Signalling Problem 3
Addition of the agonist isoproterenol to mammalian cells grown in culture leads to a reduction in the percentage of the cells that undergo programmed cell death, also called apoptosis. This increased survival phenotype was accompanied by the increased phosphorylation and enzymatic activity of a serine/threonine protein kinase called PKB (aka Akt). The effect of isoproterenol on PKB and cell survival could be blocked by the addition of the specific receptor antagonist, propranolol, suggesting that this effect was mediated by a seven transmembrane G protein-coupled b-adrenergic receptor, which is present in the plasma membrane of these cells. Activation of this receptor by isoproterenol leads to the conversion of inactive heterotrimeric GDP-bound G protein complex (GaiGDP.Bbg) into active state with the dissociation of Gbg from GaiGTP.
The above described changes to PKB activity and phosphorylation status, and the increased survival phenotype of the cells, can be abolished by a number of treatments of the cells including:
Treatment of the cells with Pertussis toxin (PTX). PTX treatment inhibits Gai subunit activation by preventing the interaction of the inactive GaiGDP.Gbg complex with the receptor.
Treatment of cells with either of two different inhibitors of PI 3-kinase (wortmannin or LY294002).
Overexpression of recombinant Gaq subunits in cells. Overexpressed Gaq binds to and inhibits any free Gbg subunits that may be present in the cell either in the resting state or following receptor mediated activation.
Treatments 1 and 3 also blocked activation of the PI 3-kinase.
Draw a flow diagram that describes the simplest signal transduction pathway(s) that fit the above data.
In the diagram indicate all the different states of each protein involved.
Show on your diagram the points at which the various inhibitors have their effects.
