Intracellular Signalling

Question 1

Define intercellular and intracellular signalling

Answer 1

Intercellular signalling signalling from one cell to another (extracellular signals) Intracellular signalling –signalling sent within the cell to stimulate a change in an activity of another or the same part of the cell

Question 2

Why do we need transmembrane receptors?

Answer 2

Proteins, peptides, charged molecules can not cross the lipid bilayer.

the signal needs to reach the inside of the cells

Transmembrane proteins span the membrane and transmit the signal into the cell

Question 3

how do ligands work to activate a receptor?

Answer 3

• Ligands may interact directly with the receptors or by binding to co-receptors or accessory molecules on the cell surface.
• Receptor activation causes a conformational change in the tertiary or quaternary structure that allows initiation of signalling

Question 4

Different Signal transduction molecules

Answer 4

Hydrophobic proteins - membrane-associated

Hydrophilic proteins - in the cytosol

Second messengers - e.g. cAMP

Ions - e.g. Ca2+

Question 5

Signal amplification explain:

Answer 5

The ligand binds to the receptor leading to the secondary messenger release

secondary messengers are a lot higher in concentration and cause an amplified reaction in the cell

divergence in pathway allows multiple effects from one stimulus

Question 6

How are signalling molecules controlled?

Answer 6

By post translational modification, e.g phosphorylation

By regulating whether a G protein has bound GDP or GTP

By provision of activators such as Ca2+ and cAMP

Question 7

Explain protein phosphorylation

Answer 7

Kinases phosphorylate proteins and phosphatases remove proteins.

This can either switch on or off the signal

Question 8

Which amino acids are phosphorylated?

Answer 8

serine, threonine and tyrosine

Question 9

Two types of kinases and example

Answer 9

Serine/Threonine kinases:

• Ca2+/calmodulin-dependent protein kinases or CaM kinases
• Protein Kinase A (PKA)
• Protein Kinase C (PKC)
• Mitogen-activated protein kinases (MAPK)

Tyrosine kinases

• Non receptor tyrosine kinases e.g. Src family kinases - Knowledge of these kinases has been fundamental to our understanding of cancer
• Receptor tyrosine kinases (RTKs) – e.g. Epidermal growth factor receptor (EGFR)

Question 10

Explain GTP binding proteins

Answer 10

Many GTP binding proteins are individual proteins (small GTPases)

Also used by heterotrimeric G proteins

Hydrolyse GTP-GDP by their intrinsic GTPase activity

Act as a molecular switch

Question 11

Signalling must be regulated

Answer 11

Cancer - Changes in cell proliferation in response to growth factors or in response to signalling errors e.g. Ras mutations (aa12 Glycine-Valine)

Question 12

What do provisional activators of proteins do and 2 examples?

Answer 12

Calcium and cAMP

Binding changes the conformation of target proteins which changes their activity

Question 13

What are the three categories of ion channels?

Answer 13

Ion channels

G proteins

Enzymes

Question 14

Ion channels features

Answer 14

ØTransport Na+, K+, Ca2+, Cl- Ions along an electrochemical gradient

ØSpecificity of the channel is defined by the amino acids lining the channel

ØChannels are formed of protein subunits

ØFast regulated opening/closing mechanism (mSec)

ØActivated by either a change in membrane voltage or by a ligand

Question 15

Voltage gated ion channel structural features?

Answer 15

a-subunit contains the pore which has 4 homologous domains, each with 6 transmembrane regions

Β subunits traffic the channel and regulate its kinetic properties

Question 16

How do voltage gated Na+ channel work?

Answer 16

Opens in response to a change in voltage

Region 4 has amino acids with positive R-groups which sense the voltage across the membrane causing movement of region 4 opening the channel

Question 17

What are ligand gated ion channels and how do they work?

Answer 17

Transmembrane proteins consisting of a receptor part and a channel which traverses the membrane

Open in response to binding of a ligand

Receptors are often classified based on which agonists they bind

Question 18

Nicotinic acetylcholine receptor

Answer 18

• Receptor has 5 subunits (2α, β, γ and δ)
• Region 2 of each subunit forms the channel
• 2 acetylcholine molecules bind the α subunits causing movement of the M2 helices opening the channel

Question 19

Name a few events regulated by Ca2+

Answer 19

Secretion, transcription factor activities, Skeletal muscle contraction

Question 20

Many effects of Ca2+ are mediated through:

Answer 20

CAM kinases calmodulin-dependent protein kinases

Calcineurin Ca2+-dependent protein phosphatase

Question 21

What is the conc of Ca2+ in the cytoplasm and how is it maintained?

Answer 21

Low concentration of 100nM and maintained by ATP dependent pumps

Question 22

How many TM domains does a GPCR have?

What are the subunits of a G-protein?

Answer 22

typically 7 TM domains - NOT a pore

G-protein consists of three subunits: a, b and g

Question 23

GPCR activation explained

Answer 23

• When a ligand binds to receptor the affinity for G-protein increases. The receptor and the G-protein may sometimes already be in a complex at the membrane.
• Receptor activation changes the conformation of the internal portion of the receptor releasing GDP
• Action of the receptor causes GDP attached to a subunit to be replaced by GTP
• G-protein splits up in bg complex an α-subunit
• Both complexes then initiate further cell signalling
• GTP is then hydrolyzed to GDP and bg recombines with a ready to associate with another GPCR

Question 24

Explain the role of cAMP and Adenylate cyclase

Answer 24

G-protein activates adenylate cyclase which produces cAMP from ATP in the cytosol

cAMP is a second messenger and works on PKA to activate it.

PKA phosphorylates many different proteins leading to increased transcription

Question 25

Phospholipase C activation and pathway

Answer 25

activated via Gaq and M1 ACh receptors

Question 26

How do GPCRs activate ion channels (muscarinic M2 receptor)

Answer 26

Acetylcholine binds GPCR, activated bg unit binds to potassium channel causing it to open

Potassium ions flow out, cells become hyperpolarized, heart slows down

Question 27

G proteins in Cholera

Answer 27

Cholera toxin

• Inhibits the GTPase activity of the subunit Gαs (stimulates adenylyl cyclase)
• Prolonged signal causes water and Cl- to move out of the cells lining the intestine
• Results in diarrhoea, dehydration and death

Question 28

G proteins in Whooping cough

Answer 28

• Bordetella pertussis releases an active adenylyl cyclase domain
• Pertussis toxin renders Gαi inactive (Gαi inhibit adenylyl cyclase)
• Modifies Gαi preventing association with GPCRs
• Prolonged signal that stimulates coughing

Question 29

GPCR signalling pathways

Answer 29

Question 30

Receptor linked to enzymes (Receptor tyrosine kinases)

Answer 30

• Ligand binding activates enzyme activity within the cytoplasmic domain
• The response usually requires receptor dimerisation
• Tyrosine residues in the intracellular domains are auto-phosphorylated in response to the signal