Genetics 8 Flashcards
Secondary Messengers
Soluble molecules that relay signals from activated receptors on the cell surface to target molecules inside the cell cytoplasm or nucleus.
Ex: cAMP, IP3, Ca2+
Primary Messenger
Extracellular molecule (like a NT or hormone) that binds as a ligand to cell surface receptors to transduce the signal inside of the cell
Effector
Molecule or protein that selectively binds to a protein that regulates its biological activity.
- The enzyme that produces the secondary messengers
- Ex: AC makes cAMP
Multifunctional protein kinase
Kinases with more than one binding domain and more than one phosphorylation site.
-ex: cAMP-dependent protein kinase, Calicum/calmodulin protein kinase
Autophosphorylation
Phosphorylates itself and for the most part, activates the kinase.
ex) Cam Kinase is fully active once it has autophosphorylated, which it is activated to do when Calcium/calmodulin binds to it.
Phosphatase
Opposite function of kinase
-Dephosphorylates molecules.
Trimeric G Proteins
Couple a receptor and an effector protein
- Both hormone/ligand and GTP are required for AC stimulation.
- Receptor agonists stimulate a low Km GTPase
- GDP and GTP modulate affinity of receptor to agonists, but not to antagonists (negative heterotropic allosteric interaction): part of cascade’s off mechanism.
Agonist binding to receptor
Causes proper signaling pathway for an active downstream enzyme
T/F, Antagonists occupy receptors, but do not yield a signal? Why or why not?
True, do not yield signal because they do not induce a conformational change that dissociates G protein’s alpha subunit.
Why is the low Km GTPase important?
Having a low Km means half the Vmax is reached with small concentration of GTP.
-GTPase can hydrolyze GTP at low concentrations within the cytosol, which helps GTP bind when GDP dissociates from the alpha subunit.
Alpha Subunit of Trimeric G protein
- Ga-GTPase: main GTPase function
- Dissociates from beta-gamma subunits when activated by receptor protein.
- 9 alpha subunit genes encode 12 different proteins
- Contains guanine binding site (GDP, GTP bind)
Beta/Gamma subunit of Trimeric G protein
- Stay associated together
- Are involved in separate signaling pathway, impact other effector molecules.
What happens to the alpha subunit when the receptor binds a ligand?
- Undergoes conformational change
- GDP it was holding onto dissociates
- low Km GTPase part of alpha grabs nearest GTP.
What is the rate limiting step of G proteins?
- The dissociation of GDP from the alpha subunit.
- Provides key regulatory feature for activation of alpha subunit.
Kinetics of GTPase activity
- In absence of ligand, kcat is 10 times faster than dissociation rate of GDP.
- This means that by default, the G protein is off (in its T form) most of the time.
Gs
- Member of As subunit.
- Activates AC
- Tissue specific types of Gold and Ggust for sensory tissues.
Go
- important in neurons with the activation of K+ channels.
- Most abundant in G protein subunit
Gq
-Subunit to activate PLC-beta= phospholipase C-Beta
Describe basic G protein Pathway from membrane receptor through generation of a 2nd messenger
- Trimeric G protein is associated with pm, GDP bound (= inactive form)
- Extracellular ligand binds to membrane receptor, causes conformational change within transmembrane receptor. H+R–> HR= activated
- Activated HR complex causes conformational changes in trimeric G protein for GDP to dissociate and GTP to bind to Gas.
- GTP binding dissociates Gas from HR and Gbetagamma. Hr–> T state, dissociates from ligand.
- Gas-GTPase travels and binds to effector (AC). Activates AC to produce cAMP
- Hydrolysis of Gas bound GTP inactivates AC.
- Inactive Gas dissociates from AC, reassociates with Gbetagamma subunits.
Intracellular concentration of GTP is _____ compared to GDP
High
-GTP is more likely to associate with Gas
What does GTP binding to Gas do?
1) Causes dissociation of G protein from Gbetagamma, to allow it to bind to the effector for the second messenger creation
2) Brings HR to T state to dissociate from ligand. Allows for cascade to be controlled based on presence of more ligands.
- More ligand to bind to the receptor? Transmits signal to continue activity.
- No more ligand? Receptor won’t be activated, cascade will stop.
What activates AC activity?
Gas-GTPase going all the way back to the activated HR
PKA
- Tetramer: 2 regulatory subunits and 2 catalytic subunits= inactive
- Regulatory subunits each have 2 cAMP binding sites, when cAMP binds, they dissociate from the catalytic subunits
- Peptide inhibitory site: has sequence that mimics protein to be activated by PKA
Functional significance of a pseudo-substrate site on a regulatory subunit or domain
- 2 types of regulatory subunit proteins, differ in how well they mimic the substrate protein of the catalytic subunit.
- Type I: Pseudo P site- occupies and inhibits, but is not substrate.
- Type II: Auto P site- occupies catalytic site, is phosphorylated (an exact match to the site). Stays bound to inhibit until removed by cAMP binding.
Peptide binding and catalytic regions mainly phosphorylate….
Serine or Threonine
Phospholipase C Beta activated by?
Gq
Beta of PLC is specific to cleaving?
PIP2, which can be cleaved into DAG, IP3, or Arachidonic Acid/Prostaglandin
DAG-Protein Kinase C Pathway
DAG cleaved off by PLPc (other half is IP3)
- Stays attached to membrane as transmembrane lipid
- Once cleaved, it goes along the membrane to activate PKC
IP3-Ca2+ Pathway
IP3 cleaved off by PLPc.
- Becomes intracellular soluble molecule, very hydrophilic.
- Once cleaved, binds IP3 dependent Ca2+ channels in ER to release Ca2+ into cytosol.
PLPcbeta
-Gaq coupled PLC. Subtype specific for PIP2 cleavage from intracellular PM
PLC-gamma
Activated by RPTK part of tyrosine kinase cascade
PKC pathway of activation
1) PLC-beta cleaves PIP2 into DAG and IP3
2) DAG stays in membrane
3) IP3= soluble, binds Ca2+ in ER
4) Ca2+ released in cytosol
5) Ca2+ binds PKC in cytosol, increases its affinity to bind DAG in PM
6) Binding of Ca2+ and DAG to PKC activate it.
Ca2+ pumps in the PM
1) Na+ driven Ca2+ exchanger: antiporter pumping Ca2+ out of the cell for every Na+
2) Ca2+ pump: ATPase pumping Ca2+ out of cell.
Ca2+ pumps in organelle membrane
- Keep Ca2+ out of cytosol, but high in organelle as a storage place
1) Ca2+ pump: ATPase pumps Ca2+ out of cytosol, into ER
2) Active Ca2+ importer in mit.
3) Ca2+ binding molecules in cytoplasm sequester Ca2+
What are the sites of IP3 dependent Ca2+ Channel in ER membrane?
1) Low km activating site for Ca2+
2) High km inhibitory site for Ca2+ (once it has been opened, or if cytoplasmic Ca2+ gets too high, Ca2+ binding will close the channel to stop the signal).
AC is activated by what subunit?
Gas
