Lecture 2: hormone action

What do hormones do on a molecular level?

Alter protein function or gene transcription

What is the endocrine axis?

• cascades in which the target tissue of one hormone is another endocrine gland

• Ex: hypothalamo-pituitary-thyroid axis

list the types of hormone receptors

cell surface receptors

intracellular receptors

what causes the creation of a hormone to be slow vs fast?

• quick: modification of something (protein) already present in cell

  • usually phosphorylating it

• slow: needs DNA

What are the types of cell surface receptors

1. receptors with in-built (inherent/intrinsic) tyrosine kinase activity

   1. insulin

   2. EGF1 (growth FACTOR)

2. receptors that recruit/activate (extrinsic) tyrosine kinase- cytokines

   1. prolactin

   2. leptin

   3. growth HORMONE

3. g-protein coupled receptors 

   1. catecholamines

what is tyrosine kinase?

an enzyme that transfers a phosphate group from ATP into a tyrosine residue in a protein

phosphorylation induces conformational changes

Outline the epidermal growth factor receptor

• extracellular receptor w/4 family members (EGF 1-4)

• Ligand-induced dimerisation

• autocrine, paracrine cell signaling

• signal transduction processes: signaling pathway becomes activated by these proteins

  • Ras

  • PI 3-kinase

  • JAK-STAT

HAS INTRINSIC KINASE ACTIVITY

What is autocrine vs paracrine cell signaling

Auto = cell produces a GF to which it also responds

Para = local cell to cell signaling in which a molecule released by one cell acts on a neighbouring target cell 

What are membrane receptors

structured molecules that cross the outer cell membrane

outline the structure of the EGF receptor

hormone binding site

2 cysteine-rich regions (for ligand binding)

a SINGLE trans-membrane region

a kinase domain

What is a hormone receptor?

a protein on/in a cell that:

1. has high affinity (binds well) with the hormone

2. binds the hormone specifically, identifying it amongst other hormones and molecules

3. binds the hormone in a reversible manner

4. is only on specific tissues

5. is saturable and has a limited number of binding sites (needs a threshold)

6. does something! mediates a biological response

outline receptor modification: post-translation modification

turning receptor on:

1. the kinase-linked hormone receptor binds with the hormone 

2. the kinase activity of the receptor phosphorylates the receptor

3. the signal can enhance itself and go through the cell

turning receptor off:

1. reverse reaction, phosphatase removes phosphate 

INTRINSIC KINASE ACTIVITY

Ras

small GTP-ase. found in two forms:

1. bound to GDP = inactive

2. bound to GTP = active 

GDP —> GTP needs co-factors

Outline post-translational modification using  EGF as an example

1. hormone binds

2. EGF receptors dimerise, meaning receptor can become active

3. conformational change occurs, allowing for kinase activity

4. kinase activity causes phosphorylation

5. phosphorylation = activation of the receptor allowing for the…

6. recruitment of GRB + SOS allows for the exchange of GDP on Ras to GTP

7. Ras + GTP activates intracellular signaling pathways such as MEK signaling pathway 

GRB

adaptor protein

SOS

exchange factor, allows for GDP to change to GTP

how does the post-translational modification of the insulin receptor differ from that of the EGF receptor?

• insulin receptors already dimerised

• uses PI 3-kinase to get activated

outline recruited tyrosine kinase activity

• receptors have no kinase domain built-in

• most common pathway = JAK-STAT pathway 

• same deal: binding to receptor, conformational change

• JAK associated w/receptor, phosphorylation allows for…

• recruitment of STAT which is also phosphorylated

• STAT takes signal to DNA, allowing transcription

only difference is how they get phosphorylated

JAK

janus-associated kinase

just a kinase :)

STAT

signal transducer and activator of transcription

a transcription factor

g-protein

a bit like small GTPases

have an inactive and active state 

how do g-protein coupled receptors work?

act via SECOND MESSENGER molecules to transfer signal into cell

relies on phosphorylation as well, and calcium flux

outline the structure of the g-protein

subunits:
1. alpha

2. beta

3. gamma

heterotrimeric (made up of 3 subunits)

activation of receptor releases alpha subunit

outline g-coupled receptor signaling

1. resting g-protein: alpha subunit associated w/GDP (inactive)

2. hormone binds, conformational change to alpha subunit

3. change allows for exchange of GDP —> GTP

4. alpha subunit released and activates 2nd messenger

what do kinases do?

phosphorylate things

outline g-coupled receptors with reference to diaglycerol (DAG), IP3 and Ca2+

end result = phosphorylated protein or modified substrate

1. hormone binds, conformational change

2. alpha subunit released after binding to GTP

3. DAG as second messenger: activates protein kinase C that phosphorylates a protein

4. IP3 as a second messenger: works through phospholipase C, enables ER to release calcium stores into the cytoplasm

   1. cytoplasmic conc. of calcium increases

   2. activate calcium-sensitive enzyme (Ca2+ sensitive enzyme) allowing for substrate to be modified to produce a modified substrate

   3. calmodulin = calcium binding protein, sensitive to calcium, calmodulin-activated protein kinase cause protein to be phosphorylated

which type/s of hormones use cell surface receptors?

peptide hormones

1st vs 2nd messenger

1st: the hormone. signal through cell surface receptor

2nd messenger: interactions between the intracellular domain and other molecules w/in the cell

list the second messengers you should know

1. PI 3-kinase

2. JAK-STAT

3. cAMP

4. DAG

5. IP3

give an overview of steroid hormone receptors

• ligands = small lipophilic molecules 

• receptor encoded by a single gene

• have an ability to bind to DNA

• function as transcription factors 

what is the issue with steroid hormones?

lipophilic = hydrophobic

blood = water

cannot move through blood

need protein carriers to move through blood, which makes them insoluble in fat

where are steroid hormone receptors found?

cytoplasm or nucleus 

outline the general steroid hormone receptor mechanism

1. hormone binds

2. conformational change

3. receptor goes from cytoplasm to nucleus to bind DNA

4. when bound to DNA, allows transcription

outline type 1 nuclear hormone receptors

• homodimers (bind two of the same together, ex 2 glucocorticoid receptors bound together)

• found in cytoplasm (usually) and in conjunction with heat-shock proteins (inactive)

• usually all steroid receptors 

type 1 nuclear hormone receptor mechanism

1. hormone binds, heat-shock protein drops off and homo-dimerises

2. protein enters nucleus, binds to DNA

3. DNA contains hormone response elements (HREs) telling it where to bind 

4. nuclear receptor/DNA complex recruits other proteins to transcribe DNA

5. new genes expressed, new proteins being made

type 2 nuclear hormone receptors

• heterodimers

• cytoplasmic or nuclear

• all have the same common factor- the retanoid X receptor

• usually involve small molecules w/similar properties to steroid hormones

• ligand-dependent transcription factors

• centrally conserved DNA binding domain, zinc fingers

• when hormones bind, AF1+Af2 work together to change cell behaviour

type 2 nuclear hormone receptor mechanism

1. already hetero-dimerised, already bound to nucleus 

2. inactive: requires repressor proteins to bind 

3. active: hormone binds, co-repressor proteins moved and replaced by co-activators 

4. co-activator complex activates gene, enhances transcription

outline the structures of nuclear hormone receptors

3 domains:

1. transactivation domain

   1. within this domain = AF1

2. DNA binding domain

   1. localises to nucleus + docks onto DNA 

3. hormone binding domain

   1. within this domain = AF2

transcriptional activator areas- when hormone is at on receptor, always will have a basal level

AFs upregulate transcription/gene expression

outline how hormones change the pattern of gene expression

promoter regions: region of DNA where DNA polymerase attaches + initiates transcription

gene = area of DNA which codes for mRNA

1. binding via its receptor 

2. hormone response elements

what are hormone response elements

• sequences (consensus) different for each of the hormone receptors

• located in regulatory regions of target gene

• usually 5’, close to core promoter

• 6bp hexamer core recognition motif

• usually 2 half sites- intervening base pairs

what are zinc fingers?

four cysteine residues that hold zinc

what is the structure of HREs?

• monomeric

• dimeric: palindrome or inverted repeat

• direct repeat

• inverted palindrome

sequence for each HRE is different for each hormone receptor- glucocorticoid has different HRE to oestrogen, etc

hexamers repeated because you have the two molecules joining onto it (2 half-sites)

how does a receptor recognise a specific HRE?

inside DNA binding area = p-box

inside p-box = zinc fingers + recognition sequences for HREs

p=box is where all the binding happens