Lecture 5 - GPCR signalling Flashcards
What are the major types of cell surface receptor that initiate signalling cascades?
G protein linked receptors and enzyme linked receptors
Activated by diverse range of extracellular ligands
Describe how conformational flexibility of the β-adrenergic receptor changes upon ligand binding
Ligand binding closes the space at the extracellular side between transmembrane helices 3, 5 and 6 , and thereby forces them apart at the cytosolic side. This induces a conformational change in the 5/6 loop prompting signal transduction.
Describe the G protein cycle
G-proteins bind tightly to their nucleotide co-factor – need a GEF (guanine nucleotide exchange factor) to release the GDP. GTP can then spontaneously bind due to its higher concentration in the cell. Nucleotide exchange taking place not phosphorylation
G-proteins are GTPases – GTP hydrolysing enzymes – with extremely low activity. A helper protein called a GTPase activating protein is needed for productive GTP hydrolysis.
G proteins are really bad GTPases
When the G protein is active in the GTP bound form it could hydrolyse the GTP but it is a bad enyzme and this would take too long physiologically - need a GTPase activating protein (GAP). Phosphate released
Describe how G-proteins form a transient protein complex with the GPCR then an effector - signal handover
After the Ga subunit receives GTP while activated by a GPCR, it can bind adenylyl cyclase. Upon GTP hydrolysis the transient Ga-adenylyl cyclase interaction is lost.
GPCR= GEF
AC=GAP
GPCR is nucleotide exchange factor so when activated will bind trimeric G protein and exchange GDP for GTP in the G@ subunit. Once this exchange has occurred, the trimeric G protein then falls apart. G@ binds adenylyl cyclase, which acts as a GAP. This is negative feedback - once activated inactivated almost immediately which helps to ensure you only signal when the GPCR is activated. Signalling only takes place when the outside stimulus is active. Once the outside stimulus is not there anymore, the trimeric G protein has been inactivated by its target adenylyl cyclase and it cannot be activated because the GPCR is not in its active state any longer. This all happens on the surface of the membrane allowing the proteins to bind each other much more easily.
What are second messengers?
Small molecules that are produced or released upon activation and can themselves activate downstream targets.
They generally amplify a signalling cascade.
Their localized production and constant destruction ensures a localized target response.
How were second messengers discovered?
Sutherland Nobel Prize 1971
Used system of glycogen biosynthesis - glycogen phosphorylase activity - which degrades glycogen
Fractionated liver tissue and separated cytoplasm and membrane. Membrane contains G-coupled receptor, G protein and adenylyl cyclase. Cytosol contains glycogen phosphorylase. Adrenaline added to membranes which will start to produce cyclic AMP because they have everything they need to produce it (G-coupled receptor, G proteins and adenylyl cyclase). Won’t activate glycogen phosphorylase because there isn’t any. Now you can take the membrane that was treated with adrenaline, remove the membrane fraction and just take the soup that is left behind, which will contain cyclic AMP. Mix that with the cytosol, which doesn’t have the receptor or adenylyl cyclase and it will activate the glycogen phosphorylase. This proves the membranes produce some type of small molecule.
How does cAMP signalling affect PKA?
cAMP binding releases PKA’s catalytic subunit and allows it to phosphorylate targets such as some nuclear receptors, CREB (cAMP responsive element binding protein), a Ca2+ channel in the ER amongst others.
There are 3 isoforms of the catalytic and 4 of the regulatory subunits.
Doesn’t just directly activate glycogen phosphorylase, there are intermediate steps:
1. Activates protein kinase A
Called PKA because it is activated by cAMP
2. Activates PKA by binding to the regulatory subunits and moving them, releasing catalytic subunits
Describe the effects of cAMP on glycogen degradation
PKA activation leads to:
Activation of glycogen phosphorylase kinase activating glycogen phosphorylase and inducing glycogen breakdown AND
Inactivation of the phosphatase that would otherwise inactivate the kinase and glycogen phosphorylase itself.
PKA inactivation leads to:
Activation of the phosphatase that leads to inactivation of glycogen phosphorylase
When adrenaline activates the G protein, adenylyl cyclase and cAMP is made, PKA is activated - PKA activates several different proteins that are important for glycogen degradation. PKA activates glycogen phosophorylase by phosphorylating a kinase called glycogen phosphorylase kinase which then phosyphorylates glycogen phosphorylase. PKA also activates a small protein which is an inhibitor protein for a protein phosphatase. This is important because protein phosphatase is the enzyme that removes the phosphate group and inactivates glycogen phosphorylase.
Describe the effects of PKA on glycogen synthesis
PKA activation leads to:
Inactivation of glycogen synthase through its phosphorylation
PKA inactivation leads to:
Activation of the phosphatase that dephosphorylates and thereby activates of glycogen synthase
Simultaneous activation of synthesis and inhibition of degradation or vice versa ensures tightened control of the selected metabolic step
PKA phosphorylates glycogen synthase. In this case, the phosphorylated form of glycogen synthase is inactive and the dephosphorylated form is active. Adrenaline needs glucose so will activate PKA to inactivate glycogen synthase. Once PKA is inactive protein phosphatase will dephosphorylate the glycogen synthase and glycogen synthesis can take place.
How are calcium ions used as a second messenger?
Ca2+ channels in the plasma membrane (CRAC, P2X) and the ER (IP3R) when activated provide a local increase in cytosolic Ca2+.
The ER Ca2+ pump (SERCA) constantly removes excess cytosolic Ca2+ into the ER.
Note: cytosolic resting [Ca2+] is 10.000-fold lower than the concentration in the ER or outside the cell. Active Ca2+ only requires a ~10-fold increase of this.
The way that calcium ions are used as a second messenger is that they are always in very low concentration in the cytosol much higher outside. This is maintained by calcium ATPases which constantly pump calcium ions out of the cell - enzymes with very high affinity for calcium ions. When the cell wants to use calcium ions as a second messenger it will open up a calcium channel creating local increase in calcium ion concentration
How is calmodulin activated?
Cytosolic Ca2+ concentration is kept low at steady state.
Various signals can trigger a local increase in Ca2+ concentration.
The increase in Ca2+ concentration activates calmodulin by inducing a large conformational change.
Calmodulin in turn activates target proteins.
Calmodulin is one of the main respondents to calcium ions. Undergoes a large conformational change when it binds calcium ions which helps it bind a kinase. This induces the activation of calmodulin, which is a kinase itself, which can go on to phosphorylate various targets.
