Lecture 15_GPCRs Flashcards
How does a metabotropic receptor work?
Neurotransmitter binding causes a conformational change in the receptor which leads to G protein binding and to the production of intracellular metabolites through enzymatic processes. These responses are of slow onset and long duration, compared to ionotropic receptors.
What’s another name for metabotropic receptors?
G-protein coupled receptors (GPCRs) or 7-transmembrane domain receptors.
First messengers vs. second messengers
Neurotransmitters, hormones and drugs that cannot cross the cell membrane (first messengers) exert their effects inside cells through molecules (second messengers such as cyclic nucleotides, ions, phospholipids, cAMP) that act intracellularly.
Most hormones and many neurotransmitters act by regulating intracellular second messengers. Just read the answer slide, a lot of info.
In most cases, there are multiple receptors for single neurotransmitters that bind to GPCRs. This was predicted from classical binding studies.
For example, there are 5 classes of muscarinic acetylcholine receptors and 6 classes of adrenergic receptors.
In many cases, the complex pharmacological responses produced by a single ligand are due to its actions on a number of different receptors.
Three components required for G-protein signaling
- Receptor (on cell surface)
- G protein (couples to receptor on intracellular side of the cell membrane)
- Effectors (usually enzymes)
All 3 are either imbedded in the cell membrane or tightly associated with it. Enzymes mediate effect of G proteins.
Physical form of GPCR
- Huge family (over 800 genes) of related receptors, each a produce of a different gene
- Each contains 7 transmembrane domains
- Neurotransmitter binds to GPCR on extracellular side of membrane
- G proteins bind to intracellular sections of GPCR
- Neurotransmitter binding likely stabilizes receptor so it can efficiently bind the G-protein trimer.
(T/F) When a GPCR is stimulated by an agonist, the effect is usually of limited duration.
TRUE.
There are two major mechanisms limiting the durations of actions of neurotransmitter agonists at GPCRs:
- Receptor desensitization: Where the extent of interaction between receptor and G proteins can be decreased. This is usually accomplished through phosphorylation of serine and threonine residues of GPCRs, by specific intracellular enzymes (kinases).
- Receptor down-regulation: Prolonged exposure to agonist leads to the internalization of receptors from cell membrane, thus decreasing the numbers of GPCRs that can interact with G proteins.
How do G proteins function?
- The family of G proteins that transduce signals from membrane receptors to effector enzymes and ion channels are known as heterotrimeric (3 different subunits) G proteins.
- The three subunits are called a, b and g in decreasing size.
- The a subunit binds guanine nucleotides and is the major mediator of the G protein’s actions on its effector. The b and g subunits primarily function to support the interactions of the a subunit with the plasma membrane and with GPCRs, but like the a subunit, they may also regulate effectors directly.
The G protein activation/inactivation cycle
In the resting state, the three G protein subunits are bound together with guanosine diphosphate (GDP) attached to the a subunit. This heterotrimer can bind to an inactive GPCR. When an agonist binds to the GPCR, a conformational change occurs, leading to the rapid dissociation of the G protein heterotrimer from the GPCR. This agonist binding also results in the release of GDP from the a subunit of the G protein heterotrimer.
The empty guanyl nucleotide binding site on the a subunit is then occupied by guanosine triphosphate (GTP) that is present at high concentrations in cytoplasm. GTP binding causes the a subunit to release from the GPCR as well as from the beta gamma dimer.
The a subunit then binds to an effector. Within a few seconds, the intrinsic GTPase activity in the a subunit hydrolyzes the bound GTP to GDP, inactivating the a subunit.
The GDP-bound a subunit dissociates from the effector, re-associates with the beta gamma dimer and is ready for another cycle of activation by GPCRs.
Gs alpha vs Gi alpha
Gs alpha stimulates adenylyl cyclase (leading to production of cAMP) and Gi alpha inhibits adenylyl cyclase (inhibiting production of cAMP).
Some G proteins are the targets of toxins
- The Gsa subunit is modified by the cholera toxin such that it binds ADP-ribose to the guanyl nucleotide binding site. This prevents the intrinsic GTPase from acting and results in persistent activation of Gsa. In the intestine this leads to marked elevations in cAMP levels, causing cells to secrete large amounts of water into the gut, leading to the severe diarrhea that is seen in cholera infections.
- Another bacterial toxin, pertussis, acts on Gia and G0a subunits, preventing their activation by GPCRs. With the disruption of the inhibitory actions of Gi on adenylyl cyclase, once again cAMP levels rise, leading to the characteristic cough seen in whooping cough.
Effector enzymes regulated by G proteins
Adenylyl cyclase and phospholipase C are the most common effector enzymes by which G proteins exert their effects.
How do adenylyl cyclase and cAMP function as a 2nd messenger?
- Adenylyl cyclase, imbedded in the cell membrane, catalyze the synthesis of cAMP from ATP.
- There are ten forms of adenylyl cyclase. All are stimulated by Gsa, but differ in their sensitivities to inhibition by Gia.
- cAMP acts by activating the cAMP-dependent protein kinases (protein kinase A; PKA) which can then phosphorylate other proteins at specific serine or threonine residues.
How do phospholipase C and phospholipid function as 2nd messengers?
- Members of the Gq family of G-proteins transduce signals between GPCRs and enzymes known as phospholipase C.
- These enzymes use phospholipids as substrates.
- GPCR occupation by ligand activates Gq. The Gqa subunit binds to the phospholipase on the inner surface of the cell membrane.
- This activated phospholipase then rapidly breaks down the membrane constituent phosphatidylinositol-biphosphate (PIP2) to inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 and DAG can both act as 2nd messengers.
