Signal Transduction - Nuclear Receptors

Question 1

Steroid and Thyroid hormones are lipophobic, which means that they can diffuse across biological membranes
TRUE OR FALSE

Answer 1

FALSE

Steroid and Thyroid hormones are LIPOPHILIC which means that they can diffuse across biological membranes

Question 2

Most of the receptors for Steroid and Thyroid hormones are soluble intracellular proteins that primarily act in the nucleus.
TRUE OR FALSE

Answer 2

TRUE

These are nuclear receptors

Question 3

Steroid hormones role in transcription:

Answer 3

Act by stimulating gene expression (slow effect, requires hours to become evident):
Example - Egg protein expression can be induced in a chick oviduct by injection of a steroid hormone known as “progesterone”

Some inhibit gene expression:
Adrenocorticotropin stimulates cortisol synthesis. Cortisol represses synthesis of a precursor of adrenocorticotropin. This is a negative feedback loop

Question 4

Steroid and Thyroid Hormone Receptors:

Answer 4

These receptors are soluble peptides bound to DNA.
In the absence of hormone they are in the cytoplasm in an inactive complex bound to chaperones.
Binding of hormones causes dissociation of chaperones, then the hormone-receptor complexes translocate to the nucleus.
Steroid receptors are related to receptors for thyroid hormones, and receptors for lipids/lipid metabolites. Together these form the nuclear-receptor superfamily.

Question 5

Structure of Nuclear Receptors:

Answer 5

At least 3 key regions:

1. A variable length N-terminal region.
2. A highly conserved, central DNA-binding domain (DBD).
3. A C-terminal ligand-binding domain (LBD), which binds to the hormone or to another ligand.

Question 6

BLANK of steroid receptor binding was used to define the exact binding sites of receptors to the BLANK DNA

Answer 6

Footprinting of steroid receptor binding was used to define the exact binding sites of receptors to the naked DNA

Question 7

Hormone Response Elements:

Answer 7

A particular hormone-receptor complex recognises a specific sequence called a hormone response element (HRE)
HREs for steroid hormone receptors contain 2 stretches of 6 bases repeated as an inverted repeat (palindrome) separated by a space containing any 3 bases.
5’-AGAACAxxxTGTTCT-3’
3’-TCTTGTxxxACAAGA-5’

Question 8

Features of steroid hormone response elements:

Answer 8

• Only in the major groove are the bases sufficiently exposed that a binding protein (e.g. DNA-binding domain of nuclear receptor) can recognize a specific sequence of bases
• The DNA double helix has ten bases per turn
• Note also the 6 base inverted repeats of steroid HREs are separated by a 3 base spacer
• So if the first 6 base repeat is exposed in the major groove, the second repeat will also be exposed in the major groove about one turn of the helix away
  being inverted, the second repeat will be a mirror image of the first

Question 9

Structures of DNA-binding domains

Answer 9

• Zinc finger domains: have two alpha-helices held roughly at right angles by loops that are stabilized by pairs of cysteine residues that bind zinc ions.
• Bind to DNA as homodimers, which have two-fold symmetry
• Side chains of the same a-helix in the other, symmetrical subunit of the homodimer interact with the inverted repeat in the major groove one turn down:

Question 10

Recognition sites for other nuclear receptors: (Not steroid hormone receptors)

Answer 10

Some contain direct repeats (not inverted repeats like steroid hormone receptors).
These repeats always have the sequence AGGTCA but are separated by spacers of variable length (From 1 to 5 bases)
5’-AGGTCAxxAGGTCA-3’
3’-TCCAGTxxTCCAGT-5’
These receptors bind as heterodimers, where the other partner is usually the retinoid X receptor.
Receptors in this class are always located in the nucleus, but ligand binding triggers conformational changes that causes binding of co-activator proteins

Question 11

How do nuclear receptors activate transcription?

Answer 11

The receptor binds to a HRE causing a conformational change that creates docking sites for proteins called co-activators.
Co-activators are proteins with various functions that allow easier access of the transcription machinery to the gene, e.g HATs, chromatin remodelling proteins
Co-activators also promote binding of RNA polymerase I

Question 12

How do nuclear receptors repress transcription?

Answer 12

Folding of the ligand-binding domains of the receptors around the ligand creates docking sites for co-repressors.
Co-repressors include factors that modify chromatin to restrict access for the transcription machinery to the gene: e.g. HDACs