Signaling Receptors Flashcards
- Draw the membrane topology a G protein-coupled receptor and identify the basic structural characteristics that mediate ligand binding and coupling to G proteins.
- GPCRs have 7 helical transmembrane domains with the N-terminus on the outside and the C-terminus on the inside.
- The 7 domains fold into a barrel and bind ligands on the outer face.
- Intracellular sequences/loops are used to interact with things on the inside of the cell.
- Ligand binds on outside of cell (to the barrel? There’s some sort of receptor domain out there) and then undergoes G protein coupling-signal transduction on the inside.
- Agonists bind the receptor and trigger activation, whereas antagonists block the receptor activation (generally bind competitively with agonist).
- 50% of non-antibiotic drugs use this mechanism.
- Explain how G protein-coupled receptors activate hetero-trimeric G proteins and diagram the GTP-hydrolysis cycle of G protein signaling.
- Agonists vs. antagonists bind the receptor.
- If it’s an agonist, you stimulate a nucleotide exchange in which GDP on Galpha unit gets switched out for a GTP (this is the rate limiting step).
- The dissociation of GDP is the rate limiting step to go from inactive to active GPCR.
- Now your Galpha protein is active to go cause signaling.
- Upon becoming active, beta-gamma and Galpha units can separate and go target different effectors.
- Effectors can be enzymes that further generate second messengers and ion channels; effectors regulated by Galpha and beta-gamma units.
- So, the receptor gets turned on, which turns on Galpha, which turns on the pathway.
- The G-protein has GTPase activity –> basically a built-in timer that allows it to turn itself off; hydrolyzes GTP–>GDP, and thus becomes inactive again.
- GAP proteins can help accelerate the turning off process.
*Just an FYI, but Switch II (Galpha) binds beta-gamma in the inactive state, and Galpha in the active state?
G proteins can be regulated by toxins:
- PTX (Pertussis) –> puts an ADP-ribose group on Galpha unit –> locks it in the inactive state so it can’t couple –> prevents a lot of downstream activity.
- CTX (Cholera) –> Galpha’s ATPase activity gets inhibited by CTX adding ADP-ribose to Galpha –> locks G protein in active state even without receptor activation.
- Describe the function of second messengers in receptor signaling and give two examples for how they are generated by activated G proteins.
- 2nd messengers can cause a lot of downstream effects in the signaling pathway that eventually lead to a specific cellular response.
- This is best understood through all those examples in the ANS (sympathetic vs. parasympathetic).
- Adrenergic receptors in sympathetic system (stimulate AC and PLC),
- Muscarinic receptors in parasympathetic system (inhibit AC and PLC).
Example 1:
-Beta1AR gets bound by NE (or epinephrine or isoproterenol) and is now in the active state.
-Subunits separate and GS-alpha goes and activates AC –> cAMP –>activates PKA –> activates L-type Ca channels,
RyR’s in the SR, and contractile proteins –> Ca influx, increased heart rate and contraction = sympathetic response.
-If you took a beta blocker, it would decrease this activity because the agonist wouldn’t be able to bind.
Antagonists = propranolol or metoprolol.
Example 2:
-Alpha1AR gets bound by NE (or epinephrine or phenylephrine), which puts it in active state.
-GQ-alpha goes and activates PLC –> cleaves PIP2 into IP3 (causes release of Ca from ER, which stimulates smooth muscle contraction) and DAG (which activates PKC, more L-Ca channels, and thus more Ca release).
-This causes peripheral vasoconstriction –>decreases blood flow to skin, increases blood pressure, and shifts blood flow to heart/lungs/muscle.
Antagonist = prazosin.
Example 3:
- M2-muscarinic receptor gets bound by Ach (or muscarine), which activates GI-alpha.
- Which antagonizes the effects of GS-alpha and shuts off AC.
- If Gi > Gs. Stimulatory pathway gets shut off and cAMP, PKA levels decrease, channels get dephosphorylated, and Ca levels decrease = decreased heart contraction.
- cAMP levels decrease due to PDE’s that degrade cAMP to AMP so it’s can’t activate PKA anymore.
- PDE inhibitors can be caffeine, theophylline, and cAMP specific Milinirone (PDE3) and Rolipram (PDE4).
- A cGMP- specific PDE inhibitor = Viagra, which inhibits PDE5.
- So drugs target PDEs to inhibit them, and prolong effects of cAMP stimulatory agonists.
Example 4:
- M2-muscarinic receptor, when bound by AChr, can also release beta-gamma subunit, which can bind a GIRK K+ channel and activate it, letting K out of cell.
- This causes membrane hyperpolarization and decreased excitability.
- This then decreases Ca influx and heart rate.
- *An m2 antagonist would increase heart rate; if you add a beta-agonist on top of that, it adds to the effect of the m2-anagtonist and you get a sympathetic response.
Example 5:
- Activation of beta2AR in the lungs by epinephrine (or isoproterenol or Albuterol) causes activation of GS-alpha.
- Which activates AC –> cAMP –> PKA –> which inhibits smooth muscle contraction –> allows bronchodilation and dilation of vasculature supplying lungs, heart and muscle.
- Whereas M3-muscarinic activation by Ach in the lungs activates GQ-alpha.
- Which activates PLC –> IP3 causes Ca release from ER and DAG (through PKC) also causes Ca influx (via L-type Ca channels) –> stimulates smooth muscle contraction and bronchoconstriction.
- Explain how receptor activation leads to signal termination through receptor desensitization and coupling to additional pathways.
-Agonist being re-uptaken or degraded can turn off the GPCR.
-2nd messengers can go away by various mechanisms (see previous lectures).
-If you desensitize the GPCR, it can turn off the pathway this way as well.
-GRK kinases can phosphorylate the receptor when it’s in an active state, and GRKs can also bind the beta-gamma unit after Galpha separates to aid binding to GPCR.
-GRK phosphorylation of GPCR helps Beta-arrestin binding –> serves as an adapter that aids with endocytosis of receptor and prevents re-coupling of a G protein so Galpha can’t get activated again.
-After it’s endocytosed, GPCR can be targeted to the lysosome or can get re-sensitized via dephosphorylation.
-Desensitization can limit the efficacy of GPCR agonist therapy, especially in chronic treatment where receptor degradation and downregulation are more likely to occur.
-Beta-arrestin couples GPCRs to activation of other downstream signals via JNK and ERK MAP kinase pathways.
-There are even biased agonist/antagonists that can preferentially activate beta-arrestin signaling over G-protein signaling.
Carvidilol = beta-blocker–> antagonizes G-protein signaling but activates B-arrestin signaling to ERK
- Give two examples of drugs that act through modulating different steps in a receptor-G protein-second messenger signaling cascade.
Carvidilol.
Albuterol in lungs?
