Lecture 2 (G-Protein-Coupled Receptors)
Describe the Fight or Flight Response:
(1) When frightened, the adrenal gland releases adrenaline. This acts as a signaling molecule and binds to G-Protein-Coupled Receptor called an adrenergic receptor. (2) The G-Protein Coupled Receptor turns on a G protein (stimulatory G-Protein "Gs"), which activates adenylyl cyclase. (3) Adenylyl cyclase converts ATP into cyclic AMP. (4) Increased cyclic AMP activates PKA. (5) Activated PKA uses hydrolysis of ATP to ADP to phosphorylate and therefore activate phosphorylase kinase. (6) Activated phosphorylase kinase uses hydrolysis of ATP to ADP to phosphorylate and therefore activate glycogen phosphorylase. (7) Active glycogen phosphorylase breaks down glycogen (which is the polymerized storage form of glucose). This increased glucose is fuel for muscles. Note: different responses to this signaling pathway in different tissues - all prepare the body for action.
What does the second messenger cCAMP activate?
-cCAMP binds to and activates protein kinase A (PKA) (Called "PKA" because it is dependent on cyclic AMP) -PKA in the inactive form consists of two catalytic subunits and two regulatory subunits. cCAMP binds at the two regulatory subunits, altering their conformation, allowing for the liberation of the two catalytic subunits. These catalyic subunits are now active. PKA then phosphorlylates specific target proteins (NOTE: PKA can phosphorylate multiple target proteins).
How is a G-Protein "Switched Off"?
A G-Protein can switch itself off by hydrolyzing GTP to GDP. This reaction can be catalyzed by the help of an RGS protein: -The α subunit of a G-protein will slowly hydrolyze its GTP to GDP via its intrinsic GTPase activity. -A RGS (Regulator of G-protein Signaling) protein can act as a GAP on the α subunit of the G protein to increase the rate of hydrolysis. Hydrolysis of GTP by the alpha subunit inactivates the subunit and will cause it to dissociate from any target protein it was associated with in its active state. The inactive alpha subunit then inactivates βγ subunit to reform an inactive trimeric G protein.
Cyclic AMP Phosphodiesterase:
A molecule that hydrolyzes cyclic AMP to form AMP. Cyclic AMP phosphodiesterase therefore normally keeps the cytosolic concentration of cyclic AMP low.
What is a "Non-hydrolyzable analog of GTP"?
A substance in which there is a substitution of a sulfur for one of the oxygens in the γ-phosphate of GTP which creates creates a nucleotide that can't be hydrolyzed.
A surge in cytosolic Ca2+ can be triggered in multiple ways (not just GPCR signaling). Give an example of this:
A surge in cytosolic Ca2+ can be triggered in multiple ways (not just GPCR signaling): For example, fertilization of an egg by a sperm triggers an increase in cytosolic Ca2+. A wave of cytosolic Ca2+ sweeps across egg from the fertilization site. This Ca2+ wave prevents entry of additional sperm and triggers embryonic development.
For each of the following conditions, would the effect of acetylcholine be increased or decreased? A) Addition of a drug that stimulates the GTPase activity of the Galpha subunit. B) Mutations in the K+ channel that keep it closed all the time C) Modification of the G alpha subunit by cholera toxin D) A mutation that decreases the affinity of the beta-gamma complex of the G protein for the K+ channel E) A mutation in the acetylcholine receptor that prevents its localization on the cell surface F) Adding acetylcholinesterase to the external environment of the cell.
A. Decrease. An increase in the GTPase activity of the Gα subunit will decrease the length of time that the G protein is active. B. Decrease. If the K+ channel remains closed, acetylcholine will not slow the heart. C. Increase. Cholera toxin inhibits the GTPase activity of the Gα subunit, keeping the subunit in an active state for a longer time. D. Decrease. The activated βγ complex binds to and activates the K+ channel; decreasing their affinity for each other will decrease the time that the K+ channel is open, effectively decreasing the effect of acetylcholine. E. Decrease. If there is no receptor on the cell surface, cells will be unable to respond to acetylcholine. F. Decrease. Acetylcholinesterase degrades acetylcholine and thus will decrease the effect of acetylcholine.
Describe the features characteristic of a G-Protein-Coupled Receptor (GPCR):
All G-Protein-Coupled Receptors (GPCRs) are structurally related: (1) All G-Protein-Coupled Receptors have seven transmembrane (alpha helix) domains. (2) Diverse ligands interact with GPCRs through the extracellular domains. (3) G-Protein-Coupled Receptors bind to trimeric G-proteins through the cytosolic domains.
Which of the following would enhance the effect of acetylcholine on heart rate? A) Addition of a high concentration of a non-hydrolyzable analog of GTP. B) Addition of a drug that prevents the alpha subunit from exchanging GDP for GTP C) Mutations in the acetylcholine receptor that weaken the interaction between the receptor and acetylcholine. D) Mutations in the acetylcholine receptor that weaken the interaction between the receptor and the G-protein.
Answer A: The addition of high concentrations of a nonhydrolyzable analog of GTP will increase the length of time that the G protein βγ complex remains free of the α subunit and able to activate the K+ channel. This will therefore enhance the effect of acetylcholine. All the other choices will make it more difficult for the signal to proceed from the GPCR to the K+ channel.
Which of the following would NOT lead to signal amplification in a G-Protein-Coupled Receptor signaling pathway? A) An extracellular signaling molecule binds and activates a G-Protein-Coupled Receptor. B) The activated G-Protein-Coupled Receptor causes Gα to separate from Gβ and Gγ. C) Adenylyl cyclase produces cyclic AMP. D) Protein kinase A phosphorylates target proteins. E) None of the above.
Answer: A The extracellular signaling molecule binding and activating a GPCR. Each signaling molecule activates only one receptor molecule.
How would the activity regulation of a trimeric G protein be affected by a mutation that caused its affinity for GDP to be reduced without significantly changing its affinity for GTP? (a) An extracellular signal would be required to initiate its activation (b) An activated GPCR would be required to activate it (c) It would be spontaneously activated without any upstream signal (d) An RGS protein could switch it off until another signal activates it (e) None of the above
Answer: C The mutant G protein would be spontaneously activated without any upstream signal. GTP is present at much higher cellular concentrations than GDP. Each time the α subunit hydrolyzed GTP to GDP, the GDP would spontaneously dissociate, allowing GTP to bind and reactivate the α subunit. This is known as a fast-cycling mutant.
What is CaM?
Calmodulin This is a Ca2+ binding protein
Describe the degradation of cyclic AMP (cCAMP):
Cyclic AMP is short-lived in the cell because it is hydrolyzed by cyclic AMP phosphodiesterase to form AMP. This process uses water.
What does the second messenger DAG activate?
DAG activates protein kinase C (PKC)
Describe how G-Proteins can directly couple G-Protein-Coupled Receptor activation to the opening of ion channels in the plasma membrane:
For example: (1) Nerves that signal a slow-down in heart rate release acetylcholine. (2) Acetylcholine acts as a signaling molecule and binds and activates a G-Protein Coupled Receptor. (3) The G-Protein Coupled Receptor activates a G protein. (4) The activated βγ complex of the G-protein binds to a K+ channel, which causes it to open. (5) K+ channel opening increases permeability to K+ (the K+ flows out of the cytosol and into the extracellular space). This results in slowing down of the heart rate because it causes a decreased rate of firing. (6) The α subunit of the G-protein hydrolyzes GTP, returning the G protein to the inactive state and closing the K+ channel.
What is the largest family of cell-surface receptors?
G-Protein-Couple Receptors (GPCRs) are the largest family of cell-surface receptors. There are over 700 types in humans
What is the relationship between G-Protein-Coupled-Receptors and second messengers?
G-Protein-Coupled Receptor Signaling Pathways act through second messengers to achieve a cellular response: Enzymes activated by G proteins increase (or in some cases decrease) the concentration of "second messengers": Each activated enzyme can generate many molecules of these second messengers. This is what leads to signal amplification and diffusion. These second messengers will then bind to specific signaling proteins (most often kinases) and influence their activity.
What is GCaMP?
Genetically encoded Ca2+ indicator
What does the second messenger IP3 do?
IP3 opens Ca2+ channels in the endoplasmic reticulum. Note: Ca2+ can also act as a second messenger!
Not only is it associated with the fight or flight response, a rise in intracellular cyclic AMP can also ___________________.
Not only is it associated with the fight or flight response, a rise in intracellular cyclic AMP can also activate gene transcription: (1) Adrenal gland releases adrenaline. This acts as a signaling molecule and binds to G-Protein-Coupled Receptor called an adrenergic receptor. (2) The G-Protein Coupled Receptor turns on a G-protein (stimulatory G-Protein "Gs"), which activates adenylyl cyclase. (3) Adenylyl cyclase converts ATP into cyclic AMP. (4) Increased cyclic AMP activates PKA. (5) PKA enters the nucleus where it phosphorylates and activates a transcription regulator (such as CREB). (6) The phosphorylated transcription regulator activates transcription of target genes.
What is a second messenger and what do they do?
Second messengers are low MW intracellular signaling molecules. These act as intracellular mediators of extracellular signals through signal amplification and diffusion.
Describe the role of G-Protein-Coupled Receptors signaling in smell:
Smell Depends on G-Protein-Coupled Receptors in Olfactory Receptor Neurons: -Modified cilia extend from each olfactory neuron in the nose. Olfactory G-Protein Coupled Receptors are highly concentrated in these specialized cilia. Note, however, each olfactory neuron produces only one type of olfactory G-Protein-Coupled Receptor. Each of these responds to specific odorants: (1) An odorant (ligand) binds to a specific olfactory G-Protein-Coupled Receptor and activates an olfactory-specific G-protein. (2) The activated olfactory-specific G-protein activates adenylyl cyclase. (3) Adenylyl cyclase converts ATP to cCAMP. (4) The increase in cCAMP opens cyclic AMP-gated ion channels. (5) The opening of these channels allows an influx of Na+ and Ca2+ (6) The influx of Na+ and Ca2+ leads to increased neurotransmitter release. This is interpreted by the brain as smell. NOTE: Ion channels are opened here, too, by G-Protein Coupled Receptors, but they DO NOT do this directly. It is instead via cyclic AMP.
What are the two most frequent target enzymes for G-proteins?
The 2 most frequent target enzymes for G proteins are: (1) Adenylyl cyclase (which produces cyclic AMP) (2) Phospholipase C (which produces inositol triphosphate & diacylglycerol)
Describe the synthesis of cyclic AMP (cCAMP):
The membrane-bound enzyme Adenylyl cyclase converts ATP into cyclic AMP (cAMP): A- Adenylyl cyclase's catalytic site is activated by the GTP-bound form of a stimulatory G-protein alpha subunit (Gαs). B - Adenylyl cyclase removes two phosphates from ATP forming AMP C- The AMP undergoes an intramolecular reaction in which the phosphate group of the 5' carbon of the ribose reacts with OH group of the 3' carbon of the ribose. This forms a cyclic AMP as well as produces water. This is an example of a cyclization reaction.
Describe the structure of an Inactive Trimeric G-Protein:
There are multiple G-protein types, but they all have a similar structure: (1) G-Proteins are called "heterotrimeric". This is because they're made up of 3 different subunits: α, β, and γ. A - Both the α and γ subunits have covalently attached lipid molecules to interact with the Plasma Membrane. B - β and γ subunits are tightly associated with each other. As a result, they function as a unit. C - Only the α subunit has GTPase activity. It undergoes a conformational switch between the GDP-bound (inactive), GTP-bound (active) forms.
Can cyclic AMP concentration in the cell increase rapidly?
Yes: cyclic AMP can increases rapidly (20 sec) in response to extracellular signal
What is a way that we can easily see the rapid increase in cyclic AMP concentrations in the cell?
cyclic AMP biosensor allows for live imaging of increase in cyclic AMP. For example: The picture shows a neuron expressing cyclic AMP biosensor
Odorant:
small chemical compound that has a smell
What are the common second messengers?
(1) 3',5'-Cyclic AMP (cCAMP) (2) 1,2-Diacylglycerol (DAG) (3) Inositol 1,4,5-triphosphate (IP3)
Describe how phospholipase C activates two signaling pathways:
(1) A signal molecule activates a G-Protein-Coupled Receptor. (2) The G-Protein-Coupled Receptor activates a G-Protein (Gq) (3) The activated G-protein activates Phospholipase C. This molecule cleaves an inositol phospholipid generating IP3 and diacylglycerol: A - IP3 diffuses through the cytosol and eventually binds and opens IP3-gated Ca2+ channels in the ER. This means that Ca2+ is released into the cytosol (Ca2+ is normally very low in the cytosol). B - Diacylglycerol (DAG) remains embedded in the Plasma Membrane where it activates protein kinase C (PKC). The increased cytosolic Ca2+ also contributes to PKC activation (called PKC because its dependent on Ca2+). PKC phosphorylates target proteins leading to a cellular response to the signal.
Describe the activation of a G-Protein-Coupled Receptor and its subsequent activation of a Trimeric G-Protein:
(1) A signaling molecule (ligand) binds to a G-Protein Coupled Receptor. (EACH SIGNALING MOLECULE ONLY BINDS ONE RECEPTOR!)This ligand binding forces the GPCR to undergoes a conformational change. (2) The activated GPCR acts like a GEF and induces the α subunit of a trimeric G-protein to release GDP (NOTE: THE GPCR CAN ACTIVATE MULTIPLE G-PROTEINS!). (3) GTP binds to the G-protein. This GTP binding induces a conformational change in the α subunit, activating both the alpha subunit and the beta-gamma complex. Note: this conformational change CAN includes dissociation of the α subunit from the βγ subunit. However, the two subunits can remain together. (4) Activated G proteins can either: A - Directly regulate ion channels in the Plasma Membrane B - Activate membrane-bound enzymes that increase (or decrease) the cellular concentration of second messenger molecules. Note: As long as the signalling receptor remains stimulated, it can continue to activate G-proteins. HOWEVER, upon prolonged stimulation, the receptors eventually become inactive even if their activating ligands remain bound. In these cases, a receptor kinase phosphorylates the cytosolic portion of the receptor. Once the receptor has been phosphorylated in this way, it binds with high affinity to an arrestin protein, which inactivates the receptor by preventing its interaction with G-proteins. The arrestin also acts as an adaptor and recruits the receptors to clathrin-coated pits, from where the receptors are endocytosed.
