Hormones, cell signalling, transport and kinetics

What is autocrine signalling?

• affects cells producing the signalling factor

What is paracrine signalling?

• Diffuse short distance to affect cells nearby

What is endocrine signalling

• acts on target cells at a distance from site of synthesis

How can autocrine signalling affect local cells?

• In a group of identical signalling cells, each cell can receive a strong autocrine signal.

• a single signalling cell receives a weak autocrine signal

what are the 4 cellular decisions post signalling?

1. survive

2. cell growth/division/proliferation

3. differentiate

4. die/apoptosis

What are the two types of hormone

1. lipid soluble

2. water soluble

Outline lipid soluble hormones:

1. how are they transported

2. how to they enter cells

3. how do they affect cells

1. carrier proteins in blood

2. diffuse through plasma membrane

3. alters expression of genes at level of nucleus

Outline water soluble hormones

1. how are they transported

2. how to they enter cells

3. how do they affect cells

1. easily travel in blood

2. bind to receptors on surface of cell

3. intracellular events

what are most water soluble signalling molecules?

• growth factors

• water soluble hormones

• MOST SIGNALLING MOLECULES

What are examples of lipid soluble hormone

• steroid hormones

• thyroid hormones

• retinoids

• vitamin D

Give 3 example locations of lipid soluble hormone production and what they produce

1. Adrenals: androgens, cortisol, corticosterone and aldosterone

2. Ovaries: estradiol

3. testes: testosterone, dihydrotestosterone

What happens when a hormone binds to its nuclear receptor?

1. hormone binds

2. receptor is activated, moves to nucleus

3. active receptor binds to DNA

4. regulates transcription of specific target genes

Outline the process of lipid soluble hormone signalling

1. hormone released from transport proteins

2. hormone enters cell

3. binds to nuclear receptor

4. bound receptor enters nucleus and binds to DNA

5. transcriptional regulation of target genes

What are the 3 domains of a nuclear receptor?

1. transcription activating domain

2. DNA-binding domain

3. hormone-binding domain

How do nuclear receptors vary?

• all have similar domains: all have 3 parts.

• vary in the N- and C- terminal regions

• this allows ligand binding specificity

How does the nuclear receptor become activated?

1. hormone binds

2. conformational change in shape, exposing the DNA binding site.

3. Protein joins with co-activator proteins which regulate activation and repress nearby target genes.

Outline water soluble hormones:

1. What do they interact with

2. what do they induce

1. membrane-bound receptors: inhibitory or stimulatory

2. intracellular signal transduction pathways

What are the 5 enzymes/pathways involves in water soluble hormone signalling

1. adenyl cyclase (cAMP)

2. guanyl cyclase (cGMP)

3. phospholipase C, IP3 and DAG

4. Tyrosine kinase

5. ion channels

what is the intra-cellular signalling pathway

1. signals sent from the cell membrane where the bound ligand binds to its complementary receptor.

2. signals are transduced through the signalling pathway: they can be amplified

Give an example of water soluble hormones for:

1. Adenyl cyclase

2. guanylyl cyclase

3. phspholipase C

4. tyrosine kinase

   1. ion channels

1. ACTH, ADH, FSH, LH and TSH

2. rare atrial natruiretic peptide

3. hypothalamic hormones, TRH, GnRH

4. insulin, prolactin, growth hormone, IGF

5. hormones using multiple pathways

What is the structure of the receptor for water soluble hormones

1. extracellular domain: binds to hormone

2. transmembrane domain

3. intracellular/cytoplasmic domain

Which domain of the cell membrane receptor changes during signalling?

• cytoplasmic domain undergoes conformational change

• this activates the signalling pathway

Growth factors vs cytokine?

• SAME THING

• growth factors are used in terms of growth

• cytokines in terms of immunology

• both perform paracrine and autocrine action

Functions of growth factors and cytokines?

1. cell division

2. cell differentiation

3. programmed cell death

4. aberrant function of growth factors/cytokines are implicated in cancer

Does a hormone have the same effect on each cell?

• NO = pleiotropy

• may have a different effect on different cell types

What is the relevance of half lives with hormones?

• must regulate responses

• signal must be withdrawn → response fades

• different hormones have different half lives depenidng on rate of destruction/removal of molecules and promptness of response when the signal is removed.

What factors contribute to a hormone’s half life?

• rate of destruction/removal of molecules

• promptness of response when a signal is turned on/off

• rate of synthesis

• modifications of proteins

Define half life

• time taken for the concentration of a signalling molecule to fall by half

what are the 4 classes of cell surface receptors

1. Ion-channel linked receptors

2. G-protein linked receptor

3. Tyrosine kinase-linked receptor

4. receptors with intrinsic enzymatic activity

What is involved with each of these signalling receptors:

1. Ion-channel linked receptors

2. G-protein linked receptor

3. Tyrosine kinase-linked receptor

4. receptors with intrinsic enzymatic activity

1. rapid synaptic signalling

2. act indirectly to activate a separate plasma membrane bound protein

3. directly associated with enzyme they activate

4. functions directly as enzyme

Outline how ion channels work

1. signal molecule binds to receptor

2. opens/closes channel

3. ions move into cell by diffusion

4. excitability of post-synaptic cell altered

Give 4 examples of ion channels

1. post-synaptic membranes

2. neuromuscular junction

3. nicotinic ACh receptor

4. GABA receptors

What are second messengers?

• small intracellular signalling molecules

• generated by receptor activation (amplification of signal)

• diffuse rapidly away

• pass signal on →activate other proteins

give 4 examples of secondary messengers

1. cAMP

2. cGMP

3. diacylglycerol - DAG, lipid soluble

4. Ca2+ ions

Name the 5 proteins involved in intracellular signalling

1. scaffolds

2. relays

3. adaptors

4. amplifiers

5. modulators

briefly outline what each of these function are:

1. scaffolds

2. relays

3. adaptors

4. amplifiers

5. modulators

1. holds proteins within pathway, not affected by signals

2. pass signal on

3. can stimulate/repress response

4. amplify signals/transduce them

5. can affect signal

What are the 2 key processes in cell signalling

1. protein phosphorylation by protein kinases

2. reversal of protein phosphorylation - phosphoprotein phosphatase

What are the two types of protein phosphorylation

1. of serine/threonine residues

2. of tyrosine residues

What can phosphorylation alter?

1. enzyme activity

2. protein stability

3. protein interactions

What is a G-protein coupled receptor

• single polypeptide chain with a central hydrophobic region that spans plasma membrane 7 times

How does G protein signalling work?

1. what type of hormone is involved

2. what other proteins are involved

3. give an example

1. water soluble

2. membrane protein, G protein, target protein (enzyme or ion channel).

3. muscarinic ACh receptor

what is a G-protein?

• guanine-nucleotide binding protein: a signal transducing protein

what are the 3 mains steps of G protein mediated signal transduction

1. no ligand = inactive receptor

2. binding of ligand recruites the G protein complex

3. GTP replaces GDP

• G alpha and beta proteins dissociate

• G alpha and beta subtunits regulate 2nd messengers: including adenylate cyclase

How can G-proteins act as inhibitors and stimulators?

• depending on the hormone that binds!

• if the hormone is stimulating → stimulating activity

• if the hormone is inhibitory → inhibition

Outline the process of the stimulatory effect mediated by adenylate cyclase - give an example of this signalling

• subunit of G protein activates adenylate cyclase

• ATP → cAMP

• cAMP acts as catalytic subunit of pKA (protein kinase A)

• pKa is part of cascade of intracellular phosphorylations.

• example = epinephrine acting through adrenergic B- receptor

How can one hormone molecule result in a large response?

• 1 hormone can trigger many cAMP molecules to be produced, which amplifies the response

Give an example of how a G-protein can inhibit adenylate cyclase

• Norepinephrine acts through the alpha-2 receptor on the pre-synaptic nerve

What are the two pathways of enzyme linked receptors

1. function directly as enzymes

2. directly associated with enzymes they activate

What are the 6 classes of enzyme linked receptors?

1. receptor tyrosine kinases

2. tyrosine-kinase-associated receptors

3. receptor like tyrosine phosphatases

4. receptor serine/threonine kinases

5. receptor guanyl cyclases

6. histidine-kinase-associated receptors (rarer)

What do each of these do:

1. receptor tyrosine kinases

2. tyrosine-kinase-associated receptors

3. receptor like tyrosine phosphatases

4. receptor serine/threonine kinases

5. receptor guanyl cyclases

6. histidine-kinase-associated receptors (rarer)

1. phosphorylate tyrosine on intracellular signalling molecules

2. associate with intracellular proteins with tyrosine kinase activity

3. remove phosphate group from tyrosine on intracellular proteins

4. phosphorylate serines/threonines on regulatory proteins

5. catalyse production of cytosolic cGMP

6. 2 parts: kinase phosphorylates itself and passes on phosphate to intracellular signalling protein

How to tyrosine kinase-linked receptors work?

• they lead to the phosphorylation of intracellular signalling proteins

• e.g. insulin and IGF-1 proteins

How do tyrosine kinase receptors activate multiple pathways?

• phosphorylation of the receptor’s intracellular domains creates a docking surface for other cell signalling proteins

Outline the principle of a signal transduction pathway

• when the ligand binds to the receptor it triggers protein kinases this leads to the activation of the desired protein(s) leading to the response.

What is meant by intracellular molecular switches and what are the two main classes of them?

They’re intracellular proteins + signal

• they recover the signalling pathway by returning active proteins to inactive

Types:

1. phosphorylation (gain or loss changes signal)

2. GTP binding

For each type of molecular switch, what is the active form and what is the inactive

Phosphorylatioin: Gain = on, Lose = off

GTP/GDP:GDP bound = inactive, GTP bound = active

How may a cell response to a hormone be delayed or prolonged in duration

• the primary response turns on secondary response proteins

• simultaneously shuts off primary response genes

Why are some hormones responses abrupt, rapid and transient?

• the response may depend on the binding of more than one intracellular molecule to a target molecule

  • nuclear receptor level: >1 activated hormone receptor complex must bind to DNA toa ctivate gene

  • cell surface level: similar binding principle may operate in a cascade

How can modulating receptor activity and density alter sensitivty to a signal? Give 5 examples

1. receptor sequestration

2. receptor down-regulation

3. receptor inactivation

4. inactivation of signalling protein

5. production of inhibitory protein

What are the 2 major communication systems

1. endocrine

2. nervous

Name the 12 endocrine glands

1. pineal

2. hypothalamus

3. pituitary

4. thyroid

5. parathyroid

6. adrenals and kidney

7. pancreas

8. testes

9. ovaries

10. skin

11. placenta

12. adipose tissue

What are the 4 organ-endocrine glands

1. heart

2. intestine

3. kidney

4. placenta

What do each of these produce as a hormone?

1. heart

2. intestine

3. kidney

4. placenta

1. Atrial natruiretic peptide (ANP)

2. cholecystokinin, gastrin

3. Active D3, EPO

4. chorionic gonadotropins (stimulate progestins)

What are the 4 characteristics of a hormone

1. chemical agents

2. synthesised and secreted by glands

3. circulate in blood to other parts of the body

4. stimulate specific tissues

what are the 3 classifications by function of hormones

1. classical (genuine) hormones

2. neurohormones

3. local hormones (paracrine signalling)

What is a classical hormone and what is an example

1. secreted from endocrine cells, diffuse into blood, transported by transporters via blood to target tissue

2. cortisol

What is a neurohormone and give 2 examples

1. synthesised within neuroendocrine cells

2. secreted from nerve terminals

3. diffuse into blood vessels and transported

examples:

1. CRH: corticotrophin releasing hormone

2. ACTH

What are local hormones and the two mechanisms by which they act?

1. diffusioni of hormone into intersistial fluid

2. paracrine and autocrine

Give an example of a water-soluble hormone and lipid-soluble hormone and how they’re transported in the blood

1. catacheolamines (epinephrine) - freely transported

2. cortisol and thyroid hormones - bound to transport proteins (cortisol binding globulin CBG)

Give 3 examples of specific lipid soluble hormone binding proteins and their hormones

1. cortisol-binding protein - cortisol

2. vitamin D-binding globulin - vit D

3. thyroid-binding globulin - T3 and T4

Give two examples of non-specific lipid soluble hormone binding proteins

1. Albumin - steroids

2. prealbumin - T3 and T4

Where are transport proteins produced and degraded, what is a potential clinical relevance?

• liver

• liver failure

how can we consider a free hormone?

• active

What is the general percentage between bound vs free hormones

• significantly more is bound than free

What can affect the amount of free hormone in the blood?

• binding protein conc

• hormone level

What are the 3 functions of transport proteins

1. serve as hormone reservoir

2. act as hormone buffers

3. reduce hormone loss via kidneys

Outline hormone reservoirs

1. free hormone diffuses into cells

2. used as it binds to receptors

3. equilibrium is reached

4. this reservoir is used to replace free hormone in the blood

5. e.g. thyroid hormones

Outline the purpose of hormone buffers

1. respond to increased hormone secretion

2. only use 50% binding capacity

3. short-term peaks in synthesis absorbed

4. raise amount of hormone in circulation

How is hormone loss reduced?

• Being bound to transport proteins - can’t filter through glomerulus in kidney

What are the plasma concentrations the result of?

• rate of secretion in blood

• rate of removal from blood

What are 3 elimination mechanisms

1. enzymatic degradation

2. within target cells after binding recpetor

3. via liver and kidneys

What is fast action elimination

e.g. epinephrine

• rapid release (from adrenal medulla)

• immediate action on tissues (heart)

• fast return to normal (few seconds)

• water soluble hormone binds receptors

• enzymatically degraded w/in cell

Outline slow elimination

e.g. thyroxine (T4)

• circulates as mostly bound or free (lipid-soluble)

• bound hormone resists degradation

• either: converted to T3 (enters cell and alters gene expression)

• OR converted into water-soluble compounds by liver → excretion in urine

what are 4 factors that affect hormone responses

1. variation by tissue (pleiotropy)

2. time (species, age, specific hormone)

3. dose and choice

4. status of target tissue

Give two examples of steroid therapy and their uses

1. prednisolone sodium succinate → ultra short acting corticosteroid for shock

2. dexamethasone - long-acting corticosteroid for immuno-suppression

How may target tissues with:

1. hypertrophy

2. atrophy

affect hormone effect?

1. more function tissue → exaggerated response

2. less function tissue → inadequate response

what is the clinical relevance of steroid treatment?

Steroid withdrawal e.g. dexamethasone (corticosteroid)

• it suppresses normal synthesis and secretion from the adrenal gland

• adrenal gland atrophies

• CRISIS if withdrawn suddenly

How do we measure hormones?

• v. small measurements:

  • picomol, nanomol

  • cortisol and thyroid: ng/mL

  • ACTH: pg/mL

  • insulin: uU/mL or mU/L

what are two assays we can use to test for hormones?

1. RIA: radioimmunoassay - highly sensitive - measures free hormone

2. ELISA - measures total

How do RIAs work?

• known concentration of hormone is radiolabelled and bound by Abto form an Ab-hormone complex

• unlabelled hormone being tested is added and competes w. labelled hormone

• the resulting complexes are precipitated using a secondary Ab and the radiolabelled measured.

• Compare known start amount of radiolabel to what we get → find conc of unknown hormone

What is the:

1. clearance rate

2. secretion rate

1. use radioactively labeled hormone or halt secretion

2. if conc remains stable, assume secretion = clearance