BIOC 222 - LECTURE 28

how do heterotrimeric G proteins associated with the membrane

• covalently attached lipid tail (post translational modification) inserts itself into the lipid bilayer

how do PLC and PKC associate with the membrane

Part of the protein recognises particular membrane lipids

describe the assocation of the alpha beta gamma unit throughout Gq signalling

• due to the lipid tail, even through the complex breaks apart with alpha breaking off from gamma and beta, they remain in the membrane

what does alpha do in Gq signalling after it dissociates from beta gamma

• binds phospholipase C

what does phospholipase C do

• it breaks apart PIP3 into IP3 and DAG

describe the structure of PIP3

• it is a glycerophospholipid with a hydrophobic tail and a hydrophillic head

• the hydrophobic part is DAG

• hydrophillic part is IP3

where do the constituents of PIP3 go after being formed

• DAG is hydrophobic so it stays in the membrane

• IP3 is hydrophillic so it doesn’t stay in the membrane and goes to the endoplasmic reticulum

what does IP3 do at the endoplasmic reticulum

• it binds calcium channels and activates calcium release>increased intracellular calcium conc

what does DAG do in the membrane

activates PKC

what are the types of lipid modification

• myristoylation

• palmitoylation

• isoprenylation

• glycosylphosphatidylinositol - GPI

what is myristoylation

• 14 carbon chain derived frrom myristic acid gets attached to an N terminal glycine

what does myristoylation require to occur

enzyme

N-myristoyltransferase

where does myristoylation occur

in the cytoplasm

what does myristoylation mediate

• membrane association

• and protein-protein interactions

what is palmitoylation

• 16 carbon chain gets attached to a cysteine residue

what is the chain attached via in palmitoylation

thioester linkage

describe the stability of the linkage in palmitoylation and the purpose of this

• it is not a very stable linkage

• therefore it can be cleaved by thioesterases

• this allows regulated membrane association - can come on and off

where do we usually see palmitoylation

• almost exclusively at the cytoplasmic membrane interface

what psot translational modfiications does alpha subunit have

palmitoylation and myristoylation

what is isoprenylation

geranyl group gets attached to c-terminus

what does isoprenylation

geranyl transferase enzyme

what kind of linkage do we see with isoprenylation and describe the stability of the linkage

• thioether linkage

• relatively stable

• more than palmitoylation

what does the enzyme that does isoprenylation recognise

CAAX box at the C terminus

where is isoprenylation usually seen

intracellular membrane or cytoplasmic face of plasma membrane

what kind of post translational modficiations do we see on gamma sub unit

isoprenylation

describe GPI

• anchors extracellular proteins onto membrane

what is a domain

structural and functional region within a protein

what can a multidomain protein contain

• catalytic domains

• bindign domains

what are the domains of PLC

• phospholipase

• PH

• C2

what does the phospholipase domain do

• cleaves PIP2

• catalytic domain

what is PH in PLC

• plekstrin homolgy

• has alpha and beta structures

• contains a postively charged binding pocket which binds to negatively charged phosphate containing groups like phosphoinositides

• PH domains can help target proteins to membranes containing particular phosphoinositides

• helps PLC bind the membrane

what does C2 do in PLC

• binds phosphotidylserine - membrane lipid

what domains does PKC have

• kinase - catalytic domain - phosphorylates stuff

• C1

• C2

what does c2 domain do in PKC

• The C2 domain shown has an eight-stranded beta-sandwich structure and loops that bind calcium.

• Calcium supports interaction with negatively charged membrane lipids, including phosphatidylserines - which is in the membrane - encourages membrane binding

• so it brings PKC to the membrane

what does C1 do in PKC

• it has an extremely hydrophobic pocket

• and DAG is very hydrophobic

• so it enables DAG binding

• allows PKC to bind DAG

how do all the domains aid in the activation of PKC

• calcium ions released due to IP3 binding ER binds to the c2 domains in PKC

• C2 domains are calcium dependant binders - they can then bind to phosphotidal serine in the membrane > bringing PKC to the membrane

• So now the PKC is bound to the membrane where the c1 domain binds DAG (which is also in the membrane)