Some Receptor Theory Questions...Constitutive or not?

[Dave]

Greetings all

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 
results.  

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 
    receptors.

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

Dave