Responses due to Changes in Gene Expression (eg. Steroid Hormones); Changes in Membrane Permeability - Richard Flashcards
Do Some other hormones (e.g. thyroid hormones, Vitamin D, retinoids) have similar modes of action, despite not having steroid chemical structures)?
Yes
What are steroid hormone receptors?
‘ligand-activated transcription factor’ proteins consisting of dimers, each consisting of 3 domains.
What are the 3 domains for steroid hormone receptor proteins?
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.
What is the zinc finger binding domain?
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
How many zinc fingers does the receptor dimer have?
2 Zn fingers
How many zinc fingers does the steroid hormone receptor dimer have?
2 adjacent Zn fingers protrude into adjacent grooves in DNA:
* 1st finger confers specificity of binding
* 2nd finger stabilises dimer structure
What does SRE stand for?
Specific response elements
What are SREs?
‘Labels’ identifying genes as targets for transcription factors.
How might SREs be displayed?
Two “½ sites”within DNA sequence – each ½ binds the Zn finger domain from one monomer of the SHR dimer.
SREs can be palindromic. What does this mean (give an example)?
receptor homodimer forms head-to-head: true steroid hormones
E.g., TGTTCT…..TCTTGT (mirror image)
-><-
SREs can also be direct repeats. What does this mean and give an example?
receptor heterodimer forms head-to-tail: thyroid hormone, Vitamin D, retinoids.
E.g., TGACCT……TGACCT
-> ->
True or false- response elements are transcriptional enhancers.
True
How are response elements transcriptional enhancers?
Explain step by step.
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.
Explain the impact of response elements as transcriptional enhancers.
Thus, impact of steroids becomes apparent within hours/days rather than min - response is ↑ synthesis of new encoded proteins (ie. ↑ gene expression).
Cell signalling responses C:
Changes in membrane permeability
All cells have an assymetric distribution of ions/molecules on either side of their PM:
Major ions: Na+, K+, Ca2+, Cl-, H+; Other relevant molecules: glucose, CO2, etc
Explain the consequence of changes in membrane permeability.
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”
What can receptors linked to ion channels that open in response to external stimuli/ligands (‘Gated channels’) bring about changes in?
Can bring about changes in ionic environment within the cell, & so alter the bio-chemical behaviour of the cell: a cellular response.
Consequences of Changes in Membrane Permeability
*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+
Can Ca2+ be a signal?
Yes
Ca2+ (ionic form; solution in body fluids; see ‘M’ [metal] in diagram
Buffered form
bound to soluble or membrane-linked “Ca2+ Buffers”
Mineral form
collagen-rich matrix onto which crystals of insoluble calcium phosphate (“hydroxyapatite”) are laid.
Co-ordination chemistry of Ca2+ enables it to promote conformational change.
This means that Ca2+s presence within cells can trigger changes in enzymatic activity/protein movement etc & behaviour of cells.
Cells allow Ca2+ concentrations to:
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
Ringer, 1883:
Ca2+ required for “excitation-contraction coupling” in the heart:
–ie. Signal transduction: nervous stimulation -> muscle contraction
Subsequently found to apply to other tissues (eg. “stimulus-secretion coupling” in the pancreas – Hermann, 1932:
ie. Signal transduction: hormonal stimulation -> enzyme secretion)
*Now acknowledged that Ca2+ signalling occurs in all eukaryotic cells.
The Ca2+ system:
- Stimulation of cell.
- Entry of calcium into localised parts of cell.
- Binding of calcium to biological molecules
- Cellular response
- Expulsion of calcium from cell
- 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.
Ca2+ Stores/Ca2+ Signaling
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.
Ca2+ released into cytoplasm via:
- channels in PM (VGCC; Glu-R; Trp)
- channels in ER membrane (IP3R; RyR)
(ie. PM/ER membrane’s permeability changes in response to signalling stimuli)
Ca2+ removed from cytoplasm via:
- “PMCA” pump (& NCX exchanger) in PM.
- “SERCA” pump in ER membrane.
Receptor occupancy coupled by G-proteins to phospholipase C-catalysed cleavage of phospholipid PIP2 to generate diacylglycerol (DAG) plus:
IP3 (head group) ‘2nd messenger’
- diffuses into cytoplasm & acts as ligand to release Ca2+ from internal stores (ER)
True or false- ER is (usually) the 1st Ca2+ signalling system activated.
True
IP3Receptor (IP3R):
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’)
Biphasic Ca2+ signalling - Capacitive Ca2+ Entry:
ER Ca2+ store has limited capacity, so can’t support sustained responses.
Zero external Ca2+: response, but only short duration
+ external Ca2+: prolonged response
“Capacitative Ca2+ Entry” (CCE):
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
Biphasic:
ER release, then influx
True or false- In other cell-types, analogous systems use cADPR (ligand) or voltage as stimulus, & RyR or TPC release channels
True
Ca2+ Imaging: Spikes, Waves & Oscillations-
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.
Different modes of Ca2+ signalling:
Release of different amounts of Ca2+ from different stores can lead to triggering of different cellular responses in the same cell
Some examples of responses involving Ca2+ Signalling:
*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)
