Lipid Microdomains

Question 1

What two groups does the lipid membrane split into?

Answer 1

Liquid ordered

Liquid disordered

Question 2

What are the characteristic of the liquid disordered membrane?

Answer 2

Loosely packed phospholipids
Rapid lateral diffusion
Made of phosphatidyl serine/ethanoamine/choline

Question 3

What are the characteristics of the liquid ordered phase?

Answer 3

Compartmentalised lipid areas
Made up of cholesterol
Bilayer assembly more rigid which prevents lateral diffusion and makes it difficult to move things

Question 4

What type of phospholipids make up the liquid ordered partition?

Answer 4

Glycosphingolipids

Sphingomyelin

Question 5

Liquid ordered areas form lipid rafts. What are the two types of raft?

Answer 5

Planar (at membrane surface)

Calveolae (depressions in membrane caused by protein caveolin)

Question 6

Molecular weight of caveolin

Answer 6

20KDa

Question 7

Structure of caveolin?

Answer 7

Cytosolic N terminus
Enters membrane
Intramembrane fold
Cytosolic C terminus which is palmitolyated allowing anchoring to membrane (post translational modification)

Question 8

Where a calveolae found?

Answer 8

Many cells types especially pulmonary vascular endothelial cells

Question 9

How many genes encode for caveolin?

Answer 9

3 (Cav1,2 and 3)

Question 10

Where is CaV1 expressed?

Answer 10

Ubiquitous but high levels on endothelial and SM cells

Question 11

Where is Cav2 expressed?

Answer 11

Ubiquitous but high levels of endothelial and SM cells

Question 12

Where is Cav3 expressed - why?

Answer 12

Muscle specific as promote region responds to myogenic transcription factors

Question 13

What othe rproteins are important for caveolae formation?

Answer 13

Calvin (wraps around caveolin and coats caveolar surface (KO = no caveolae)
EHD2 - dynamin like protein important for invagination

Question 14

What experiment can be done to separate liquid ordered and liquid disordered?

Answer 14

Fractionalisation
Add detergent at 4 degrees and rafts will fractionate in a sucrose density gradient (low to high in test tube)
Liquid disorder will not fractionated and will fall to bottom

Question 15

Why do rafts show more ‘buoyantcy’?

Answer 15

Detergents will not degrade rafts therefore they will remain in the lower density fractions of the test tube

Question 16

What test can be done to analyse the proteins in the various fractions of the sucrose gradient?

Answer 16

Western blotting

Shows that caveolin is present in high amounts in th lower density fraction

Question 17

What scientist did the fractional analysis of the sucrose gradient?

Answer 17

Lisanti et al 1994

Question 18

What other proteins are found within the lower density fractions (lisanti et al 1994)

Answer 18

Stains heavily for signalling proteins such as Gas, JAK2, Lyn and c-Src

Question 19

Can some signalling proteins be found in the high density liquid disorder area?

Answer 19

Yes, some can be found in the liquid ordered and isorded e.g. RhoA and RhoB

Question 20

Since many signalling proteins can be found in the liquid order area what does this mean?

Answer 20

Lipid rafts may be important for signalling microdomain formation

Question 21

What is a signalling microdomain?

Answer 21

Specialised regions of membrane which compartmentalise signalling molecules which relies on interactions with lipid rafts and/or caveolin

Question 22

Wha domain is important for the scaffolding properties of caveolin?

Answer 22

The caveolin scaffolding domain (CSD)

Question 23

Who showed that the CSD was important for signalling regulation?

Answer 23

Li et al 1996

Question 24

What proteins interact with the CSD?

Answer 24

PKA
Scr
AC (3+5)
Ga

Question 25

When bound tot he CSD what happens to the bound signalling molecules?

Answer 25

The CSD holds them in an inactive state i.e. Src activity is increased when caveolin is not expressed and mains GPCRs in GDP bound state

Question 26

What types of aminoacids makes up the CSD?

Answer 26

Aromatic aminoacids

Question 27

What happens when the signalling cascade of the CSD bound protein is activated?

Answer 27

The bound protein unbinds from the CSD due to conformational change

Question 28

What proteins may aid in microdomain formation?

Answer 28

EDH2 and cavin

Question 29

Give an example how may caveolin microdomains are useful to cells?

Answer 29

Calcium channels localise to calveolae/cholesterol rich areas of membrane These caveolae are also in close proximity to peripheral SER which serves to bring calcium current close to the effector

Question 30

What did Ostrom et al 2000 study?

Answer 30

Overexpression of AC6 increases AC activity (measured by increase in cAMP per unit time) on addition of isoprenylin (B2AR agonist) but not on addition of PGE2 (EP2R agonist). Both act through AC. However, this shows that B2AR signalling efficiently activates AC6 but PGE2 does not.

Question 31

What does western blotting in ostrom et al 2001 show?

Answer 31

AC6 is found in caveolae along side B1AR | EP2R are below the threshold of detection in caveolae but they are found in non caveolae areas (AC6 not found here)

Question 32

Why might Ep2R not be about to effieicently couple to AC6?

Answer 32

They exist in the liquid disordered membrane area so lateral diffusion prevents this coupling BAR more efficient at producing AC6 activity as localised to same environment.

Question 33

What type of study supports that L type VGCC are co-localised to Cav3?

Answer 33

Electronmicroscopy with antibody to both proteins

Question 34

What compound removes cholesterol to membrane?

Answer 34

M(beta)CD

Question 35

What does M(beta)CD do so GPCR modulation of VGCC?

Answer 35

It attenuates it | Normally salbutamol increases calcium current but M(beta)CD will decrease the calcium current

Question 36

How can rafts/ lack of cause disease?

Answer 36

Efficiency in intracellular signalling can be altered

Question 37

Give an example of pathological consequences of letting micro-domain dependant signalling

Answer 37

CaV1+3 KO = cardiomyopathic consequences Ma cause HF and hypertension May be due to non inhibited signalling pathway as no CSD e.g eNOS mediated nitrosative stress

Question 38

What conclusion can be drawn from the fact that pulmonary hypertension occurs in CaV1 KO but not CaV2 KO?

Answer 38

Different Cav subtypes may have different roles in protecting against disease and regulatory pathways