Lecture 13 - Intracellular signalling II: Heterotrimeric G-proteins and Small GTPases - G-proteins = Guanosine nucleotide-binding proteins ATP - Functions: 'molecular switches' > On/ Off Switches O - Activity regulated by binding and hydrolysis o G proteins ffunction by GTP binding O O NaO -0 ONa NHÀ GTP 0 OH O Naj NH 0 OH GTP + protein Two Types: 1. Heterotrimeric G- Proteins o Three different subunits o G- alpha (binds guanosine nucleotide), G- beta and G- Gamma 2. Small GTPases o 1 amino acid sequence > one protein Heterotrimeric G proteins - Key signal relay ffor a large ffamily off serpetine receptors (G protein coupled receptors) - 7 transmembrane protein where the N terminal is on the exterior and is a ligand binding domain - Contain intracellular loops > C3 - C-Terminal is inside the cytosol How do they function? Exterior Cytosol GTP - protein NH2+ E1 E2 E3 E4 H1 H2 H3 H4 H5 H6 H7 COO C1 C2 C4 C3 G protein interaction 1. Start with : hormone, inactive receptor, inactive G protein bound to GDP and inactive effector 2. G protein complex receptor is inactive as it is detached ffrom G protein-coupled receptor 3. Extracellular signal binds to G- protein channel receptor -> activates the receptor and opens a binding site ffor alpha subunit on G protein complex 4. GDP is released ffrom alpha subunit > replaced by GTP 5. Whole G- Protein complex dissociates ffrom receptor and individual components separate o Beta and gamma subunit dissociate ffrom alpha o GTP-alpha subunit and beta-gamma complex act as secondary messengers 6. Beta-gamma complex activates an ion channel 7. Alpha- GTP activates an effector which is an enzyme 8. Finally, GTP hydrolyses into GDP + Phosphate molecule Acetylcholine K+ channel Properties: Exterior - Heterotrimers made up off: Cytosol o a-subunit off 40-45 KDa G Active muscarinic acetylcholine receptor P o B-subunit off ~35 KDa o y-subunit off 7 KDa - Ga-subunits are post-translationally modiffied by myristoylated at N-terminal glycine residue - GBy-subunits are post-translationally modiffied by geranyl /ffarnesyl side chain GDP GTP K' + + + - + + 3 GBy
Location and activation - Located at the cytoplasmic fface off the plasma membrane - Anchored to the membrane by a covalenty attached lipid anchor Ga-subunits - Post-translationally modiffied o myristoylation at N-terminal glycine residue Gßy-subunits - Post-translationally modiffied o geranyl/ffarnesyl side chain Heterotrimetric G- Protein binding to GPCRs (G- Protein chanel S H 0 O Hormone receptors) CH2 O Protein Trimeric Gs protein - Heterotrimeric G-proteins bind to GPCRs at a speciffic region - TM5, TM6, C3 and C4 important Exterior Inactive receptor Cytosol 3 Gy GB RESTING STATE Down-stream effectors off Heterotrimeric G- proteins Gaz G D P - G proteins trigger several second messenger systems: - CAMP, cGMP, Protein Kinase C, opens calcium channels in the endoplasmic reticulum NH 0 N 2 N NH 5 N 0-CH2 5 O-CH, PO 2 N Nº NH2 CH3 -(CH2), -C-O-CH2 OPO 2- 0 16 0 2 CH3-(CH2),-C-O-CH