Some Receptor Theory Questions...Constitutive or not?
DAVID at chempath.uct.ac.za
Mon Oct 3 15:11:00 EST 1994
I am working on a G-protein coupled receptor and have created a mutant using
site-directed mutagenesis. I have done inositol phosphate assays and
binding assays on this mutant and have had some apparently contradictory
In the inositol phosphate (IP) assay, I have an increased basal (without
ligand), but wildtype maximal response. This would imply a constitutively
active mutant. However, I then did a competition binding assay, and a
Scatchard plot of the data indicated that there were 4x more receptors in
the mutant compared to the wildtype, and the Kd's were the same. This would
indicate that the mutant is simply overexpressed, and beacuse there are more
receptors, there is an increased inositol phosphate basal.
The main problem with all of this is that there is no antibody available yet
to quantitate the number of receptors on the cell surface.
There is also the question of spare receptors in the system. We have been
trying to make an expt that would tell us whether or not there are spare
receptors. We thought that by transfecting with different amounts of DNA,
we would be changing the amount of receptors in any given cell. Going on
this idea, we did the expt using DNA conc.'s from 0.1ug/well to 10ug/well
for the wildtype and doing a dose-response for each DNA conc. What we got
was a decrease in the max IP response, with no change in the ED50. This
implies no spare receptors (spare receptors should've given a right shift in
ED50 with decreasing amounts of DNA, with the same max).
I did an IP assay on mutant and wt using different amounts of DNA, and
stimulated with a dose of ligand that gives a max IP response. My reasoning
was that at the DNA conc. I usually used, the IP response was saturated so
that even four times more receptor could not increase it. Thus, by lowering
the amount of DNA (and theoretically, the number of receptors per cell), I
might reach a point where the response is not saturated. If the mutant was
over-expressed, then at this point it should give a higher max than the wild
type. However, the responses for mutant and wt dropped off at the same
time. In fact the mutant max was always slightly below that of the wildtype.
My questions (after all that build up) are the following:
1) Is our thinking right that by decreasing the DNA we decrease the number
of receptors per cell?
2) The binding was done at 4øC, which apparently only measures high affinity
receptors and not low affinity (some people think that ALL receptors are
forced into a G-protein indepedent high affinity state at 4øC). I am
unsure as to whether I am measuring all or only the high affinity
3) Is there some way of proving, beyond a doubt, if a receptor is
constitutively active, without having an antibody?
I am sorry for the long explanation, but I think it was necessary to explain
the situation. If you would like to talk in more detail, you can e-mail me
directly. I would be grateful for any ideas on the matter.
Thanks for your time
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