Endocrinology/Objectives/Lecture 8
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Hormone receptors
LIST the overall structure of membrane and nuclear receptors.
Membrane receptors have extracellular hormone-binding and dimerization domains and intracellular modulation/phosphorylation/enzymatic domains. Nuclear receptors must have at least two regions: a hormone-binding domain and a DNA-binding domain. In addition, nuclear hormone receptors have dimerization, transcription, modulation, and phosphorylation domains.
DEFINE a receptor as understood by endocrinologists.
A receptor is a protein that transduces a signal. As such, all receptors must contain a hormone-binding domain and a transduction domain.
STATE the members of the nuclear receptor superfamily.
| Hormone | Receptor |
|---|---|
| Androgen | AR |
| Estrogen | ERα, ERβ |
| Glucocorticoid | GR (type II GR) |
| Mineralocorticoid | MR (type I GR) |
| Progestin | PR |
| Peroxisome proliferator | PPARα, PPARβ, PPARγ |
| All-trans-retinoic acid | RARα, RARβ, RARγ |
| 9-cis-retinoic acid | RXRα, RXRβ, RXRγ |
| Thyroid hormone | TRα, TRβ |
| Vitamin D | VDR |
LIST the dimerization partners for RXR.
All hormones aside from steroid hormones are able to dimerized with RXR:
- PPAR
- RAR(α,β,γ)
- RXR
- TR
- VDR
DESCRIBE the major groups of receptors in this superfamily.
Steroid receptors
- Androgens
- Estrogens
- Glucocorticoids
- Mineralocorticoids
Non-steroid receptors
- TR
- RAR
- VDR
- RXR
STATE the role of hormone binding for steroid and other nuclear receptors.
In the absence of hormone, steroid receptors are bound to HSP90, making them unable to bind DNA and therefore inactive. Inactive receptors translocate between cytoplasm and nucleus in some equilibrium. Hormone binding changes the conformation of the receptor, HSP90 is released, and the receptor can now bind DNA, stablizing the nuclear form of the receptor. Dimerization usually occurs to increase the DNA-binding affinity of the receptor. DNA-bound receptor binds transcriptional coactivators that activate transcription.
Non-steroid receptors do not bind HSP90 in the absense of hormone. Some, like TR, are DNA-bound even in the absence of hormone. Hormone alters the conformation of the receptor, revealing transactivational domains facilitating the interaction with transcriptional coactivators.
DESCRIBE the overall structure of the DNA-binding domain of these receptors.
The DNA-binding domain is rich in Arg, Lys, and Cys, and forms two zinc fingers. It encodes the specificity for DNA-binding in the knuckle of the fingers (the P-box). This region contains an α-helix (a recognition helix) capable of binding the major groove of DNA containing specific sequences.
STATE the DNA-binding motifs used by these receptors.
The DNA-binding motif of a receptor is contained within its P-box. There are three different P-box amino acid sequences:
| Receptors | P-box sequence | DNA sequence |
|---|---|---|
| GR, MR, PR, AR | cGSckV | AGAACAnnnTGTTCT |
| ER | cEGckA | AGGTCAnnnTGACCT |
| TR | cEGckG | AGGTCATGACCT |
| VDR | cEGckG | AGGTCAnnnAGGTCA |
| TR | cEGckG | AGGTCAnnnnAGGTCA |
| RAR | AGGTCAnnnnnAGGTCA |
Single-letter abbreviations are used for amino acids; capitals are used for amino acids required to obtain the listed DNA sequence specificity.
TR binds inverted repeats with no separation between inverted repeats.
Note the 3, 4, 5 rule for spacing between direct repeats in VDR, TR, and RAR. These are the only nuclear hormone receptors that bind direct repeats instead of inverted repeats.
DESCRIBE the role of transcriptional coactivators in nuclear receptor actions.
Transcriptional coactivators bind to the hormone-binding domain in the presence of hormone (so hormone binding must have changed the conformation of the receptor to allow binding of the coactivator). Coactivators do not directly bind DNA, but take advantage of the DNA-binding capabilities of the nuclear receptors with which they associate.
DRAW a Scatchard plot.
(Maybe later.)
USE a Scatchard plot to determine the Kd and max number of receptors.
Kd can be obtained from the slope of a Scatchard plot. Kd would equal -1/m, where m is the slope of the curve. The number of receptors is obtained from the x-intercept.
EXPLAIN a curved Scatchard plot.
Curved Scatchard plots result from two or more underlying affinities for hormone (ie, two or more hormone-binding domains present, perhaps by having multiple receptors present with distinct hormone-binding affinities). The underlying affinities are very difficult to elucidate.
DEFINE the term Kd.
Kd is the concentration of hormone necessary to achieve half-maximal saturation of hormone. It is the concentration of hormone that results in half of the receptors being bound to hormone, half unbound.
INTERPRET a hormone binding semi-log plot.
A semi-log plot is a modern alternative to the Scatchard plot in which relative binding (ie, bound receptor concentration divided by total receptor concentration) is plotted against the log of the hormone concentration. The result is a sigmoidal plot that crosses the origin. The curve's point of inflection occurs at half-maximal saturation and is therefore used to identify Kd. A left shift results from a decreased Kd, which indicates an increase in affinity between hormone and receptor; a right shift results from an increased Kd, indicative of a decrease in hormone-receptor affinity.
