L7 - Rho GTPases in cell migration (Rho family of small GTPases)

what are small GTPases

• small proteins

• members of one of the largest groups of signalling proteins: Ras superfamily

• they change conformation once activated

• they bind to and activate downstream effectors

what does the activity state of small GTPases depend on?

• GTPase signalling depends on the bound nucleotide

what is GTP?

• guanosine triphosphate (GTP)

• nucleotide

what is the basic structure of GTP?

• guanine base

• ribose sugar

• three phosphate groups - alpha (a), beta (B), gamma (y)

GTP = Guanine + Ribose (Guanosine) + 3 phosphatases

what happens during GTP hydrolysis?

• the high energy phosphate bonds (especially beta-gamma) are hydrolysed during GTPase activity

• this hydrolysis of GTP → GDP + Pi turns GTP binding proteins from active to inactive

• the gamma phosphate is cleaved off (GTP + Pi)

cyclic regulation of GTPases - how GTPases are activated and inactivated

1. inactive state (OFF) - GTPase bound to GDP

2. activation - a GEF (Guanine nucleotide exchange factor) helps GTPase release GDP and bind GTP

3. active state (ON) - GTPase bound to GTP - can now interact with and activate downstream effector proteins

4. inactivation - a GAP (GTPase activating protein) speeds up GTP hydrolysis - GTP → GDP + Pi

   —> GTPase returns to inactive GDP-bound state

what are the two structural features of small GTPases?

1. p-loop

2. switch regions (interact with effectors)

what is the P-loop of small GTPases?

• phosphate binding loop

• coordinates the phosphate groups of GTP/GDP

• requires Mg2+ (magnesium ions) to stabilise the nucleotide binding

What are the switch regions of small GTPases?

• switch 1 and switch 2

• these regions change shape depending on whether GTP or GDP is bound

• GTP-bound state - they form correct shape to bind to effector proteins

• GDP-bound state - switch changes shape and release effector - turning signal off

what is catalysis of GTP hydrolysis?

• the process by which an enzyme speeds up the chemical reaction that breaks down GTP to GDP

What does catalysis of GTP hydrolysis involve in order to work?

• a catalytic glutamine (Q61 - glutamine amino acid) helps position the water molecule

• this is necessary to hydrolyse GTP

what happens if there’s a mutation is Q61 (or G12V)?

• it will disrupt the positioning

• the enzyme cannot break down GTP

• the GTPase will remain active for too long

What role does the negative charge of GTP play in GTP hydrolysis?

• GTP has many negative charges from its phosphate groups which are stabilised by: the p-loop which forms hydrogen bonds and lysine amino acid

• this ensures GTP fits correctly and allows hydrolysis into GDP

what are GAPs?

GAPs - GTPase Activating Protein

what do GAPs do?

• these proteins help speed up hydrolysis of GTP to GDP on small GTPases

• they switch off signalling as they switch GTPases from active state (GTP bound) to inactive state (GDP bound)

  —> turning OFF signalling

what are GEFs?

GEFs = Guanine Nucleotide Exchange Factors

what do GEFs do?

• they accelerate exchange of GDP for GTP

• small GTPases now GTP bound (active state)

• stabilises nucleotide form - stabilise GTPase in a state without GDP and without Mg2+ = making it easier for GTP to bind

what’s the T17N mutant?

• dominant negative mutant which traps the GTPase in an inactive form

what are GEF domains?

• specific domains on GEF proteins responsible for helping GTPases swap GDP for GTP

what are the 3 major families of GEFs?

• Dbl-homology domain

• Dock-family

• Sec7 domain

what are the 3 key members of the Rho family?

• RhoA, Rac1, Cdc42

• balance between them is vital

what do the members if the Rho family (RhoA, Rac1, Cdc42) do?

• they are small GTPases that regulate actin organisation (actin based motility)

• they control all components of cell migration

explain an example of how Rho causes contractions through interaction with downstream effectors:

1. active RhoA (GTP-RhoA) will bind to its effector Rho kinase

2. this will phosphorylate the myosin light chain

3. this will drive the actomyosin contraction

what type of signals are Cdc42 and Rac1?

• protrusive signals (pushing out)

• promote actin polymerisation at front of the cell

• from filopodia (Cdc42) and lamellipodia (Rac1)

• they push membrane forward - used to explore environment and start movement

what type of signal is RhoA?

• contractile signal (pulling in)

• promotes stress fiber formation

• activated myosin II for rear contraction

• retraction

what are the 2 types of cell migration environments?

2D and 3D

2D cell migration environment

• random movement

• flat surface

• fast movement

  (don’t go where they need to)

3D cell migration environment

• directional movement

• integrated - cells respond to multiple cues

• slower movement

  (goes where it needs to be)

what is the freedom of cells restricted by?

• restricted by regulatory systems

• to ensure cells behave correctly and don’t cause harm

what does increasing Rac1 activity do?

• changes migration rate/speed

• but it depends on the context/situation

what does migration require?

localised signals

• signals activate specific pathways only at certain parts of the cell

• ensures directional movement rather than random movement