.1) General principles of cell communication AND membrane receptors

paracrine signalling

paracrine signalling

secreting cell secretes to nearby cells

Autocrine signalling

Secreting cell is same as target cell

Hormone exits cell then enters cell

Endocrine signalling

Secrete into circulation to target cell e.g. pancreas beta cells secreting insulin

Juxtacrine signalling

Secreting cell targets adjacent cell

Intracrine signalling

Hormone targets secreting cell and stays within cell

Milestones in receptor biology

Otto Loewi experiment

Evidence for receptors

• Isolated two frog hearts and placed each of them in a chamber filled with saline

• Connect them with a tube

• Measure heart rate

• Stimulate vagus nerve of one heart to slow down

• After a delay the second heart gets stimulated

Hypothesis: electrical stimulation of the vagus nerve released a chemical into the fluid of chamber 1 that flowed into chamber 2. He called the chemical “Vagustoff” which we now know is the neurotransmitter ACh

Henry dale

Isolated ACh from ergot (fungus)

Isolated ACh from human body

Proved with Loewi that ACh is a neurotransmitter

ACh receptors

ACh receptors can have different effects

Can become ion channels

Agonist - compounds that produce the same effects as the neurotransmitter

Antagonist - blocks active site but has no response

Nicotinic AChR

Nicotinic AChR - two extracellular domains (binding sites) for ACh

Pentametric ion channel - 5 different subunits

transmembrane portion - alpha helices and intracellular domain

muscle stripe

Radioligand binding assay

incubate to allow binding

wash excess ligands

radioactivity proportional to number of receptors that have ligands bound

can measure affinity at a concentration of ligand

Affinity

How strongly a certain compound binds to a receptor

Affinity = 1 / KD

KD (equilibrium dissociation constant) - 50% of receptors occupied by ligand

Lower KD has a greater affinity

Types of receptors

Ligand gated ion channels (ionotropic)

• milliseconds

• Nicotinic ACh Receptors

G coupled protein receptors (metabotropic)

• seconds

• Muscarinic ACh receptors

• largest family of membrane receptors

Kinase linked receptors

• hours

• Cytokine receptors

Nuclear receptors

• hours

• Oestrogen receptor

Kinase linked receptors

Summary: 

1. GF (1^st messenger) binds to receptor

2. Causes dimerisation of receptor

3. Tyrosine kinase regions are activated and become phosphorylated (6 x ATP)

4. (Receptor tyrosine kinase) RTK is now fully activated

5. Cellular proteins become activated which causes a cascade which initiates a cellular response

• Speed: Slow

• Example: Tyrosine kinase receptor

• If these receptors are mutated, they cause cell proliferation and so are often implicated in various form of cancer.

kinase

Phosphatase

Kinase - adds phosphate

Phosphatase - removes phosphate

GPCRs

Mediate effects of many hormones and neurotransmitters

When G proteins are inactive they contain a nucleotide GDP

Alpha subunit contains enzymes which hydrolyse GTP to GDP to stop the reaction

GPCR typically have 7 alpha helixes and spans the plasma membrane 7 times

have alpha, beta and gamma subunits

Types of G proteins

G alpha s - activates adenylyl cyclase (AC)

- opens calcium ion channels in some tissues

• ATP —AC→ cAMP—cAMP phosphodiesterase → AMP

G alpha q - activates phospholipase C

G alpha i- inhibits AC

G alpha 12/13 - regulates small g proteins for cytoskeleton contraction

G beta gamma - regulate ion channels and enzymes like PLCb

GPCR mechanism

agonist binds to receptors causing a conformational change allowing it to bind with the heterotrimetric G proteins

causes G protein to open and GTP binds to alpha subunit displacing GDP

G protein dissociated with receptor and alpha subunit separates from beta and gamma

subunits interact with other effectors initiating a biological response

alpha subunit hydrolyses GTP to GDP……

Phospholipase C (PLC)

PLC cleaves a membrane phospholipid, PIP2, to give two secondary messengers (IP3 and DAG)

DAG remains in cell membrane and attracts and activates protein kinase C

Calcium signalling

G protein dependent opening of ligand gated ion channels

Release of intracellular stores of calcium in ER mediated by release of IP3

Calcium acts as a secondary messenger activating calcium/calmodulin dependent protein kinase

Example 1

Hormonal regulation of glycogen metabolism

Glucagon or Adrenaline binds to G alpha s

Activates AC

converts ATP to cAMP

Causes release of protein kinase A

Cascade of reactions converts glycogen to glucose

Same process inactivates glycogen synthase

cAMP phosphodiesterase keeps the reaction controlled by degrading cAMP to prevent the reaction from reoccurring, cAMP to AMP

Example 2

Regulation of smooth muscle tone

Calcium bind with calmodulin and the complex stimulates myosin light chain kinase (MLCK)

Phosphorylates myosin light chain

Myosin light chain Phosphatase (MLCP) removes phosphate group

G alpha s induces muscle relaxation

Example 3 - real life

TSH receptor signalling in the thyroid - Gαs

1) TSH (released via pituitary gland) binds to Gs-protein coupled receptors on the surface of thyroid cells leading to the production of cAMP which activates PKA 🡪 CREB activation (TF)

2) This leads to an increased replication of thyroid cells and it also induces the synthesis and release of thyroid hormones into the bloodstream.

cAMP/PKA pathway

alpha subunit interacts with AC.

AC converts ATP into cyclic AMP which binds to protein kinase activating Protein kinase A (PKA)

The activated PKA enters into the nucleus and helps activate CREB (transcription factor)

This induces a response which results in the secretion of hormones or cell replication.

PDE also degrades cAMP converting it into AMP to shut off signalling cascade.

Effects of cAMP/PKA pathway in endocrine cells

Increase hormone secretion

Under prolonged stimulation - increased cell proliferation

TSH receptor

Autonomous thyroid adenoma (ATA)

TSH is secreted by anterior pituitary

ATA - cause hyperthyroidism

- benign tumours caused by activating mutations in TSHR gene (somatic mutation)

TSH resistance - inactivating TSHR mutations

• complete (very rare) - homozygous, severe congenital hypothyroidism

• Partial (common) - heterozygous, mild hypothyroidism

Cushing syndrome

Mutation in PKA C subunit

Adrenal tumour

GPCR animation