7 - Intracellular Receptors, Steroid Hormones and Auxin - Gray

Question 1

Give an overview of steroid hormones, what they control and their receptors. Draw a diagram of their movement through the body

Answer 1

• lipophilic
• transported by carrier proteins
• diffuse through membranes where they bind their cytosolic receptor and move into nucleus
• influence growth and differentiation
• effective for hours or days, control things eg growth, puberty

Question 2

state how steroid hormones and related lipid soluble signal signals are similar. and how they differ. give examples of each

Answer 2

• similar; same mode of action, similar receptors
• differ; steroid hormone Rs present in cytoplasm then move into nucleus. related lipid soluble signal Rs reside in nucleus
• steroid eg estrogen, glucocorticoid
• lipid soluble, thyroxine, retinoid acid (replacing skin, controlling growth and development in embryogenesis)

Question 3

draw the OVERALL domain structure of nuclear receptors (both steroid andrelated lipid soluble signal)

Answer 3

343 - 7 word

Question 4

how can the domain structure of receptors be compared?

Answer 4

through receptor cloning

Question 5

what are orphan receptors?

Answer 5

receptors we do not know the function of, nor what ligand binds. there are many orphan nuclear receptors

Question 6

name some nuclear receptors stating whether they are steroid receptors or related lipid soluble signal Rs

Answer 6

steroid;
estrogen, progesterone, glucocorticoid receptors

lipid soluble;
retinoid acid receptor (RAR), thyroxine receptor (TR)

Question 7

describe the experiment that was used to show nuclear localisation of steroid receptors. draw a diagram

Answer 7

• dexamethasone (synthetic glucocorticoid)
• cultured animal cells transfected with different expression vectors
• fluorescent labelled Ab used to identify where B galactosidase expressed
• eventually showed that only the ligand biding domain required for nuclear localisation
  343 - 7 word

Question 8

describe the different DNA response elements in the nucleus for both steroid and related lipid soluble signal Rs and state the structure of the Rs that bind

Answer 8

STEROID;

• 6bp inverted repeats separated by 3bp
• receptor binds to response element as a dimer

RELATED LIPID SOLUBLE SIGNALS;

• vary depending on receptor
• however ALL repeat elements
• bind heterodimer of the nuclear receptor + RXR (retinoiC X receptor) (receptor monomer common to several pathways)

Question 9

describe (& draw diagram) showing how the glucocorticoid receptor causes transcription of genes in nucleus

Answer 9

• hormone (glucocorticoid) enters the cell and binds to the ligand binding domain of GR
• displaces the inhibitor (eg heat shock protein)
• exposes NLS and the GR enters the nucleus
• binds to the glucocorticoid response element on DNA as a homodimer causing transcription by altering acetylation of histones

Question 10

draw diagrams and summarise the binding of both types of lipid soluble receptors to DNA

Answer 10

STEROID;
- bind in cytoplasm, translocate to nucleus
- interact as homodimer and bind to inverted repeats
THYROXINE AND RETINOIC ACID;
- bind ligand in nucleus
- heterodimer with nuclear R : RXR with direct repeat response element

Question 11

state how both steroid receptors and related lipid soluble signal receptors activate transcription. draw a diagram

Answer 11

STEROID;
- activation domain of steroid Rs activates HAT (histoneacetyltransferase) causing the acetylation of Lys residues of histone tails causing nucleosomes to open up allowing the transcription of DNA and activation of gene expression

RELATED LIPID SIGNAL;
- inhibit HDAC (histonedeacetylase) therefore preventing removal of acetyl groups so the DNA stays unwound

Question 12

state some of the differences and similarities seen between plant and animal cellular signalling

Answer 12

similarities;
- receptors are proteins
- specificity for receptors allows downstream signalling
differences;
- types of receptors use are different , no RTK/RSK only pelle receptor kinases and Raf

Question 13

describe the 4 processes that auxin regulates and give an alternative name for auxin

Answer 13

• cell growth, division, differentiation
• controlling gravi/phototropism
• apical dominance
• root branching

Indoleacetic acid (Trp derivative)

Question 14

how do plants respond to a light stimulus?

Answer 14

• phototropic signal perceived @ shoot tip
• auxin found at tip diffuses down the dark/shaded side of the plant shoot
• promotes cell growth(cell division and expansion) down the dark side so bends towards the light source

Question 15

draw a diagram to show how auxin was found only at the shoot tip

Answer 15

343 - 7

Question 16

draw a diagram of the auxin related genes promoter elements and the different things that bind. explain each promoter too

Answer 16

343 - 7

Question 17

how was the auxin receptor discovered and give the name for it

Answer 17

• TIR1 - auxin receptor
• mutagenesis done of Arabidopsis thaliana
• sprayed with 24D (high conc of auxin, too much auxin kills the plant)
• mutants in tir1 gene were insensitive/resistant to 24D because couldn’t “see” the auxin

Question 18

what does TIR1 encode ? give the function of this

Answer 18

TIR1 encodes F box protein that selectively targets specific proteins for degradation by the proteasome

Question 19

draw a diagram of the proteasome

Answer 19

343 - 7 word

Question 20

describe and draw a diagram showing how E1/2/3 labels specific proteins for proteasome degradation

Answer 20

• E1 activates a Ub by bonding it to Cys residue
• Ub attached to E2 Cys residue
• E3 transfers the Ub onto a Lys residue of the target protein
• polyUb proteins degraded by proteasome

Question 21

give other uses of the proteasome degradation pathway in eukaryotes. how can this be targeted for medical use?

Answer 21

• regulating cell cycle and transcription
  CELL CYCLE;
• targets cyclins for degradation during anaphase. allowing cytokinesis and cell division
  TRANSCRIPTION;
• once IkB P (when in complex with NFkB) it can be targeted to proteasome leaving NFkB free to move into nucleus and activate transcription of inflammatory genes

MEDICAL;
- proteasome inhibitors cause apoptosis in cells as proteasome plays an. important regulatory role. cancer therapy

Question 22

ubiquitin mediated proteolysis may be as important as _____

Answer 22

phosphorylation

Question 23

give an example of an E3 Ub ligase and state how many E3 ligases are encoded in the eukaryote genome

Answer 23

• SCF ubiquitin ligases

- 600 E3 ligases encoded in genome

Question 24

state the name and function of all the components in an SCF ub ligase. draw a diagram of this complex

Answer 24

• Skp1 - bridge
• Cullin - scaffold
• F box - specificity for target
  343 - 7 word

Question 25

draw a diagram and explain the complex that targets Aux/IAA repressors for degradation in Arabidopsis thaliana

Answer 25

• auxin = molecular glue that binds the Aux repressors to the F box/TIR1 protein that targets the protein for degradation
• auxin promotes SCF-TIR1 and Aux interaction
• auxin binds to cleft within TIR1 creating a hydrophobic patch for Aux to bind (no conformational change seen upon this ligand binding - unusual)
• becomes polyUb and is targetted to proteasome

Question 26

explain what happens once SCF-TIR1 has targeted the Aux repressor for degradation

Answer 26

• the ARFs are released and can activate the expression of auxin-response genes
• auxin mediated events are triggered
• the auxin response genes also encode Aux repressor
• when auxin is still present, the Aux is degraded however when no auxin the Aux can repress the auxin response genes
• known as feedback inhibition and restores pathway repression