Physiology 3140A Lecture Notes - Lecture 8: Gs Alpha Subunit, Adenylyl Cyclase, Pertussis Toxin
22 May 2018
School
Department
Course
Professor
Physiology 3140
Dr. Rylett
Lecture 8
Example: role of PK-A and CREB in regulation of proinsulin gene transcription in pancreatic beta cells
- CREB is a transcription factor that regulates the transcription of the proinsulin gene (proinsulin
is then degraded to make insulin)
o Only one CREB is shown in the image but remember that CREB functions as a dimer
o
- transcriptional activities of CREB:
- regulated by cAMP-dependent signalling pathway
- represents final communication link in regulation of gene expression by activation of cAMP-
dependent signalling systems
- Know the substances which are used experimentally to study cAMP and PKA in biological
processes
- Cell gets all kinds of information from all different molecules and uses that info in a net way so
the response of the cell is a summation of all of those things
o Some of those thing may be excitatory while others is inhibitory
- In this image,
o GLP and Galanin (hormones)
o GLP binds to its receptor which is coupled to the Gs protein to stimulate adenylate
cyclase
o Galanin binds to its receptor which is coupled to a Gi protein to inhibit adenylate cyclase
o Net effect on adenylate cyclase will depend on the relative balance of information
coming from those
▪ More GLP = more stimulatory info
▪ More Galanin = more inhibitory info
Cholera and pertussis toxin actions
Cholera toxin:
- Catalyzes ADP-ribosylation of Gs alpha subunit
- Gs alpha loses catalytic activity for inactivating GTP to GDP
- Increased adenylate cyclase activity, increased cAMP, prolonged PKA
- Prolongs the activity bc it blocks the GTPase activity
Pertussis toxin
- Modifies the alpha subunit of the Gi protein
- Catalyzes ADP-ribosylation of Gi alpha subunit
- Gi alpha locked in GDP-bound form and inactive, so unable to inactivate adenylate cyclase to
block cAMP formation
- Net effect could be increased cAMP, prolonged PKA
Is adenylate cyclase involved in a certain outcome in the cell?
- Foreskelin (drug that is often used experimentally)
- Activator of adenylate cyclase
- Can directly activate adenylate cyclase
- You could do an experiment where you have a certain cell type and a certain outcome measure
- Does cAMP and PKA drive that outcome measure?
- You could add foreskillin to you experiment that would artificially activate adenylate cyclase –
leading to an increase in cAMP, activating PKA and may or may not lead to the outcome measure
find more resources at oneclass.com
find more resources at oneclass.com
that youre looking at (that is one way to ask directly if this part of the pathway is directly
involved)
- AKAP= A kinase anchor protein (anchors the regulatory subunits to the cytoskeleton)
Is cAMP driving this biological measure that I want to understand?
- If this is your question immediately, you could add cAMP to your cells ro tissues
- Problem: cAMP is a highly charged molecule that cant get into the cells
- So there have been several cAMP analogues developed that you can use experimentally and they
diffuse across the membrane
- One of these is called 8 Br
o Used to answers strictly about cAMP
o Can passively diffuse across plasma membrane allowing you to measure whether it has
an affect
What happens if you prolong the concentration of cAMP
- cAMP has a relatively short half life
- phosphodiesterase converts activate cAMP into the inactive 5’ AMP
- So now there is a situation where you have activation of adenylate cyclase, production of cAMP
the step function where you have an increase in cAMP but you don’t want it to be chopped up
by phosphodiesterase
- You want it to be raised and prolong the concentration of cAMP
- You can do this by inhibiting the phosphodiesterase
- One of the compounds to do this is IBMX (iso butyl methyl zanthene)
- Caffeine is a methyl zanthene
o so when we drink caffeine, we are reducing our phosphodiesterase activity and
prolonging our cAMP concentration
- MAIN POINT FROM THE SLIDE: know the compounds and how these compounds could be used
in an experimental setting and what the outcome would be if you block phosphodieseterase
Glucose phosphorylase and glucose metabolism
- There are multiple targets for a PKA, PKA then inhibits something and activates other things
- The sum total of all of this is to yield an increase in metabolic energy that can be used by skeletal
muscle for example
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Example: role of pk-a and creb in regulation of proinsulin gene transcription in pancreatic beta cells. Know the substances which are used experimentally to study camp and pka in biological processes. Gs alpha loses catalytic activity for inactivating gtp to gdp. Prolongs the activity bc it blocks the gtpase activity. Increased adenylate cyclase activity, increased camp, prolonged pka. Modifies the alpha subunit of the gi protein. Gi alpha locked in gdp-bound form and inactive, so unable to inactivate adenylate cyclase to block camp formation. Net effect could be increased camp, prolonged pka. You could do an experiment where you have a certain cell type and a certain outcome measure. Akap= a kinase anchor protein (anchors the regulatory subunits to the cytoskeleton) If this is your question immediately, you could add camp to your cells ro tissues. Problem: camp is a highly charged molecule that cant get into the cells.
