Hormone Receptors

Question 1

Two main functional domains

Answer 1

1 Hormone binding domain

• Determined by binding assays (Scatchard plots)
• Agonists bring a repsonse; antagonists inhibit
• HIGH AFFINITY BINDING
• **Nuclear receptors: C terminus **
• **Membrane receptors: outside of cell **

2. Transducing domain

• Induction of second messengers, activation of transcription
• Nuclear receptors: txn machinery interaction
• **Membrane receptors: cytoplasmic side **

*Also, all undergo dimerization and may be modified by phosphate

Question 2

NUCLEAR RECEPTORS

Answer 2

1. All contain DNA binding domain
2. Can either turn off or turn on transcription

Question 3

MEMBRANE RECEPTORS

Answer 3

1. Can be glycosylated

Question 4

Peroxisome proliferator hormone

Answer 4

1. Synthetic hypolipidemic compounds
2. Long chain FA
3. Arachidonic acid metabolites may be the physiological ligands

Question 5

Hormone binding domain

Answer 5

1. 15-50% homology btwn receptor families
2. 11-12 alpha helices
3. Helix 12 seals binding cavity and exposes a surface for interaction with transcriptional coactivators
   * amphipathic

Some cross reactivity can occur with hormone binding

-GR and MR

Question 6

DNA Binding Domain

Answer 6

1. Rich in arginine, lysine, and cysteine (4 for each Zn)
2. 2 zinc fingers that stabilize structure to form alpha helix
3. P-box/knuckle: DNA recognition helix
4. GR, MR, PR, AR bind to same sequence
   * inverted repeat of binding site (nnn)
5. VDR, TR, RAR, RXR bind to same sequence

• direct repeats w/ 3,4,5 spacings
• TR can also bind to inverted repeat with no spaces

6. ER binds to its own sequence

Question 7

Steroid receptor activation

(hGR, hMR, hAR, hPR, hER)

Answer 7

1. Presence of hormone causes release of HSP90 from DNA sequence and receptors can bind to DNA
2. DNA bound receptor binds to transcriptional coactivators that active txn
   * Receptor binding sequence: LxxLL
3. Hormone shuttles between cytoplasm and nucleus. Presence of hormone stabilizes nuclear form (binds tightly to nucleus)

Question 8

TR, RAR, VDR, RXR Activation

Answer 8

1. Some are bound to DNA at all times
   * Suppressor is bound to DNA
2. Hormone alters conformation of receptor so that it can interact with transcriptional activators; knocks of suppressor

• Only bind to receptor at hormone binding domain when in the presence of hormone
• Interact with transcriptional machinery

Question 9

Hormone Binding Assays

(4 purposes)

Answer 9

1. Determine which cells/tissues contain a receptor
2. Determine whether a cell/tisssue has multiple subtypes of receptor with differential hormone binding
3. Determine affinity of receptor to a ligand
4. Determine whether lack of response to hormone is due to absence of receptors.

Question 10

Scatchard plot

Answer 10

1. Soluble extract produced by homogenization & centrifugation
2. Extract is incubated with increasing concentrations of radioactive hormone
3. hormone that binds to R is separated from unbound hormone using chromatography or activated charcoal
4. Affinity determined by slope of line

Curved line: two receptors with different affinities

Question 11

Dissociation Constant

&

Hormone bound

Answer 11

1. Concentration of hormone that half saturates the receptor
2. Alternative way of determine Kd is to use a semi-log plot of binding against Log [hormone].

Question 12

Class 1 family receptors

Answer 12

1. GR, MR, AR, PR
2. Recognize same DNA sequence; pallindrome/inverse repeat
3. Homodimerize
4. Steroids; nuclear receptor w/ HSP90

Question 13

Class 2 receptors

Answer 13

1. Estrogen Receptors
2. Recognize pallindrome/inverse repeat
3. homodimerize
4. Steroid: nuclear receptor w/ HSP90

Question 14

Class 3 family receptors

Answer 14

1. VDR, TR, RAR, RXR
2. DNA binding: direct repeats

VDR: 3, TR:4, RAR: 5

RXR: heterodimerizes

*TR: can also bind pallindrome w/ no spaces

2. hydrophobic; bind nuclear receptors

*can bind to DNA in presence or absence of hormone