Responses due to Changes in Gene Expression (eg. Steroid Hormones); Changes in Membrane Permeability - Richard

Question 1

Do Some other hormones (e.g. thyroid hormones, Vitamin D, retinoids) have similar modes of action, despite not having steroid chemical structures)?

Answer 1

Yes

Question 2

What are steroid hormone receptors?

Answer 2

‘ligand-activated transcription factor’ proteins consisting of dimers, each consisting of 3 domains.

Question 3

What are the 3 domains for steroid hormone receptor proteins?

Answer 3

1.domain for binding hormone (& also binding the 2nd unit of the dimer).
2.zinc-finger domain needed for DNA binding (to a steroid response element [SRE]), with high affinity; Kd ~1nM).
3.domain needed for the receptor to activate the promoters of the genes being controlled (eg. unravel chromatin, recruit RNA polymerase), & thus to bring about changes in gene expression.

Question 4

What is the zinc finger binding domain?

Answer 4

Cys-X-X-Cys-X(13)–Cys –X-X-Cys
*(rich in basic Aas: Cys, Arg, Lys; hence, well suited to bind [acidic] DNA)
*Ligand-binding -> conformation change in receptor -> Zn finger assembly

Question 5

How many zinc fingers does the receptor dimer have?

Answer 5

2 Zn fingers

Question 6

How many zinc fingers does the steroid hormone receptor dimer have?

Answer 6

2 adjacent Zn fingers protrude into adjacent grooves in DNA:
* 1st finger confers specificity of binding
* 2nd finger stabilises dimer structure

Question 7

What does SRE stand for?

Answer 7

Specific response elements

Question 8

What are SREs?

Answer 8

‘Labels’ identifying genes as targets for transcription factors.

Question 9

How might SREs be displayed?

Answer 9

Two “½ sites”within DNA sequence – each ½ binds the Zn finger domain from one monomer of the SHR dimer.

Question 10

SREs can be palindromic. What does this mean (give an example)?

Answer 10

receptor homodimer forms head-to-head: true steroid hormones
E.g., TGTTCT…..TCTTGT (mirror image)
-><-

Question 11

SREs can also be direct repeats. What does this mean and give an example?

Answer 11

receptor heterodimer forms head-to-tail: thyroid hormone, Vitamin D, retinoids.
E.g., TGACCT……TGACCT
-> ->

Question 12

True or false- response elements are transcriptional enhancers.

Answer 12

True

Question 13

How are response elements transcriptional enhancers?
Explain step by step.

Answer 13

Steroid hormones & their receptors (“SHRs”) move to the nucleus.
*Unravel chromosomes by recruiting Histone Acetylases (“HAT”s) /removing Histone Deacetylases (“HDAC”s).
*Bind to SRE (or ‘enhancer’) DNA sequence located in the vicinity of a gene (up to several kB up/downstream of its promoter & coding region).
*On binding of receptor to SRE, form complex to which RNA polymerase is recruited; nearby promoter is activated & transcription initiated/enhanced.

Question 14

Explain the impact of response elements as transcriptional enhancers.

Answer 14

Thus, impact of steroids becomes apparent within hours/days rather than min - response is ↑ synthesis of new encoded proteins (ie. ↑ gene expression).

Question 15

Cell signalling responses C:

Answer 15

Changes in membrane permeability

Question 16

All cells have an assymetric distribution of ions/molecules on either side of their PM:

Answer 16

Major ions: Na+, K+, Ca2+, Cl-, H+; Other relevant molecules: glucose, CO2, etc

Question 17

Explain the consequence of changes in membrane permeability.

Answer 17

Thus, ionic concentration gradients exist across PM, and opening channels in response to stimuli result in movements of ions into/out of cells: “electrical (or NERVOUS) signalling”

Question 18

What can receptors linked to ion channels that open in response to external stimuli/ligands (‘Gated channels’) bring about changes in?

Answer 18

Can bring about changes in ionic environment within the cell, & so alter the bio-chemical behaviour of the cell: a cellular response.

Question 19

Consequences of Changes in Membrane Permeability

Answer 19

*Changes in pH or oxidation state within cells
–Changed membrane permeability to H+
*Supplying proteins with ionic co-factors (eg. NRAMP channels allow Fe2+ (co-factor, with haem, for many proteins) across PM)
–Due to changed membrane permeability to Fe2+
*Supplying cells with energy (eg. GLUT4 moves glucose into cells)
–Due to changed membrane permeability to glucose
*Changes in potential difference across membrane (eg. depolarisation, repolarisation in nerve impulses)
–Due to changed membrane permeability to Na+ & K+

Question 20

Can Ca2+ be a signal?

Answer 20

Yes
Ca2+ (ionic form; solution in body fluids; see ‘M’ [metal] in diagram

Question 21

Buffered form

Answer 21

bound to soluble or membrane-linked “Ca2+ Buffers”

Question 22

Mineral form

Answer 22

collagen-rich matrix onto which crystals of insoluble calcium phosphate (“hydroxyapatite”) are laid.

Question 23

Co-ordination chemistry of Ca2+ enables it to promote conformational change.

Answer 23

This means that Ca2+s presence within cells can trigger changes in enzymatic activity/protein movement etc & behaviour of cells.

Question 24

Cells allow Ca2+ concentrations to:

Answer 24

Rise to levels at which proteins/phospholipids bind (reversibly)
Trigger conformational changes (&  changes in function)
And then be reduced back to levels at which this process is reversed.
REGULATED discrete localised releases of Ca2+ are used as signals to trigger a range of cellular responses

Question 25

Ringer, 1883:

Answer 25

Ca2+ required for “excitation-contraction coupling” in the heart: –ie. Signal transduction: nervous stimulation -> muscle contraction

Question 26

Subsequently found to apply to other tissues (eg. “stimulus-secretion coupling” in the pancreas – Hermann, 1932:

Answer 26

ie. Signal transduction: hormonal stimulation -> enzyme secretion) *Now acknowledged that Ca2+ signalling occurs in all eukaryotic cells.

Question 27

The Ca2+ system:

Answer 27

1. Stimulation of cell. 2. Entry of calcium into localised parts of cell. 3. Binding of calcium to biological molecules 4. Cellular response 5. Expulsion of calcium from cell 6. Cell recovery NB. Ca2+ forms insoluble complexes with phosphorylated or carboxylated compounds (Proteins, phospholipids, nucleic acids), so cytoplasmic levels of Ca2+ are kept low to avoid precipitation of biological compounds.

Question 28

Ca2+ Stores/Ca2+ Signaling

Answer 28

ER (which contains specialised Ca2+ buffer proteins) has larger surface area than PM -> mops/pumps up Ca2+ that has leaked into cytoplasm & -> acts as Ca2+store -> space for pumps in ER allows countering of passive Ca2+ leak, & ‘refilling’ of stores. Cells have 2 parallel Ca2+ mobilisation systems.

Question 29

Ca2+ released into cytoplasm via:

Answer 29

1. channels in PM (VGCC; Glu-R; Trp) 2. channels in ER membrane (IP3R; RyR) (ie. PM/ER membrane’s permeability changes in response to signalling stimuli)

Question 30

Ca2+ removed from cytoplasm via:

Answer 30

1. “PMCA” pump (& NCX exchanger) in PM. 2. “SERCA” pump in ER membrane.

Question 31

Receptor occupancy coupled by G-proteins to phospholipase C-catalysed cleavage of phospholipid PIP2 to generate diacylglycerol (DAG) plus:

Answer 31

IP3 (head group) ‘2nd messenger’ - diffuses into cytoplasm & acts as ligand to release Ca2+ from internal stores (ER)

Question 32

True or false- ER is (usually) the 1st Ca2+ signalling system activated.

Answer 32

True

Question 33

IP3Receptor (IP3R):

Answer 33

ligand-gated Ca2+ channel in ER memb. Ligand [ie. IP3 ]-> Ca2+ release (‘Ca2+ spark’) IP3R can also be activated by Ca2+ itself -> starts chain reactn (‘Ca2+ wave’)

Question 34

Biphasic Ca2+ signalling - Capacitive Ca2+ Entry:

Answer 34

ER Ca2+ store has limited capacity, so can’t support sustained responses. Zero external Ca2+: response, but only short duration + external Ca2+: prolonged response

Question 35

“Capacitative Ca2+ Entry” (CCE):

Answer 35

If stimulus requires prolonged response & [Ca2+]ER decreases, ER protein STIM migrates to PM. At PM, STIM interacts with ‘Orai1’ & ‘Trpc’ proteins (form PM channels: “Ca2+ release-activated channel” (CRAC)), triggering opening &  Ca2+ entry within ~5min

Question 36

Biphasic:

Answer 36

ER release, then influx

Question 37

True or false- In other cell-types, analogous systems use cADPR (ligand) or voltage as stimulus, & RyR or TPC release channels

Answer 37

True

Question 38

Ca2+ Imaging: Spikes, Waves & Oscillations-

Answer 38

Cytosolic Ca2+ can be monitored using indicator dyes that change their luminescence or fluorescence when they bind Ca2+. –Eg. FURA-2 *Fluorescent indicators can be loaded into cells & used with confocal fluorescence microscopy to provide imaging and quantitation of Ca2+ within cells. *This has revealed different modes of Ca2+ signalling: spikes, oscillations and waves.

Question 39

Different modes of Ca2+ signalling:

Answer 39

Release of different amounts of Ca2+ from different stores can lead to triggering of different cellular responses in the same cell

Question 40

Some examples of responses involving Ca2+ Signalling:

Answer 40

*Muscle contraction *Membrane fusion *Calmodulin *PKC/DAG *TCR & calcineurin/NF-AT 1&2) Physical Movement, Endocytosis, Exocytosis 3,4&5) Phosphorylation (eg. of transcription factors)