Neuronal and Cellular Signalling Lecture 7 Notes: Second Messengers Part 2 Small GTPases - Small GTPases are simple, single proteins which bind GTP. GTPases are also known as monomeric G-proteins, Rho proteins or Ras family proteins. Unlike HGPs, monomeric GTPases can hydrolyse GTP to GDP (i.e. have intrinsic GTP hydrolysis activity). 1 GTPase activity is regulated by GTPase activating proteins (GAP) to hydrolyse GTP (inactivation), as well as guanine nucleotide exchange factors (GEF) which activate GTPases. -Chemical signaling molecule Receptor 3TP GDP Active Ra Ras GDE GTP - GEF and GAP activity can be regulated by external or internal signalling. Guanosine nucleotide dissociate inhibitors (GDI) lock the protein in an inactive state. Inactive GAP - An extracellular signal binds to and activates a TM receptor. The receptor activates GEF, which swaps GDP for GTP, thus activating GTPase. GAP proteins will enhance hydrolysis of GTP to GDP, inhibiting GTPase activity. - Small GTPase/Ras/Rho/monomeric G-protein superfamily comprises over 150 mammalian proteins in several families. Family members all function via cycling between inactive GDP- bound and active GTP-bound forms. Members include: (a) Ras (rat sarcoma, oncogene) (b) Rho (c) Rac (d) Cdc42 (e) Arf (f) Rap - Many small GTPase structure . Growth factor LPA ·- proteins function in cell regulation. For example, an Cdc GTP 42 extracellular growth factor can bind to a TM receptor, activating Rac GTP Rho GTP Active ("on") GTPase Rho kinase Formin ParB WASP WAVE Activator protein Inactivator protein Myosin LC phosphatase 4 GEF Guanine nucleotide exchange factor) -GTP GDF P GAP Polarity Arp2/3 (GTPase accelerating protein) Arp2/3 a GTPase Myosin LC-P 1 Inactive ("off"} (e.g. Cdc42 or Rac) by swapping the GDP for GTP. This then functions to Actin Actin Myosin activity Actin polymerization polymerization polymerization P: 1 1 Filopodia formation Lamellipodia formation Stress fiber formation and contraction D P modulate the polarity of cells and the polymerisation of actin. For example, in a moving cell this stimulates actin polymerisation for filopodium formation. Alternatively, it may activate a Rac protein for actin polymerisation to aid lamellipodia formation. Finally, Rho protein may be activated leading to the activation of Rho kinase (forms myosin) and Formin (actin polymerisation) which is thought to move the front of the cell forward (by pushing out actin) whilst the back of the cell is pulled up to maintain cell volume (via myosin activity). - Small GTPases regulate many cellular functions, family member functions include:
(a) Ras (regulates cell growth, proliferation and differentiation) (b) Rho (actin cytoskeleton, cell proliferation) (c) Rab (intracellular vesicle trafficking) (d) Ran (regulates transport between nucleus/cytoplasm, S and M phase transitions) (e) Arf (activation of PLD, vesicle formation in trafficking and endocytosis) (f) Kir/Rem/Rad (cytoskeleton, cell shape, voltage-dependent channel regulation) Mutant forms (e.g. cannot release GTP) of small GTPases are used to investigate cellular function. For example, if Rho proteins are activated this results in large actin structures within the cytoskeleton. If Cdc42 is made permanently active, this results in filopodia formation. Finally, if Rac is permanently active, lamellipodia form