Signalling cascades

Question 1

What are the main functions of Receptor Signalling Cascades?

Answer 1

• Can detect a single molecule of ligand e.g. a photon.
• Transduce this signal across the membrane.
• Rapidly amplify this signal.
• Activate a cascade of proteins that bring about appropriate cell behaviours.

Question 2

Explain the rhodopsin signalling cascade in vision

Answer 2

1. One rhodopsin molecule absobs one photon and this causes a conformational change between the cis and trans state in retinol (the ligand bound within rhodopsin)
2. The activates (500) of the G-protein Transducin molecules which consists of an alpha a beta and a gamma subunit
3. In the inactive state, transducin is bound to GDP but activation by the receptor results in the release of GDP and binding of GTP
4. The alpha subunit activates (500) cyclic GMP phosphodiesterase which cleaves (1000000) cyclic GMP to GMP
5. This causes (250) Na⁺ channels to be closed within the membrane
6. 10⁶-10⁷ Na⁺ ions per second are prevented from entering the cell for about 1 second
7. The membrane potential is altered by 1mV

Question 3

Describe or draw a diagram to show which type of signalling is appropriate in different biological contexts (the kind of response that is required)

Answer 3

• Altered protein function signalling cascades are necessesary when rapid (<1 sec-mins) responses are required e.g. for vision
• Changes to transcriptional activity is useful when the cell needs to be modified for in the long term (mins-hrs) e.g. embryo patterning

Question 4

Name the 4 main types of receptors

Answer 4

• Ligand gated ion channels
• G-Protein coupled receptors
• Receptor tyosine kinases
• Intreacellular receptors

Question 5

Types of receptors: Describe Ligand gated ion channels

Answer 5

Ligand gated ion channels→ A protein within the membrane (usually tetrameric- 4 subunits). When a lignad binds it causes a flux of ions cossing the membrane therefore changing membrane polarity

Question 6

Types of receptors: Describe G protein coupled receptors

Answer 6

G protein coupled receptors→ These are the largest class of cell surface receptors. They are characterised by 7 transmembrane domains. G-Protein activation►Generation of second messenger►activation of cell signalling

Question 7

Types of receptors: Describe Receptor tyrosine kinases

Answer 7

Receptor tyrosine kinases→ They have enzymatic activity. When the ligand binds, dimer formation and autophosphorylation occurs between the domains. They can also phosphylate other molecules to activate cell signalling

Question 8

Types of receptors: Describe Intracellular receptors

Answer 8

Intracellular receptors→ Small hydrophobic molecules such as steroid hormones diffuse across the membrane and bind to receptors in the cytoplasm. The receptor become activated and transports them to the nucleus. This leads to the activation/alteration of transcription and translation

Question 9

Name some common Second Messengers

Answer 9

• IP₃
• cAMP

Both of these are closely linked to G protein signalling

• NO
• cGMP
• Ca²⁺

Question 10

Describe Nitric Oxide (NO) as a signalling molecule

Answer 10

• Free radical gas
• Quite unusual to have a gas as a signalling molecule.
• Gas second messengers can diffuse through both cell membrane and cytoplasm.

Makes them excellent paracrine and autocrine signalling molecules

Question 11

Descibe the physiological Relevance of NO

Answer 11

NO is involved in:

• Relaxation of vascular and gastrointestinal smooth muscle.
• Inhibition of platelet aggregation.
• Reduction in cardiac hypertrophy.
• Protection against ischemia/reperfusion heart damage.
• Improved cognitive functions.

And many more…..

Question 12

Describe the biosynthesis of NO

Answer 12

It is formed within the body and not naturally occuring within our internal environment

It is made by a family of enzymes called Nitric Oxide Synthase (NOS)

These enzymes catalyse the production of NO and the by-product citruline from L-arginine

Arginine+NADPH+O₂→ Nitric oxide+NADP+Citruline

Question 13

What are the 3 different NOS enzymes?

Answer 13

• nNOS►Neuronal NOS
• eNOS►Endothelial NOS
• iNOS► Inducible NOS

Question 14

Where is Neuronal NOS (nNOS) located?

Answer 14

In the nervous system and type II skeletal muscles

Question 15

Where is Endothelial NOS (eNOS) located?

Answer 15

In the endothelium

(also present in cardiac myocytes, renal mesangial cells, osteoblasts and osteoclasts)

Question 16

Where is inducible NOS (iNOS) located?

Answer 16

In the immune system and cardiovascular system

Question 17

TRUE or FALSE: The general function of nNOS is cell communication

Answer 17

TRUE

Question 18

TRUE OR FALSE: The general function of eNOS is vasoconstriction

Answer 18

FALSE

The general function of eNOS is vasodilation

Question 19

TRUE or FALSE: The general function of iNOS is immune defence

Answer 19

TRUE

Question 20

TRUE or FALSE: nNOS and eNOS are constitutive (always present and always active)

Answer 20

TRUE

Question 21

TRUE or FALSE: nNOS and eNOS generate large amounts of NO

Answer 21

FALSE

nNOS and eNOS generate low amounts of NO.

Question 22

TRUE or FALSE: iNOS is 100x more active than nNOS and eNOS

Answer 22

FALSE

iNOS is 1000x more active

Question 23

TRUE or FALSE: iNOS is expressed only upon immunological challenge

Answer 23

TRUE

iNOS helps macrophages destroy pathogens

Question 24

Draw a table that compares the 3 types of NO synthases (NOS)

Answer 24

Question 25

Explain eNOS Activation in Vascular Tissue

Answer 25

1. In a blood vessel an acetylcholine signal will result in a complex of Ca²⁺ and calmodulin

This activates NO synthase which converts arginine +O₂ into citruline +NO

1. NO is relesed and diffuses across the cell membrane to enter smooth muscle cells
2. Here it acts on the enzyme Guanylate cyclase which converts GTP to cGMP with the release of PP_i
3. This results in vasodilation as the smooth muscle relaxes to expand the volume of the blood vessel.

Question 26

Draw the pathway of eNOS Activation in Vascular Tissue

Answer 26

Question 27

What are the cellular Targets of cGMP?

(Draw a spider diagram)

Answer 27

• cGMP-dependent kinases (Protein kinase G-PKG)
• cGMP-gated cation channels
• Cyclic nucleotide phosphodiesterases (PDEs) (break down cGMP and turn off the signal)
• Transporters

Question 28

What are the Pharmacological Targets of NO and cGMP?

Answer 28

•NO donors:

–Nitro-vasodilators, sodium nitroprusside

•NOS inhibitors

–L-NG-nitroarginine

•GC inhibitors

–Methylene blue

•PDE inhibitors (Type V specific)

–IBMX, dipyridamole, zaprinast, sildenafil (Viagra)

Question 29

Describe calcium ions as signalling molecuels

Answer 29

• Extremely important signalling molecule
• Can alter local electrostatics due to shape and charge
• Can cause conformational changes in proteins through electrostatic interactions
• Important to control concentration because it can react with phosphate and when ATP is used as an energy source, a lot of free phosphate is available- Produces calcium phosphate (white solids)

Question 30

Explain why free [Ca²⁺] is tightly regulated in the cytosol.

Answer 30

• Extremely high levels are toxic = cell death.
• Reduces background noise to allow Ca²⁺ to act as a second messenger.

[Ca²⁺] Is lowest in the cytoplasm , followed by the ER and outside the cell its the highest

Question 31

Name some calcium pumps

Answer 31

These are for moving Ca²⁺ against its concentration gradient:

–Plasma membrane Ca²⁺ ATPase (PMCA).

–Smooth endoplasmic Ca²⁺ ATPase (SERCA).

Question 32

Name some calcium exchangers

Answer 32

These are for moving Ca²⁺ along its concentration gradient using an ion thats more concentrated outside the cell:

–Na+/Ca²⁺ exchanger (NCX)

–Na+/Ca²⁺/K⁺ exchanger (NCKX)

Question 33

Explain Calcium Oscillation

Answer 33

Ca²⁺ signalling can diffuse like a wave due to biphasic response of IP₃R.

1. IP₃ binds its receptor and IP₃R becomes sensitive to Ca²⁺.
2. Initially, IP₃R allows Ca²⁺ into the cytosol .
3. Ca²⁺ induced Ca²⁺ release (CICR).
4. At high [Ca²⁺] IP₃R activity is inhibited by Ca²⁺.
5. Prevents further release of Ca²⁺ into the cytosol.

–Negative feedback.

•This positive / negative feedback propagates along the cell membrane.

Question 34

Describe the properties of Calmodulin

Answer 34

• Small (148 amino acid) protein.
• “Dumbell” shaped.
• Flexible central hinge.
• Coordinates Ca²⁺ through 4 E-F hand motifs.
• Acts as an _adaptor protein (_can allow other proteins to interact via calmodulin therefore without directly binding to each other).
• When Ca²⁺ binds to calmodulin it changes the conformation of the ends of calmodulin allowing it to interact with other proteins