Cell Surface Hormone Receptors

How are receptors modified?

• Post-translational modification

• Forward reaction - kinase

• Reverse reaction - Phosphatase (removes phosphate group)

How do steroid hormones activate gene transcription via a nuclear receptor?

1) Hormone binds to its receptor

• steroid hormone diffuses through phospholipid bilayer of cell membrane as it is lipid-soluble

• inside cytoplasm, it binds to its specific intracellular receptor (which is inactive and attached to heat shock proteins)

• binding causes conformational changes to receptor and the release of inhibitory proteins

2) The receptor dimerises

• once activated two receptor molecules form a dimer (same = homodimer, different = heterodimer)

• dimeristion essential for stable DNA binding

3) Zinc fingers (especially the first) bind the DNA at an HRE

4) The P-box inside the zinc finger 'reads' the DNA to ensure it's the right gene

• P-box determines which sequence the receptor can recognise and read

5) The complex recruits transcription machinery - gene expression changes

• once bound, hormone-receptor-DNA complex acts as a transcription factor

• Recruits other proteins to the gene's promoter region

• Leads to transcription of mRNA, then translation into protein - producing the hormone's effect

How do steroid hormones work? (detailed explanation)

1) Most hydrophobic steroids are bound to plasma protein carriers - only unbound hormones can diffuse into the target cell
2) Steroid hormone receptors are typically in the cytoplasm or nucleus
3) Some steroid hormones also bind to membrane receptors that use second messenger systems to create rapid cellular responses
4) The receptor-hormone complex binds to DNA and activates or represses one or more genes
5) Activated gene create new mRNA that moves into the cytoplasm
6) Translation produces new proteins for cell processes

How do steroid hormones work? (simplified explanation)

1) Hormone travels in blood to carrier
2) Free hormone diffuses into target cell
3) Hormone-receptor binds to DNA
4) Transcription produces mRNA
5) Translation produces new proteins

How do Type I nuclear receptors work?

• When the hormone (ligand) binds in the cytosol, results in the dissociation of heat shock proteins, homo-dimerisation, translocation (active transport) from the cytoplasm into the cell nucleus and binding specific sequences of DNA known as hormone response elements (HREs)

• The nuclear receptor/DNA complex then recruits other proteins which transcribe DAN downstream from the HRE into mRNA and eventually protein causing a change in cell function

How do type II receptors work?

• Type II receptors stay in nucleus regardless of ligand binding status

• They bind as heterodimers with RXR to DNA

• In absence of ligand (hormone), receptors are often in complexes with co-repressor proteins

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

• When coactivator binds, receptor complex activates transcription of the target gene

• Additional proteins including RNA polymerase are then recruited to the NR/DNA complex which translate DNA into mRNA

How does a hormone alter gene transcription?

• 1st step (binding via its receptor, to target sequences of DNA)

• Hormone Response Elements

• Located in regulatory regions of target gene

• Usually 5', close to core promoter (can be elsewhere)

• 6bp hexamer (core recognition motif)

• Usually 2 half-sites - intervening base pairs

How does a receptor recognise its specific HRE?

• P-box with DNA binding site - P-box contains AA that recognise the specific DNA base sequence (the HRE)

• P-box contains zinc fingers and recognition sequences for hormone

How does EGF act as an extracellular receptor?

• Autocrine or Paracrine signalling causes EGF to be released

• Ligand-induced dimerisation

• EGF (ligand) binds to EGFR outside the cell

• Ligand-induced dimerisation - binding causes a conformational change in EGFR

• Causes the EGFR to pair with another EGF-bound EGFR to form a dimer - dimerisation

• The dimer causes the intracellular tyrosine kinase domains to phosphorylate each other (add phosphate groups to tyrosine residues)

• Causing the EGFR receptors to be activated

• Signal transduction processes occur
  -> Ras
  -> PI 3-kinase)
  -> JAK-STAT

How does G Coupled Receptor signalling occur?

• Resting G protein α subunit associated with GDP

• Activation of receptor by hormone induces conformational change to receptor

• Induces conformation change to α subunit allowing for the exchange of GDP for GTP

• α subunit is released and activates second messenger

How is gene expression regulated?

• By proteins binding to promoter and regulatory regions

• Regulatory proteins (e.g hormones) can turn on and off genes according to other factors

• Ligated steroid receptor dimers bind to unique regions in the promoter region of genes

How is JAK involved in recruited tyrosine kinase activity? (detailed explanation)

• Receptors that need RTKA don't have the tyrosine kinase enzyme built in
  1) The receptors exist as monomers in cell membrane and each receptor's intracellular domain is bound to an inactive JAK
  2) When hormone binds to two receptor monomers - brings them together by dimerisation
  3) Each receptor brings its own JAK so there are two JAK molecules side by side
  4) Due to dimerisation of receptors, JAK enzymes come close to together and each one add phosphate groups on the tyrosine residues on the other - autophosphorylation
  5) Phosphorylation activates JAK enzymes to make them now active kinases
  6) Active kinases phosphorylate the tyrosine residues on the intracellular domains of receptors
  7) Phosphorylated tyrosines act as binding sites for STAT
  8) Binding of STAT causes its activation through JAK phosphorylating STAT
  9) STATs detach and dimerise and travel to nucleus
  10) STAT dimers bind to DNA and turn specific genes on or off which leads to changes in gene expression

How is JAK involved in recruited tyrosine kinase activity? (simplified explanation)

1) Hormone binds receptor - receptors dimerise
2) JAK molecules come together - autophosphorylation activates them
3) JAK phosphorylates receptor - creates docking sites
4) STAT binds and is phosphorylated - STAT activated
5) STAT unbinds and the two STAT molecules dimerise
6) STAT dimer moves to nucleus and binds to DNA - gene expression changes

How is the alpha subunit released?

• Activation of receptor releases α subunit

What activates Ras?

• GTP

What are corepressors?

• proteins that keep DNA tightly wound and prevent RNA polymerase from working (inhibit transcription)

• transcription factor

What are examples of type I receptors?

• Glucocorticoid receptors

• Mineralocorticoid receptors

• Androgen receptors

• Oestrogen receptors

• Progesterone receptors

What are examples of type II receptors?

• Thyroid hormone receptor (TR)

• Retinoic acid receptor (RAR)

• Vitamin D receptor (VDR)
  Note: all of these receptors heterodimerise with RXR

What are features of Steroid Hormone Receptors?

• Ligands are small lipophilic molecules

• The receptor is encoded by a single gene

• Tble to bind to DNA

• Function as transcription factors

• Many receptors have been identified that have no known ligand (orphan receptors)

What are Hormone Response Elements (HREs)?

• Short DNA sequences that receptors bind to

• Determine which genes the receptor can turn on or off

What are important features of signal transduction?

• Phosphorylation - caused by enzymes PKA (from cAMP) and PKC (from DAG)

• Calcium flux - IP3 causes calcium release from endoplasmic reticulum
  --> calcium acts as another second messenger - activates calcium-dependent enzymes and proteins (calmodulin) - leads to muscle contraction, secretion, metabolism changes

What are the family of EGF receptors?

• EGFR1

• EGFR2

• EGFR3

• EGFR4

What are the key domains of steroid receptors?

• LBD (ligand binding domain) - binds the hormone

• DBD (DNA binding domain) - binds to DNA using zinc finger motifs

What are the subfamilies of alpha subunits?

• Gαs → stimulates adenylyl cyclase → ↑ cAMP

• Gαi → inhibits adenylyl cyclase → ↓ cAMP

• Gαq → activates PLC → IP₃ + DAG → ↑ Ca²⁺ + PKC

• Gα12/13 → activates Rho GTPases → cytoskeletal effects

What are the two types of nuclear receptors?

• Type I (cytoplasmic)

• Type II (nuclear)

What are the two types of tyrosine kinase activity?

• Intrinsic

• Recruited

What are the types of hormone receptors?

• Cell surface receptors

• Intracellular receptors

What are the types of Hormone Response Elements?

(1) Monomeric HRE - one receptor binds alone
(Dimeric HRE - two receptors bind):
(2) -> Direct Repeat - repeats in the same direction, separated by a few bases
(3) -> Palindromic HRE - inverted repeats with no gap
(4) -> Inverted Palindrome - inverted repeats with a small gap

What are two examples of receptors with intrinsic tyrosine kinase activity?

• Epidermal growth factor receptor (EGF receptor)

• Insulin receptor

What do DNA Binding Domains (DBDs) contain?

• two zinc fingers
  1) first zinc finger --> binds to the specific DNA sequence (HRE)
  2) Second zinc finger --> helps receptor dimerise

What do heat shock proteins do?

• Prevent the receptor from entering the nucleus or bind DNA prematurely

What do steroid receptor dimers bind to to regulate gene expression?

• Bind to unique hormone response elements (HREs) in the promoter region of genes

What do the type II receptors heterodimerise with?

• Retanoid X receptor (RXR)

What do thyroid hormones do?

• Increase basal metabolic rate

What does G Protein Coupled Receptor do?

• Act via second messenger molecules to transfer signal into the cell:

• Activate intracellular G proteins

• G proteins stimulate enzymes to produce second messengers

• second messengers amplify the signal and trigger specific cellular effects

What does JAK do?

• Add phosphate groups to tyrosine residues on other protein

What does phosphorylation induce?

• Conformational changes

What does retinoic acid do?

• Regulates cell growth and differentiation

What does the EGFR contain?

• Hormone binding site

• 2 cysteine-rich regions

• single trans-membrane region

• kinase domain

What does Vitamin D do?

• Increase calcium reabsorption

What is a gene?

• Area of DNA which codes mRNA

What is a hormone receptor?

• A protein on or inside a cell that binds to a specific hormone

• With a complementary binding site for specific hormone

What is are examples of receptors with Recruited Tyrosine Kinase Activity?

• Growth Hormone Receptor (GHR)

• Prolactin Receptor (PRLR)

What is autocrine signalling?

• When a cell secretes a hormone that binds to receptors o it's own surface, triggering a response within that same cell

• A form of cell signalling

What is EGFR?

• It is a receptor tyrosine kinase (RTK) that sits in the cell membrane

What is JAK?

• Kinase

What is ligand-induced dimerisation?

• When a ligand (signalling molecule) binds to a receptor, it causes two receptor molecules to come together (form a dimer) and activates them

What is paracrine signalling?

• When a neighbouring/nearby cell secretes a hormone on another cell that triggers a response within that other cell

What is STAT?

• Signal Transducer and Activator of Transcription

• Signal Transducer - carries signal from receptor at cell surface to cell interior

• Activator of Transcription - turns specific genes on or off once in the nucleus

What is the difference between homodimers and heterodimers?

• Homodimers - two identical receptors join (e.g two glucocorticoid receptors)

• Heterodimers - two different receptors join (e.g thyroid receptor and Retinoid X receptor (RXR))

What is the promoter?

• Region of DNA where RNA polymerase attaches and initiates transcription

What is tyrosine kinase?

• An enzyme that transfers a phosphate group from ATP to a tyrosine residue in a protein

What properties must a hormone receptor have?

• High enough affinity to detect the hormone in the blood

• Saturable and must have a limited number of binding sites

• must bind in a reversible manner (affinity should not be too high so that hormone can unbind)

• The receptor must mediate some biological response

What second messengers does G Protein Coupled Receptor act via?

• Cyclic AMP (cAMP)

• IP3 (inositol 1,4,5-triphosphate)

• DAG (diacylglycerol

Which subunits form a single functional unit?

• β and γ subunits

• so strongly associated that they function as one complex - move and act on target proteins together

• they NEVER act independently

Why are G proteins heterotrimeric?

• made up of three different subunits

• α (alpha), β (beta) and γ (gamma) subunits