Physiology 3140A Lecture Notes - Lecture 5: Heterotrimeric G Protein, Protein Kinase, Phosphorylation
22 May 2018
School
Department
Course
Professor
Physiology 3140
Dr. Rylett
Lecture 5
GPCR Activation (Recap)
- Unstimulated state: trimeric G protein inactive
- Upon ligand binding to GPCR, GPCR conformation changes that allows GPCR
to bind heterotrimeric G protein
- GPCRs function as guanine nucleotide exchange factors GEF: switch G
into an activated (GTP) bound state
- GTP binding: G activating conformational
o releasing the G protein from GPCR
o triggering dissociation of the G from γ : effector activation
G Protein Activation/Activation
Diversity of GPCRs Signaling
G Protein-coupled Receptors II
- cells are good at adapting to the amount of receptor that they need at the cell surface
- ½ of the drugs that are prescribed to patient are ligands that bind to GPCRs
- regulation of GPCRs involved desensitization and down regulation
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Definitions
- Desensitization:
o Occurs so that the receptor can no longer signal to the cell
o Ligand binds to receptor →something happens →some type of second messenger
molecule gets sent to the cell
o This process can be turned off (by either the ligand dissociating from the receptor or
there are other processes that can take place to stop the receptor from being able to
signal again
▪ Ex: theres lots of ligand floating outside of the cell, it binds to any receptor,
initiates a process and sends a signal into the cell so the cell modifies its
behavior (ex: by changing it metabolism or its cytoskeleton or it secreted some
other molecule)
▪ Now, once this has happened, cell recognizes that it did what it needed to do,
and it may not want to do it again (there may be a refractory period before it
wants to do that again)
▪ And desensitization is a way for the cell to do this
o Desensitization decline in response to continuous or repeated application of agonist(aka
ligand)
o mechanism for protecting cells against receptor overstimulation
o this can happen through a number of processes which involved down regulation
- Down-regulation:
o persistent loss of receptors after long-term exposure to agonist
o occurs during long-term administration of receptor agonist (tolerance)
o receptors degraded (lysosomes)
o decreased receptor synthesis: gene transcription/post-transcriptional events
- so desensitization is when the receptor are no longer sensitive to the ligand/agonist white
downregulation is the process by which that takes place
- note: there can be a persistent loss of receptors if there is long term exposure to the agonist
GPCR Desensitization
- Receptor Inactivation: exposure of GPCRs to agonists often results in a rapid attenuation of
receptor responsiveness
- Note: hormone, agonist and ligand all mean the same thing (interchangeable)
- Hormone binding to receptor leads to activation of the receptor
- Conformational change in shape of receptor allows receptor to couple to the G protein (affinity
for binding G protein goes up)
- G protein binds: GEF changes GDP to GTP and alpha subunit
dissociates from beta/gamma subunit
- These go off and they have different effectors that they bind
to
- Alpha subunit often mediates cellular processes in the cell
but beta/gamma can too
- If there is a lot of ligand present, it goes into desensitization
mode:
o G protein uncouples and is not able to recouple to the receptor
o Some mechanisms that mediate this over diff time frames are:
▪ Phosphorylation: this has a negative impact to desensitize and uncouple the G
protein (rapid effect)
▪ Sequestration: receptors get taken into cell so they are no longer at het cell
surface so they cant see the ligand anymore (longer term effect)
▪ Down regulation: degradation of receptor, decreased receptor synthesis and
tolerance (long term effect)
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find more resources at oneclass.com
Receptor Inactivation
- exposure of GPCRs to agonists results in rapid attenuation(loss) of receptor responsiveness
- process, termed desensitization, can be achieved by four mechanisms
- inactivation occurs when there is a lot of ligand present
- cells have adapted to these kinds of diff processes and makes a decision based on how much
ligand is present outside the cell
o small amount of ligand = cell doesn’t want to perform that function anymore
o large amount of ligand = cells has to do something more drastic
1. Phosphorylation:
o GPCRs are covalently modified by phosphorylation of intracellular serine residues
ser/thr protein kinases
o This results in the inability of receptor to couple to heterotrimeric G protein and
stimulate the exchange of GDP for GTP on the -subunit
o when the receptor becomes phosphorylated, it is now in a conformation that has a low
binding affinity for the alpha subunit
o note: a lot of these biological signaling processes are highly dependent on the
conformation of the individual proteins involved – which dictates the binding affinity it
has for other proteins
o normally, the ligand binds to the GPCR, causes a conformation shape change in the
receptor and now the affinity of the receptor for the G protein increases
o if the ligand is not bound, and the conformation has not changed, it has a low binding
affinity →so the whole thing is dictated by this change in conformation of the receptor
and the change in affinity that the receptor now has for the alpha subunit
o phosphorylation is most rapid means by which GPCR desensitization can be achieved
o GPCRs phosphorylated within seconds to minutes following ligand binding
o Mediated by diff classes of kinases:
▪ The mediation depends on the amount of ligand outside the cell
▪ If theres a low amount of ligand outside the cell:
• Second messenger-dependent protein kinase
• cAMP-dependent protein kinase (PKA)
• protein kinase C (PKC)
• signaling pathways need to feedback onto the receptors to tell them that
the job was done and to not do it anymore
▪ if there is a lot of ligand outside the cell:
• Receptor-specific G protein-coupled receptor kinase (GRKs) are
recruited
• GRKs only come into action when they are really needed as a backup
system
2. Internalization:
o Sometimes phosphorylation is not enough and the cell wants to decrease the number of
receptors on the cell surface so internalization happens (receptors move into the cell)
o GPCRs can be temporarily removed into interior of cell so they are no longer available at
cell surface for ligand binding and unable to interact with heterotrimeric G protein to
mediate signaling
o Receptors that go into the cell can recycle back to the cell membrane
o Ligand binds, sends signal into cell, cell behavior changes, cell does what its supposed to
do, the cell doesn’t want to do it anymore and so some of the receptors gets pulled in so
they cant find so you cant get signaling anymore
o Now you want a situation where you want that specific thing to happen again so the
receptors can recycle to the cell surface
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Upon ligand binding to gpcr, gpcr conformation changes that allows gpcr. Gpcrs function as guanine nucleotide exchange factors (cid:523)gef(cid:524): switch g(cid:573) Gtp binding: g(cid:573) activating conformational triggering dissociation of the g(cid:573) from (cid:574) : effector activation: releasing the g protein from gpcr to bind heterotrimeric g protein into an activated (gtp) bound state. G protein-coupled receptors ii cells are good at adapting to the amount of receptor that they need at the cell surface. Of the drugs that are prescribed to patient are ligands that bind to gpcrs regulation of gpcrs involved desensitization and down regulation. Note: there can be a persistent loss of receptors if there is long term exposure to the agonist. Receptor inactivation: exposure of gpcrs to agonists often results in a rapid attenuation of receptor responsiveness. Note: hormone, agonist and ligand all mean the same thing (interchangeable) Hormone binding to receptor leads to activation of the receptor.
