-activators and co-repressors)

2) DBD (zinc finger DNA binding/TF binding)

3) LBD (hsp90, AF-2 binds to co-activators and co-repressors)

zinc finger protein

specialized protein that interacts with DNA in the form of a dimer

D-box domain interacts with one another

P-box domain interacts with the DNA (NT- has a point mutation in the P-box)

reporter gene assay

identifies whether or not a system is working; expression or lack of expression of activity

can be used to analyze steroid receptor structure/function, target gene promoter activity and ligand specificity

example experiment:

cells + WT GR gene and GRE-luciferase (reporter) --> -/+ dex --> assay for reporter (luciferase)

w/ WT GR gene

in Dex- cells, no luciferase activitiy

in Dex+ cells, high luciferase activity

can be used to identify agonist/antagonist function (ex: use RU486 instead of Dex, would have low luciferase activity for both low and high RU486 because it is an antagonist of the GR

ligand binding domain

site on the receptor to which a ligand binds

acts as a repressor when inactive; needs a ligand to activate the repressor

found that the ligand binding domain was a repressor using gene reporter assay that showed that luciferase was constitutively expressed and the receptor was constitutively active (with or without dex) in LBD KO cells

LBD + HSP90 represses the rest of the receptor, in active conformation, HSP90 cannot bind to the receptor

can adding a LBD to other transcription factors repress protein function

experiment:

Myc --> transcription factor that facilitates the change of elongated fibroblasts to rounded fibroblasts

1) create chimeric protein with ER LBD attached to Myc

2) transfect Myc, Myc-ER

3) add various concentrations of radiolabeled E, -/+ E100x

4) saturation binding assay

5) scatchard plot (to see if estrogen even binds to the receptor)

then going back after transfection, -/+ E, then look at cell shape to see if there is a difference between Myc with LBD and Myc w/o LBD

found that Myc w/ LBD and no E = long cells, Myc w/ LBD and E = round cells

(control is Myc without LBD = all round cells with and without estrogen)

HSP90 interacts with LBD on the receptor

Vit D resistant rickets

mutant zinc finger receptor, looks like Nt- GR; this receptor has a point mutation in the DNA binding domain (P-box) (Nt- = no DNA binding)

mutation in receptor that prevents response to vitamin D

gel shift assay

Electrophoretic technique that separates molecules based on their size; may be used to identify proteins bound to DNA

have end labeled DNA

GRE --> -/+ receptor --> electrophoresis (native conditions) --> separates by size

experiment:

add just GRE (just the DNA target of the receptor) = just get a band for the size DNA fragment in the gel

GR + GRE = band for DNA fragment + GR in the gel

NT- + GRE = band for DNA in the gel because no DNA binding

S-(no LBD) + GRE = band for DNA in the gel because receptor is inactive

ER + GRE = band for DNA because no binding between ER and GRE (different targets)

DNase footprinting

assay that determines the sequence of the binding site

add receptor to the sample, receptor should protect the DNA from the dnase so that the dnase will cleave DNA everywhere that the receptor is not

will see a missing hole in pattern which is where the receptor has bound

electron microscopy

use electrons to image presence of receptor

mouse mammary tumor virus (MMTV)

causative agent for mouse mammary tumors, transmitted through milk; lactation hormone is a glucocorticoid

Dex treatment increases transcription levels of MMTV RNA and multiplication of the virus

- promoter is highly responsive to glucocorticoid receptors due to the presence of the glucocorticoid response element

- found TGTTCT in binding site

- palindromic sequence where two GRs bind to the GRE palindromic sequence

found that dnase footprint (gap in labeled DNA) showed up whenever there was no mutation/a mutation outside of TGTTCT binding sequence, but gap in labeled DNA did not show up when there was a mutation in the binding sequence OR spacer size was n = 2 or n = 4 (frameshift mutation)

- Dex reuptake activity is lowered with mutations as well

estrogen receptor palindromic sequence

using gel shift assay, found that there was specificity for hormones and their receptors

however, with GRE, AR, PR, and MR were able to bind to the receptor sequence