path(ways) The to enlightenment: understanding intracellular signaling
Announcements
Quiz 9 will come out today and will be due next Wednesday at noon. It will cover genomics, proteomics, and cell trafficking (midway through Mondays lecture). Clear G-protein animation at:
http://www.youtube.com/watch?v=V_0EcUr_txk
Nuthin but a G thang: G-protein coupled receptors
1000s of G-protein coupled receptors (GPCRs) identified 7 transmembrane domain proteins with extracellular receptor coupled to trimeric (3 subunit) G-protein in the membrane Ligand (first messenger) binds within three extracellular loops Activation of G-protein by the activated GPCR eventually acts on effectors, which generate intracellular second messengers that potentially initiate multiple signaling pathways (signal cascades)
Ligand binding to the GPCR shifts the receptor conformation
Once the ligand is bound, the affinity for the G-protein increases At this point the G-protein has a bound GDP. The G-protein is bound to a trimeric complex consisting of alpha, beta, and gamma subunits Once bound to the receptor, GTP displaces GDP. A single activated receptor can activate many GDP-G-protein complexes, which can amplify a signal
With GTP bound, the alpha subunit dissociates and moves in the membrane to an effector molecule
Adenylyl cyclase
The alpha and beta/gamma subunits break into 2 Subunits are held in membrane by covalent linkages to fatty acids Alpha subunit (Galpha) +GTP move to effector, which is adenylyl cylclase in this example Gbetagamma can also bind to a separate effector in some cases
ATP alteration creates a second messenger
Adenylyl cycles converts ATP to cyclic AMP (cAMP) cAMP is a common second messenger While the Galpha unit is bound to adenylyl cyclase, it will continue to form cAMP, which is another amplification step
GTP eventually reverts back to GDP
GTP hydrolysis usually occurs by a weak auto-catalysis This terminates formation of cAMP
GDP+Galpha then dissociates from adenylyl cyclase and reforms with the Gbetagamma
G-protein receptor kinases (GRK) are serinethreonine kinases that desensitize GPCRs
Kinases are a major class of proteins that phosphorylate molecules, often via ATP hydrolysis GRKs use ATP to phosphorylate the activated receptor. Desensitization is the cessation of the receptor response despite having bound ligand
Arrestin binds to the two phosphates and prevents G-protein activation
Arrestin prevents association of G-proteins with the activated receptor, so the G-protein cant be activated by GTP. Arrestin bound receptors are often removed from the membrane by endocytosis. This is not an obligatory part of the cycle.
Summary of G-protein signaling steps for adenylyl cyclase/cAMP pathway
1) Ligand binding to receptor causes conformational changes that increase affinity for G-protein 2) G-protein binds to receptor 3) GTP replaces GDP in G-protein 4) G-protein, with GTP, dissociates from the receptor and splits into a Galpha subunit and a Gbetagamma subunit 5) Galpha moves in membrane to an effector, such as adenylyl cyclase 6) Adenylyl cyclase converts ATP to cAMP, which is the second messenger in this pathway 7) Galpha is weakly autocatalytic and hydrolyzes GTP to GDP+P 8) The full trimeric G-protein reforms with GDP 9) Two amplification steps: multiple activated G-proteins can be created while a ligand is bound and multiple cAMP are created for each activated G-protein 10) Desensitization can occur when GRKs phosphorylate the bound GPCR and allow Arrestin to bind, preventing G-protein association
Further regulation of the G-protein cycle
Guanosine dissociation inhibitor (GDI) can hold G-protein inactive Guanine exchange factors (GEF) facilitate the GDP to GTP switch The time period of the auto-GTPase activity can be regulated by GTPase activating protein (GAP) to form a complex with the Gprotein and target protein
Second messengers
Activated by effector that has been activated by first messenger (ligand) or the transduction of the ligand binding, such as activation of a G-protein by a ligand-activated receptor. Examples include: cAMP, Ca++, Phosphoinositides, such as PIP2 Inositol trisphosphate (IP3) Diacylgycerol (DAG) Cyclic GMP (cGMP, mostly in vision) Nitric oxide
http://www.youtube.com/watch?v=iGb93jCKVXs&feature=related kinase Protein A (PKA) is a heterotetramer, with two catalytic subunits and two regulatory subunits cAMP binding to the regulatory subunits releases the PKA catalytic subunits, which then go to phosphorylate multiple cellular targets.
Many processes are influenced by [cAMP] activating protein kinase A (PKA)
The same receptor and beginning of the pathway (cAMP formation and PKA activation) can have different downstream effects depending on cell type
Sensitization of the gill withdrawal reflex reveals three phases of memory
Non-associative memory Light touch evokes a small withdrawal. A single shock will sensitize the gill withdrawal to a touch for about 1 hour 4 shock will sensitize the withdrawal for 1 day, and 4 sets of 4 shocks for 4 days will sensitize the withdrawal for weeks This gives 3 different time windows for sensitization that have distinct molecular mechanisms Kandel et al. 2001
Tail shock causes serotonin (5HT) release and activates excitatory interneurons that modulate withdrawal
In this example, serotonin (5HT) was directly applied to the sensory neuron Figure shows temporal spread of [cAMP], assessed indirectly through fluorescent labeling of PKA subunits with two different fluorescent markers whose proximity was measured by FRET. Despite continued presence of 5HT, [cAMP] reduced due to phosphodiesterase activity that degrades cAMP to AMP
Steps in short-term sensitization in Aplysia
5HT release activates G-protein coupled 5HT receptors Activated Gs forms cAMP cAMP binds to regulatory PKA subunits, allowing catalytic PKA to act PKA phosphorylates K channels, leading to increased transmitter release and a stronger withdrawal
Repeated 5HT stimulation allows PKA to go to nucleus and activates CREB that allows formation of a persistent PKA to maintain sensisitization for hours instead of minutes
Kandel et al. 2001
Activated G-proteins or other paths can activate kinases that phosphorylate phospholipids
PI DAG
Four main types of phospholipases that cleave fatty acids at the ester bonds linking the different components, of which phospholipase C (PLC) is the best studied A kinase often phosphorylates the phospholipid to activate it. PLC cleaves phosphoinositol 4,5 bisphosphate (PIP2) into diacylglycerol (DAG) and inositol triphosphate (IP3)
Phosphoinositide signaling path, part 1
Two kinases regulate the formation of phosphoinositide-4,5bisphosphate (PIP2, step 2 above) In the above example, ligand-binding activates a GPCR. The activated Galpha then binds to phospholipase-C-beta (PLCbeta) and allows it to become functional
Phosphoinositide signaling path, part 2
PLC , on cytoplasmic membrane, cleaves PIP2 to make DAG and IP3 DAG activates protein kinase C (PKC) IP3 binds to smooth endoplasmic reticulum (ER), which causes Ca release from ER through a channel
Fertilization of an egg (oocyte) induces a calcium wave by release from IP3 sensitive Ca channels
Example shown is from a flatworm oocyte, but very similar in mammals Arrow is where sperm fertilizes egg An initial Ca flash occurs due to calcium influx near fertilization site. This triggers the onset of a Ca wave due to release from IP3 sensitive channels Calcium can also be used as an indirect measure of neural excitation
Stricker 1999
Protein-tyrosine kinases are another main class of intracellular signaling pathway
>90 PTK in human genome Receptor binding induces receptor dimerization Either one ligand can bind to two receptors and they dimerize (left) Or two ligands bind to change receptors and they dimerize (rt.)
Protein tyrosine kinases, part 2
Trans-autophosphorylation means that one subunit will phosphorylate the other subunit in the dimer The phosphorylated receptors will now bind to target proteins with SH2 or PTB domains and activate them Growth factors such as epidermal growth factor and platelet derived growth factor use protein tyrosine kinases
Youve activated the receptor, now what? Different signaling proteins
a) b) c) d)
Adaptor proteins: link signaling proteins with SH2 domain Docking proteins: can add tyrosine phosphorylation sites Transcription factors: Activated factor goes to nucleus to regulate genes (STAT protein shown in example, involved in immune function) Other signaling enzymes, such as PLC-gamma, PI3K, phosphatase