Lecture 6 - Exam 1

Question 1

What are some types of ligands that nuclear receptors can recognize?

Answer 1

Hormones, pro-hormones, metabolites (fatty acid derivatives), estrogens, progesterone, glucocorticocids

Question 2

What is the main purpose of nuclear receptors?

Answer 2

Modulate gene transcriptions - they are a family of ligand activated transcription factors - important for development, reproduction, metabolism, cardiac and vascular function, tissue growth

Question 3

Give an overview of nuclear receptor pathway?

Answer 3

Ligand is carried in blood to inside cytoplasm where it forms an HSP complex, which bring ligand to nuclear receptor (in cytoplasm) and HSP dissociates so NR and hormone complex can dimerize (either homo or heterodimer) - dimer travels through nuclear pore into nucleus where it binds the hormone response element (HRE) on a target gene and co-activators help with transcription

Question 4

What makes up Class I NRs?

Answer 4

Thyroid hormone receptors, retinoic acid receptors, vitamin D receptors, peroxisome proliferation-activated receptors, orphan receptors

Question 5

What makes up Class II NRs?

Answer 5

Retinoid X acid receptors, chicken ovalbumin upstream stimulators, hepatocyte nuclear factor 4, testis receptors and eye development receptors

Question 6

What makes up Class III NRs?

Answer 6

Steroid receptors and estrogen-related receptors

Question 7

What makes up Class IV, V, and VI NRs?

Answer 7

Orphan receptors

Question 8

What are the domain on a NR?

Answer 8

A/B, C (DBD), D, E (LBD), AF-2, F

Question 9

What is the A/B domain of a NR?

Answer 9

A variable NH-2 terminal region, containing the ligand independent AF-1 transactivation domain

Question 10

What is the C domain of a NR?

Answer 10

DBD - It is the conserved DNA-binding domain responsible for recognition of specific DNA sequences

Question 11

What is the D domain of a NR?

Answer 11

It is the variable linker region, the hinge that connects DBD and E/F

Question 12

What is the E domain of a NR?

Answer 12

LBD - it is the ligand binding domain and the dimerization surface, where HSP binding occurs

Question 13

What is the F domain of a NR?

Answer 13

It is the variable C terminal domain

Question 14

What is the overall structure of LBD of NR?

Answer 14

Structure is similar for different NRs and contains 12 conserved alpha helices H1-H12 and folded into 3-layered antiparallel sandwhich, hydrophobic ligand binding pocket accommodates ligand and after binding undergoes conformational change

Question 15

Which domain of a NR is highly conserved?

Answer 15

C - two zinc fingers formed by four cysteine residues

Question 16

Which domain of a NR is least conserved?

Answer 16

A/B domain

Question 17

What are HREs?

Answer 17

Hormone response elements located within promoters or enhancers of target genes to give specific DNA sequences that NRs recognize to bind to - most NRs bind to enhancer elements and repress transcription unless they are in the presence of hormones and form complexes that function as activators

Question 18

In what forms can NRs bind to HREs?

Answer 18

They can bind as monomers, homodiemrs, or heterodimers. Steroid receptors bind as homodimers to palindromic elements. Monomeric binding binds AT rich sequence. Heterodimers recognize palindrome, direct repeats or inverted repeats.

Question 19

What is the difference in permissive and non-permissive heterodimers?

Answer 19

In non-permissive ligand of one monomer (RAR) must bind before the ligand of the other monomer (RXR) can bind to initiate transcription. In permissive either ligand can bind first but trx activity is low, if both bind trx activity is high.

Question 20

What are negative HREs?

Answer 20

Mediate negative regulation when bound to the receptor - unoccupied ligand increases transcription, but when ligand is bound it reverses the stimulation - positioned downstream of the TATA box, usually close to transcription initiation site - examples: glucocorticoids and thyroid hormone

Question 21

What are HREs dependent on?

Answer 21

Adjacent transcription factors (TATA box, TFII, THII),

Question 22

What do coactivators do in NRs?

Answer 22

Mediate the interaction of transcription factors with the basal transcriptional machinery - bridge molecules

Question 23

What are coactivator families of NRs?

Answer 23

p160, PPARgamma, RNA coactivator

Question 24

What are cointegrator proteins?

Answer 24

CBP, p300, TRAP/DRIP complex

Question 25

What are the receptor interacting proteins for a NR and what do they do?

Answer 25

General transcription factors like TATA or RNA poly subunits, sequence-specific transcription factors, coactivators and cointegrators - help to carry out NR functions

Question 26

What is a requirement of coactivators and cointegrators for NRs?

Answer 26

Receptor must be bound to ligand to interact with coactivators AND COINTEGRATORS

Question 27

What are the family members of the p160 coactivator family for NRs?

Answer 27

SRC-1, SRC-2, SRC-3 (steroid receptor coactivators)

Question 28

What is the general structure of the p160 family of coactivators?

Answer 28

AF-2 binding is ligand dependent, a conserved helix-loop-helix motif (HLH) in n terminus, LXXLL motif in center that is highly conserved for other protein to protein interaction and HAT activity in AD1 and AD2 in C terminus

Question 29

What happens if there are mutations in the LXXLL motif of p160 coactivators?

Answer 29

Receptor and coactivator activity is abolished but cointegrator CREB binding protein (CBP) is not effected

Question 30

What happens if the HAT domain is mutated in p160 coactivators?

Answer 30

Coregulators can become disabled

Question 31

What is the general structure of cointegrator proteins - CBP and p300?

Answer 31

LXXLL domain with three CH zinc fingers in center, KIX domain interacts with CREB, HAT in center and SRC-1 at C terminus - cointegrators associate with p160

Question 32

What is the SWI/SNF family?

Answer 32

Genes that affect the mating-type switching and sucrose fermentation pathways in yeast - similar to RSC structure also identified in yeast and all are conserved in eukaryotes - ATP dependent chromatin remodeling complexes that restructure the nucleosome to make DNA accessible - multi-component complex

Question 33

What is the TRAP/DRIP complex?

Answer 33

Multiprotein complexes with ligand-dependent interaction to vitamin D receptor (DRIP) or to the thyroid hormone receptor (TRAP) to enhance activity by recruiting polymerase to target promoter

Question 34

What are corepressors?

Answer 34

Proteins that bind transcriptional activators to inhibit formation of transcriptionally active complexes

Question 35

What are NCoR and SMRT?

Answer 35

Corepressors - nuclear corepressor (NCoR) and silencing mediator for retinoic and thyroid hormone (SMRT) structurally similar and repress transcription when bound to TR homodimeres - bind in absence of ligand on receptor which is bound to HRE and silences activity

Question 36

What is the general structure of SMRT and NCoR?

Answer 36

Repressor domains RD1, 2 and 3, receptor interacting domains (RID) at C terminus, RD1 interacts with mSin3A which recruit HDAC Class I and Class II bind at RD3, HDAC3 interacts at RD2

Question 37

What happens when ligand binds NRs that have attached corepressors?

Answer 37

Receptors recruit coactivator complexes with HAT activity (p160), SWI/SNF complex remodels chromatin using ATP, TRAP/DRIP complex recruits RNA polymerase II holoenzymes - may be sequential or combined and factors relieve repression and allow for activation

Question 38

What are the NR subtypes and their ligands?

Answer 38

Subtypes based on ligands and possible functions Endocrine receptors like steroid receptors (glucocorticoid, estrogen) and heterodimeric receptors (thyroid, vitamin D) - ligands are vitamins and hormones Adopted orphan receptors like PXR, LXR, PPAR, RXR - ligands are lipids and xenobiotics Orphan receptors - SHP, TLX, PNR - do not know ligand

Question 39

What does the glucocorticoid receptor do (GR)?

Answer 39

Binds cortisol and glucocorticoids - in almost all cells to regulate genes controlling immune rsponse, development and metabolism

Question 40

When are glucocorticoids produced?

Answer 40

Starvation and stress

Question 41

What is the mechanism of glucocorticoid receptors (GR)?

Answer 41

Receptor resides in cytosol complexed with other proteins like HSP - when cortisol diffuses through membrane and binds these other proteins dissociate and it becomes activated - it is able to homodimerize and translocate to nucleus and bind to specific response elements and activate transcription, iin repression the GR can complex with other transcription factors and prevent their activation of genes

Question 42

What is a mineralcorticoid receptor (MR)?

Answer 42

AKA aldosterone receptor but has equal affinity for aldosterone and cortisol - induces expression of proteins regulating electrolyte and water balance - found in kidney, colon, heart, CNS, induce Na/K channels to open

Question 43

What are the two estrogen receptors (ER)?

Answer 43

ERalpha and beta - encoded by separate genes and have preferential ligand binding

Question 44

How do ERs signal?

Answer 44

Estrogen enters cell binds to ER causing activation which leads to homo and heterodimerization - binds to estrogen response elements (EREs) - specific DNA sequences - binds coactivators SRC-1, RIP140, CBP, that link it to general transcription factors and RNA polymerase to alter gene transcription

Question 45

What cell types do ERs signal to?

Answer 45

Endothelial, neural, breast cancer and osteoblast

Question 46

How do thyroid hormone receptors work (TRs)?

Answer 46

Hormone binding induces conformational changge in receptor that causes it to function as an activator - alpha and beta receptors - bind to repeated sequence T3 response elements (TRE) as monomers or homo/heterodimers- expression varies by tissue and developmental stage - binding with ligand allows for coactivators to bind and HAT activity to open chromatin