Park Lecture 1/08/06

Lecture is posted for today and tomorrow. He is working on a grant right now, so things are hectic.
Theme of day: signaling to nucleus. RK talked about receptors at cell membrane. 2 kinds of hormones work in nucleus:
Growth factors, cytokines are proteins that bind to receptors then go to nucleus to activate transcription factors to lead to increased or decreased gene expression.
Steroid hormones bypass a receptor and go straight to nucleus to activate nuclear receptor. to lead to increased or decreased gene expression.
Today working on steroid hormones. We will look at glucocorticoids (retinoids, est progesterone, thyroid hormone). He has picked thyroid hormone and glucocorticoids. Thyroid hormone is like vitamin D and retinoic acid. Glucocorticoids are in class of classic steroids.
Sl 2: objectives
Looking mostly at promoter level. Receptor structure,, etc.
3: Remember this slide and you are halfway home for this lecture. Free thyroid hormone moves into cell. Thyroid receptor is in nucleus, bound to DNA. Corepressors leave, coactivators bind, increased RNA leads to increased proteins. Cortisol binds to receptor in cytosol. Binds promoter of gene. Get increased mRNA and increased protein.
4:
Structures of ligands. Do not memorize. Text is weak, so rely on his words.
Shown is T4. Two Tyr hooked together with iodine. Cortisol is a glucocorticoid your body makes.
5: Regulation of T3 Study- focus on mechanism of signaling rather than physiology
Hypothalamus releases TRH to activate TSH. TSH increases production of thyroid hormone in thyroid gland. Has huge blood flow. TSH acts by cAMP to stimulate production of T3 and T4. Very little free- travels bound to thyroid binding protein. T3 is more active. Deiodinases do conversion. Iodine is important. Thyroid hormone inhibits production of TSH and TRH via feedback inhibition. The anterior pituitary becomes insensitive to inhibitory effects, get high levels thyroid hormone.
6:
TH increases metabolic rate. Once used for weight loss, but had bad cardiovascular effects. T3 more active. Slow onset action and long half life. Activates gene expression.
7: Graves Disease
8: Hypothyroid- gland gets bigger trying to extract more iodine from blood.
9: Hypothyroid patient needing iodine. Thyroid can get big enough to occlude breathing.
10: Hyperthyroid- look at patient’s eyes.
11:
Ch 11 Lodish fig 41
All nuclear receptors have DNA binding domain consisting of 2 zinc fingers. Has ligand binding as well. Ligand binding domain forms a pocket.
12: Fig 11-44
For glucocorticoid receptor- amino terminus is a ligand binding domain and activation domain. Receptor binds as a dimer.
13:
Be aware of this.
C domain, hinge region, ligand, then AF2 region which recruits coactivators. Glucocorticoids bind as homodimers. Orphan receptors- we don’t know ligand. General principles apply to all receptors.
14:
15: properties of thyroid hormone receptor:
B domain not much function. DNA binding domain has 2 zinc fingers- 1st for specificity, second contributes, but is not main contributor. Hinge acts as binding site for corepressors. Ligand binding domain is pocket and conformational change results in activation. AF2 holds ligand in 6-12 hrs.
16:
TR binds as heterodimer with retinoid X receptor (RXR on slide). Binds to direct repeats separated by 4 nucleotides. TR not needed for DNA binding. Unliganded , bound receptor will repress transcription.
17:
zinc fingers- P box determines what DNA sequence the receptor recognizes. D box is involved in dimerization with RXR. Ligand binding domains are also important. The P box does not determine this alone. (Lots of what he is telling u today is oversimplified so we will understand it) Other things help. AGGTCA is important for recognition.
18: RXR
RXR is universal heterodimerization partner. Has ligand, but the ligand’s importance in thyroid hormone function is not clear. RXR needs TR to bind.
19:
3,4,5 Rule- vitamin D receptor motif separated by 3 nucleotides, Tr by 4 nucleotides, Retinoic acid receptor motif repeats by 5 nucleotides.
20
Nuclear receptors can bind to different sequences and assume different conformations.
21:
The field (the people who study these receptors) is interested in role of corepressors and coactivators. TR binds DNA without ligand. Associated with corepressors in absence of ligand. Corepressors and activators do not contact DNA. Ligand in pocket, results in a conformational change, corepressors leave, AF2 recruits coactivators by intraction with LXXLL motif.
22:
Coactivators- very simplistic model.
Remember that coactivators have enzymatic activity. 1st one was HAT, histone acetyltransferase activity. DNA is wound around histones. When transcription factors acetyate the histones, DNA unwinds and promoter region is more accessible. The coactivators aceylate histones. The “Field” is interested iin coactivators and how they work.
23:
Coactivators can come on and off. Different ones associate at different times. They do not touch the DNA.
24: Mangeldorf model
Coactivation helps activate RNA polymerase.
25:
True for TR and glucocorticoid receptors. AF2 domain interacts with LXXLL domain. Coactivators form large complexes.Remember SRC, CBP, TRAP.
26: Model of coactivation. Has Receptor interactive domain and domain to interact with CBP. CBP structure also shown.
27:
This is more complicated than what he has said. DNA shown wound around histones…Coactivators make DNA accessible to transcription factors, recruit polymerase, promote transcription.
28:
Corepressors have HDAC (histone deacetylase) activity. Silence transcription.
29:
Mechanism of activation of gene expression.
30:
Another model same thing.
31:
Activate a gene- may be 100 proteins involved.

Glucocorticoids 1
Similar, but main difference is that receptor sits in cytosol. Then receptor in cytosol goes into nucleus. Actions of glucocorticoids:
Cortisol is amtiinflammatory and immune suppressive.
2:
clinical uses- see slide.
3:
Glucocorticoid made in adrenal gland.
4: too much glucocorticoid in asthma or RA- get muscle weaknesss and wasting, central obesity, cataracts, see slide.
6:
2 isoforms- focus on alpha. Has DNA Binding domain in middle of protein, ligand binding domain with AF2 domain. Has NLS and domain to bind heat shock protein and domain for dimerization.
7:
Glucocorticoids travel associated with cortisol binding globulin. Ligand binding causes dissociation of HSP. The NLS is exposed. Last 3 steps same as TR- keep differences in mind.
8:
9:
Mechanism of antiinflammatory actions: positive side. Inhibits transcription by binding Fos-Jun to inhibit transcription.
10:
Glucocorticoids suppress production of cytokines and TNF. Bind to fos-jun and KFkappaB heterodimer. Physically interact with these factors without binding DNA.
11: PEPCK gene used to discuss how glucocorticoids interact in diabetic liver. PEPCK important for gluconeogenesis.

ChIP assays are used to study regulation of gene expression.
PEPCK promoter has various transcription factors bound. Cortisol in, binds, accessory factors and coactivators necessary to activate gene.

ChIP used to show what proteins are bound to promoter in vivo at a specific time point. Chromatin immunoprecipitation assay.
Formaldehyde forms crosslinks between the nuclear receptors and DNA and nuclear receptors and coactivators. Crosslink few minutes, lyse cells, sonicate DNA into 500 bp fragments, pull down with antibody. Reverse crosslink with heat, take DNA and identify gene of interest by PCR amplification. Tells whether a particular protein or coactivator is associated with your gene of interest.
Be able to compare TR and GR. (He said this SEVERAL TIMES! EXAM QUESTION!)

Generalized thyroid hormone resistance is rare, but does exist. Have high levels of thyroid hormone without effect. 2 ways to get- no receptor or TR with AF2 domain mutation. Mutant has worse effect. TR binds to DNA. AF2 mutation is associated constitutively with corepressors to act as dominant negative molecule. No TR, get no positive and no dominant negative effects. Patients mutant in 1 copy show severe effects.

Tomorrow in nucleus with growth factors binding to membrane receptors.