E2

• Hormones

  • Alter protein function (FAST- sec to mins)
  • Alter gene expression (SLOW mins to hours)
  • Altered cytoplasmic machinery= altered cell behaviour
  • Must bind with high enough affinity to receptors= cause a reaction
• Types of hormone receptors

  • Cell surface receptors- involve tyrosine kinase cascade
  • Intracellular receptors
• Tyrosine kinase

  • Enzyme that transfers a phosphate group from ATP to a tyrosine residue in a protein
  • Phosphorylation induces conformational changes
  • Can have INTRINSIC or RECRUITED activity
• Intrinsic TK activity

  • Epidermal Growth Receptor (EGR)
  • Insulin receptor
• EGF

  • Family of receptors (EGF 1-4)
  • Ligand induced dimerization
  • Peptide ligands (encoded by specific genes)- cleaved to yield active hormone
  • Autocrine, paracrine cell signalling
  • Signal transduction processes: Ras, Phosphatidylinositide 3-kinase (PI 3-kinase), JAK-STAT
• EGF receptor

  • Extracellular receptor
  • Membrane receptors= structured molecules that cross outer PCM
  • Has a hormone binding site, 2 cysteine rich regions, 1 trans-membrane region, Kinase domain
• Receptor modification

  Post translational modification
• G protein coupled receptors

  • More adenylate cyclase and PLC
  • 7 cross membrane helices
  • Act via 2nd messenger molecules to transfer signal into the cell
  • 2nd messengers: cAMP, inositol 1,4,5 triphosphate (IP3), diacylglycerol (DAG)
  • Phosphorylation and Ca influx involved in signal transduction
• G subunits

  • G proteins are heterotrimeric- a, B, y subunits
  • B/y subunits form a single functional unit
  • Many isoforms of α subunits
  • 4 subfamilies (Gsα, Giα, Gqα, Goα)
  • Activation of receptor releases alpha subunit
• 1. coupled receptor signalling

  1. Resting G protein α subunit associated with GDP

  2. Activation of receptor by hormone-> conformational change to receptor

  3. -> conformational change to α subunit

  4. Allows exchange of GDP for GTP

  5. Α subunit is released and activates 2nd messenger
• Peptide hormone receptors

  • Bind to a cell surface receptor- linked to Tyrosine Kinase or G protein coupled
  • 1st messenger is the hormone- signal through cell surface receptor
  • 2nd messenger- interactions between the intracellular domain and other molecules within the cell
  • 2nd messengers: PI 3-kinase, JAK-STAT, cAMP, inositol, IP3, DAG
• Steroid hormones and other small molecules

  • Easily pass though plasma membrane- because they're lipophilic- includes glucocorticoids and sex hormones
  • Other small molecules- thyroid hormone, vitamin D
  • Receptors are inside target cells: Cytoplasm- glucocorticoids, sex hormones, Nucleus- thyroid, vitamin D
• Steroid hormone receptors

  • Ligands are small lipophilic molecules
  • The receptor is encoded by a single gene
  • Have an ability to bind DNA
  • Function as transcription factors
  • Many have no known ligand (orphan receptors)
• Getting into the cell

  1. Most hydrophobic steroids are bound to plasma protein carriers

  2. Only unbound hormones can diffuse into the target cell

  3. Steroid hormone receptors are in cytoplasm or nucleus

  4. Some steroid hormones also bind to membrane receptors that use second messenger systems to create rapid cellular responses

  5. The receptor-hormone complex binds to DNA and activates or represses 1 or more genes

  6. Activated genes create new mRNA that moves into the cytoplasm

  7. Translation produces new proteins for cell processes
• Type 1 receptors

  • Glucocorticoid, mineralocorticoid, progesterone, oestrogen and androgen receptors
• Type 1 receptors

  1. Ligand binding to type I nuclear receptors in the cytosol

  2. Results in the dissociation of heat shock proteins, homo-dimerization, translocation (i.e., active transport) from the cytoplasm into the cell nucleus, and binding to specific sequences of DNA known as hormone response elements (HRE's)

  3. The nuclear receptor/DNA complex then recruits other proteins which transcribe DNA downstream from the HRE into mRNA-> protein-> change in cell function
• Type 2 receptors

  • VDR, RAR and TR heterodimerise with RXR
• Type 2 receptors

  1. Retained in the nucleus regardless of the ligand binding status

  2. Bind as heterodimers (usually with Retinoid X Receptor (RXR) to DNA

  3. In absence of a ligand- receptors are in complexes with co-repressor proteins

  4. Ligand binding to the nuclear receptor causes dissociation of co-repressor and recruitment of co-activator proteins

  5. Additional proteins including RNA polymerase are then recruited to the Nuclear Receptor/DNA complex which translate DNA into mRNA
• Nuclear receptor proteins

  • ARE transcription factors
  • Cause phenotypic change
• Hormones change the pattern of gene expression

  • Promoter- DNA region where RNA polymerase attaches and starts transcription
  • Gene- DNA area which codes mRNA
  • Gene expression- regulated by proteins binding to promoter and regulatory proteins
  • Regulatory proteins/hormones can turn on/off genes according to other factors
• Ligated steroid receptor dimers

  Bind to unique regions in the promoter region of genes
• How does a hormone alter gene transcription

  1. Binding via its receptor to target sequences of DNA

  2. Hormone response elements (HRE)

  3. Located in regulatory regions of target gene

  4. Usually 5', close to core promoter- can be elsewhere

  5. 6pm hexamer- core recognition motif

  6. Usually 2 half-sites- intervening base pairs

  7. 1ST Zinc finger domain binds DNA

  8. The second zinc finger domain is involved in dimerization
• Structure of HREs

  • Monomeric HRE- A/T HRE- 5' A/T rich sequence precedes half-site
  • Dimeric HRE- Palindromic HRE, DR HRE- direct repeat, IP HRE- inverted palindrome
• How does a receptor recognise a specific HRE?

  1. DNA sequence recognition- protein contains a DNA-binding domain which is recognised

  2. Conformational changes

  3. Transcriptional activation