L8: Rho GTPases in cell migration

what are small GTPases

small 21kDa proteins

state the significance of the ras superfamily

one of the largest groups of signalling proteins

name the divisions of the ras superfamily

Ras

Rab

Rho

Arf

what does GTPase signalling depend upon

bound nucleotide

describe cyclical regulation of GTPases

GDI regulates GDP→GTP exchange reactions by slowing the rate of exchange

GEF facilitate the exchange of GDP→GTP

GAP accelerates hydrolysis of GTP→GDP to turn off signalling pathways

describe the structure of a G protein

2 switch regions to bind effectors

P-loop to bind phosphate

Mg²⁺ essential for nucleotide binding

what is the most effective way of monitoring GTPase ability

monitoring effector binding

how does a small GTPase catalyse GTP hydrolysis

glutamine brings in water molecule + holds it in the correct position

also counteracting of -ve charge at phosphates via the P loop (lysine residues from H bonds w/ phosphates)

name a mutation in a small GTPase and explain how this effects hydrolysis

Q61L (glutamine → leucine) - water molecule not brought in and hydrolysis cant occur - GTPase constitutively on

or

G12V (glycine → valine) - pushes Q61 (glutamine) out of position so water molecule brought into the wrong place + hydrolysis cant occur - GTPase constitutively on 

how do GAPs activate hydrolysis in small GTPases

Arg residue +vely charged so offsets some -ve charge to facillitate hydrolosis

Arg also stabilises glutamine to stabilise water

GAPs stabilise position of GTPase to ensure Mg2+ in correct position + stabilises water

how do GEFs accelerate exchange of GDP for GTP

binds to GTPase + induces conformational change in nucleotide-binding site - specifically sidrupting Mg2+ ion coordination to destabilise GDP binding

how do GEF mutants affect their usual function

prevent binding to GTPase so nonfunctional - dominant negative mutation

how many families of GEFs are there

70

how is RhoA involved in actin-based motility

drives stress fibre assembly

how is Cdc42 involved in Actin based motility

involved in actin branching and organisation, and regulation of filopodium assembly

how is Rac1 involved in actin-based motility

regulates lamellipodium assembly

describe RhoA downstream activation of myosin

RhoA-GTP activates Rho kinase which phosphorylates the myosin light chain to cause actomyosin contraction

what kind of signals are Cdc42 + Rac

protrusive signals

what kind of signal is RhoA

contractile signals

how do Cdc42/Rac + RhoA coordinate 

suppression of RhoA during protrusion to ensure directional movement

how is migration guided

fibres provide migrational cues + localised signals required