Nuclear receptors

Hormones mechanisms of action can be split into two groups
1. Hormones that do not enter cells act via cell surface receptors
2. Hormones that enter cells (small, lipophilic) act on nuclear receptors that regulate transcription of homone-specific genes
  1. Nuclear receptor ligands
    1. Not encoded in the genome (theyre steroids mostly, except thyroid related hormones - modified amino acids)
    2. Small, lipophilic -> passive uptake
    3. All deirved from dietary, environmental anc metabolic precursors
    4. Cause changes in gene expression
    5. Diverse ligands and functions
      1. Gonadal steroids (androgens, progestogens, oestrogens)
        Growth, differentiation
      2. Glucocorticoids (cortisol)
        Gluconeogenesis, carbohydrate metabolism
      3. Mineralocorticoids (aldosterone)
        Sodium transport
      4. Vitamins e.g. (Vit D3)
        Vit D3 - calcium transport
      5. Thyroid hormones
        Development and metabolic behaviour of the cell
      6. Retinoids (retinoic acid)
        Differentiation and growth (embryonic)
Families of hormones / receptors
1. How?
  1. Ancestral gene coding for ancestral protein with ancestral function
    1. Mutation results in a duplication of the gene
      1. These genes evolve separately and result in two related genes with different products
  2. Nuclear receptor families (Suprrfamily has 6 subfamilies)
    1. Steroiods
    2. Thyroid hormone receptors
    3. Vitamin D
    4. Vitamin A-like
    5. Sub families NR5 and NR6 are orphan receptors and their ligans unknown
Ligand specificity
1. For steroids
  1. A-ring gives receptor specificity
  2. D-ring responsible for receptor activity
  3. Nuclear receptors first discovered for oestrogens - target organs (uterus and vagina) take up and retain the ligand againstconcentration gradient
    1. Discorvered using radiolabelled oestradiol
      1. Incubating in rat uterus and them homogenising and seperating the nuclear and cytosolic material
        Oestradiol is cytosolic at 2 degrees C
        Oestradiol is nuclear at 37 degrees C
    2. Hormone ilicits a response at very low concentrations
    3. The hormone does not change after causing the response
    4. The ER
      1. Three molecular forms separated by sucrose gradient centrifugation
        8S - multimer (inactive receptor)
        Bound to associated heat shock proteins (HSP) - including HSP90, form a coplex that inactivates the recptor
        4S - monomer
        Oestradiol (or other oestrogenic compound) causes diasassociation of the HSP90 complex - monomer is now free
        5S - dimer (active receptor)
        Two active 4S monomers dimersie and bind to chromatin at the oestrogen response element (ERE)
        This promotes basal TF formation at the near promoter region (including RNA pol II)
      2. Structure
        Phosphoprotein with sulphydryl groups (S-H bonds) - usually from cysteine residues - important for ligand binding
        Methods used to study the structure
        Limited proteolysis
        Proteases only recognise specific sites
        Indicated areas of little structure (allow enzyme to bind to protein)
        Can be coupled with function studies to map functional domains of the protein - loss of certain region causes loss of certain function?
        Immunochemical mapping - using Ab's to identify sites similar between other proteins
        cDNA sequencing
        Then BLAST analysis
        Five distinct domains of the ER
        A/B - modulator
        Most variable between steroid receptors
        Features transactivatn function (via AF-1 domain)
        Target for regulatory kinases (e.g. Akt) - ER can be activated without ligand (seen in breast cancer)
        Interacts with tissue specific co-activators
        C - DNA binding
        Most conserved domain amongst steroid receptors
        Rich in Cys and basic amino acids (DNA is acidic)
        Contains two zinc fingers in two regions; C1 and C2
        Recognises hormone response elements (HRE)
        C-terminal extension domain separates it from the D-hinge region
        D - hinge region between DBD and LBD
        Variable in length between different receptors
        Allows rotation between DBD and LBD
        Necessary for dimerisation and lining up on the response element
        Interacts with co-regulators
        May be involved in nuclear localisation
        E - ligand binding (LBD)
        Moderately conserved amongst steroid receptors
        Features transactivation domain AF-2 - activated by ligand binding
        11-13 alpha helices in hydrophobic ligand-binding pocket
        Ligand binding = conformational change (helix 12 moves and provides a surface for co-activator binding)
        Ligand binds in hydrophobic pocket and interacts with alpha helices; 3, 4 and 5
        Interacts with HSP's - blocks dimerisation site
        Tamoxifen exploits this domain
        F - unknown
        Not found in all receptors
        Possibly binds co-repressor?
        C and E domains and ER funciton
        C (DBD)
        Features two zinc fingers (C1 and C2)
        C1 features a P-box which targets the receptor to a HRE halfsite
        C2 features D-box which is involved in dimerisation - interacts with D-box on other receptor
        Each receptor monomer (in the dimer) binds to a HRE half-site
        Each HRE is specific to the dimer it binds
        Half-site 1: binds receptor monomer 1
        3-base gap
        Half-site 2: binds receptor monomer 2
        Ensures the dimer sits on the same side of the DNA
        Both half sites are reverse pallindromes of each other - e.g. AGAACAnnnTGTTCT
        E (LBD)
        Ligand phenolic A-ring (characteristic of oestrogens) interacts with; glutamate353 and Arginine394
        Ligand D-ring with OH-group interacts with receptor histidine 524
        Causes a conformation change involving alpha helix no.12 - this presents a binding surface for co-activators (stabilise basal TF machinery +/- HAT)
        Antagonists bind via phenolic A-ring structures but lack a structure corresponding to the OH- of the D-ring
        Coactivators bind via their NR box
        Unbound receptor hides the co-activator site and presents a co-repressor (e.g. HDAC) site instead
        Corepressors bind via their CoRNR box
        Stabilised by van der waals
        Agonists and antagonists require a structure corresponding to the phenolic A-ring
Receptor regulation of gene transcription
1. Ligand-dependent gene activation
  1. Mediated by the LBD (ligand binding domain)
  2. Gene transcription by RNA pol II
  3. Ligand bound receptor recruits coactivators that stabilise general/basal TFs and promote transcription
2. Repression by un-liganded receptor
  1. Active repression of transcription
  2. Recruits corressors - e.g. HDAC
3. Also, ligand-dependent repression
  1. Not well understood
    1. Ligand binding recruits negatively acting regulators
4. Tissue specificity
  1. Receptor concentration in responsive tissues
  2. Ligand concentration
  3. Ligand action - may be agonist, partial agonist or antagonist in different tissues
  4. Tissue specific coactivators/repressors - allow for gene regulation in different tissues
  5. Phosphorylation of the NR
    1. Has different effects
      1. PI3K/Akt signalling can activate ER by phosphorylation
        Seen in hormone treatment-resistent breast cancer
Steroid hormone membrane receptors
1. Oestradiol causes rapid generation of cAMP -> PKA activation -> activation of CREB (TF)
  1. G protein-coupled receptor
2. Activation of PLC -> increase in IP3 -> calcium flux
  1. G protein-coupled receptor
3. Activation of TFs via MAPK
  1. G protein-coupled receptor

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Nuclear receptors

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