lecture 3 Flashcards
how do nuclear receptors work in signal transduction
binding of the messenger to a receptor protein
most messengers are hydrophillic so the membrane is impermeable to then , these receptors have a cell surface location
steroid and thyroid hormones are lipophilic substances and can diffused across biological membranes
receptors for hormones are soluble intracellular proteins that primarily act on the nucleus
these have a major medical importance:
breast cancer cells can only grow in the presence of oestrogen, tamoxifen blocks oestrogen binding to the receptor and thus stops growth of cancer cells
cortisol represses the activation of cells of the immune system, cortisol analogues such as dexamethasone are used to reduce inflammation
how do steroid hormones regulate transcription
effects of steroid or thyroid hormones are slow and take hours to be come evident
hormones stimulate gene expression
eg. egg laying ducts of immature chicks dont express egg proteins such as ovalbumin but this can be induced by injection of steroid hormone projesterone
in some cases steroid hormones inhibit gene expression, in the pituitary gland synthesis of pro-opiomelanocortin is repressed by glucocorticoids such as cortisol
adrenocorticotropin synthesis by cortisol therefore represents a classical neg feedback loop
how were steroid and hormone receptors identified
by binding of radioactively labelled steroid or a synthetic analogue and were laboriously purified by biochemical purification techniques
receptors were soluble polypeptides of 50-100kDa which bound to dna
where are steroid and hormone receptors found in absence of a hormone
in the cytoplasm where they are retained as complexes bound to chaperones
what does the binding of a hormone do to steroid and hormone receptors
causes dissociation of the chaperone and the hormone receptor complexes then translocate to the nucleus
what forms the nuclear receptor family
thyroid hormone receptors and receptors for lipids or lipid metabolites - their relations was shown by cDNA sequencing
some members of the family have no known hormone or ligand and are therefore currently names orphan receptors
always found in the nucleus even in absence of hormone (unlike steroid hormone receptors)
3 key regions of nuclear receptors
variable n terminal region, highly conserved central dna binding domain and c terminal ligand binding domain that binds the hormone or other ligand
what is footprinting of steroid hormone receptors
clong of several genes regulated by steroid hormones showed that the steroid receptor complexes bound directly to naked dna
binding sites were defined by the technique of footprinting
the steroid complex was bound to the dna radioactively labelled at one end
dna was digested with an amount of endonuclease that randomly cleaved once per dna molecule
products of digestion were run on a sequencing gel
series of labelled fragments were obtains - clear area showed where the dna was protected from digestion by the hormone receptor complex
how were hormone receptor complexes discovered
comparison of dna binding sites on several steroid regulated genes showed that a particular hormone receptor complex recognises a hormone response element or HRE
what do hormone receptor complexes for steroid receptors contain
2 stretches of 6 bases repeated as an inverted repeat separated by a spacer containing only 3 bases
what is a dna palindrome
a sequence that is the same when read on 2 complimentary strands in 5’ and 3’ way
what are the features of steroid hormone response element
dna double helix has a major groove and a minor groove
bases are in the centre of the helix, phosphate/ribose backbone on the outside
on major grooves bases are sufficiently exposed so binding proteins can recognise specific sequences of bases
6 bases inverted repeats of steroid HREs are separated by a 3 base spacer
first 6 base repeats is exposed in the major groove, second will be exposed in the major groove almost 1 turn of the helix down
as it is an inverted repeat the second repeat will be an exact mirror of the first
the minor groove is more narrow so bases are harder to distinguish
structures of dna binding domains
how are they determined
determined by crystallography
called zinc finger domains, 2 alpha helix held roughly at right angles by loops that are stabilised by pairs of cysteine residues that bind zinc ions
cysteine side chains do not usually form disulphide bridges inside cells because the cells interior has a strongly reducing environment
zinc crosslinking cysteines is the equivalent of disulphide bridges to stabilise extracellular domains or proteins
structures of dna binding domains when bound to dna
nuclear receptor dna binding domain is bound to synthetic dna containing a hormone response element have been determined
steroid hormone dna binding domains bing to hres as homodimers
side chains of the same alpha helix in the symmetrical subunit within the homodimer interact with the inverted repeat in the major groove one turn down
recognition sites for other nuclear receptors
recognition sites for other nuclear receptors contain direct repeats
repeats always have the sequence AGGTCA separated by spaced of variable length from1 to 5 bases
therefore it is difficult to see how they bind as homodimers
as heterodimers the other partener is usually the retinoid x receptor (a nuclear receptor that bind the 9-cis retinoic acid
receptors are always in the nucleus, ligand binding triggers conformational change that causes binding of co activator proteins
