Lipid Microdomains Flashcards
What two groups does the lipid membrane split into?
Liquid ordered
Liquid disordered
What are the characteristic of the liquid disordered membrane?
Loosely packed phospholipids
Rapid lateral diffusion
Made of phosphatidyl serine/ethanoamine/choline
What are the characteristics of the liquid ordered phase?
Compartmentalised lipid areas
Made up of cholesterol
Bilayer assembly more rigid which prevents lateral diffusion and makes it difficult to move things
What type of phospholipids make up the liquid ordered partition?
Glycosphingolipids
Sphingomyelin
Liquid ordered areas form lipid rafts. What are the two types of raft?
Planar (at membrane surface)
Calveolae (depressions in membrane caused by protein caveolin)
Molecular weight of caveolin
20KDa
Structure of caveolin?
Cytosolic N terminus
Enters membrane
Intramembrane fold
Cytosolic C terminus which is palmitolyated allowing anchoring to membrane (post translational modification)
Where a calveolae found?
Many cells types especially pulmonary vascular endothelial cells
How many genes encode for caveolin?
3 (Cav1,2 and 3)
Where is CaV1 expressed?
Ubiquitous but high levels on endothelial and SM cells
Where is Cav2 expressed?
Ubiquitous but high levels of endothelial and SM cells
Where is Cav3 expressed - why?
Muscle specific as promote region responds to myogenic transcription factors
What othe rproteins are important for caveolae formation?
Calvin (wraps around caveolin and coats caveolar surface (KO = no caveolae)
EHD2 - dynamin like protein important for invagination
What experiment can be done to separate liquid ordered and liquid disordered?
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
Why do rafts show more ‘buoyantcy’?
Detergents will not degrade rafts therefore they will remain in the lower density fractions of the test tube
What test can be done to analyse the proteins in the various fractions of the sucrose gradient?
Western blotting
Shows that caveolin is present in high amounts in th lower density fraction
What scientist did the fractional analysis of the sucrose gradient?
Lisanti et al 1994
What other proteins are found within the lower density fractions (lisanti et al 1994)
Stains heavily for signalling proteins such as Gas, JAK2, Lyn and c-Src
Can some signalling proteins be found in the high density liquid disorder area?
Yes, some can be found in the liquid ordered and isorded e.g. RhoA and RhoB
Since many signalling proteins can be found in the liquid order area what does this mean?
Lipid rafts may be important for signalling microdomain formation
What is a signalling microdomain?
Specialised regions of membrane which compartmentalise signalling molecules which relies on interactions with lipid rafts and/or caveolin
Wha domain is important for the scaffolding properties of caveolin?
The caveolin scaffolding domain (CSD)
Who showed that the CSD was important for signalling regulation?
Li et al 1996
What proteins interact with the CSD?
PKA
Scr
AC (3+5)
Ga
When bound tot he CSD what happens to the bound signalling molecules?
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
What types of aminoacids makes up the CSD?
Aromatic aminoacids
What happens when the signalling cascade of the CSD bound protein is activated?
The bound protein unbinds from the CSD due to conformational change
What proteins may aid in microdomain formation?
EDH2 and cavin
Give an example how may caveolin microdomains are useful to cells?
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
What did Ostrom et al 2000 study?
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.
What does western blotting in ostrom et al 2001 show?
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)
Why might Ep2R not be about to effieicently couple to AC6?
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.
What type of study supports that L type VGCC are co-localised to Cav3?
Electronmicroscopy with antibody to both proteins
What compound removes cholesterol to membrane?
M(beta)CD
What does M(beta)CD do so GPCR modulation of VGCC?
It attenuates it
Normally salbutamol increases calcium current but M(beta)CD will decrease the calcium current
How can rafts/ lack of cause disease?
Efficiency in intracellular signalling can be altered
Give an example of pathological consequences of letting micro-domain dependant signalling
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
What conclusion can be drawn from the fact that pulmonary hypertension occurs in CaV1 KO but not CaV2 KO?
Different Cav subtypes may have different roles in protecting against disease and regulatory pathways
