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of the gonadotropin receptors in intact, living cells (Guan et al.
2009,
2010
;
Urizar et al.
2005
) .
Studies with both the hLHR and the hFSHR demonstrated saturable BRET
2
titration curves when cells are co-transfected with a fixed concentration of receptor-
Rluc and increasing concentrations of receptor-GFP
2
(Guan et al.
2009,
2010
;
Urizar et al.
2005
). These findings, in the context of several control experiments,
suggested that the interactions promoting receptor self-association are specific and
not spurious. To examine whether newly formed gonadotropin receptors homodi-
merize/oligomerize, cell fractionation studies were performed on cells co-expressing
receptor-Rluc and receptor-GFP
2
. Fractions enriched for plasma membranes or
for ER membranes were examined for receptor dimerization by assaying for
speci fi c BRET
2
activity and were also probed for monomeric, dimeric, and oligo-
meric forms of the receptor by Western blotting. For both the hLHR and hFSHR,
speci fi c BRET
2
activities consistent with receptor dimerization, as well as Western
blot dimers and oligomers, were detected in plasma membrane-enriched as well as
ER-enriched fractions (Guan et al.
2009,
2010
). These results suggest that, similar
to other GPCRs (Hebert et al.
2006
; Bai
2004
; Angers et al.
2002
; Terrillon et al.
2003
; Salahpour et al.
2004
; Herrick-Davis et al.
2006
; Issafras et al.
2002
; Overton
and Blumer
2002
), the dimerization/oligomerization of the gonadotropin receptors
is a constitutive process where the receptor self-associate early in the biosynthetic
process. As such, the receptors exiting the ER to be trafficked to the Golgi and then
plasma membrane are already in self-associated forms.
Quantification of the dimeric and oligomeric forms of the hLHR on Western
blots relative to monomeric forms of the hLHR as a function of pretreatment of the
cells with or without hCG indicated a greater abundance of the dimeric and oligo-
meric forms after hormone treatment (Tao et al.
2004
). While these results could be
due to an agonist-dependent increase in dimerization/oligomerization of the hLHR,
they might also be observed if hCG occupancy of the hLHR in intact cells stabilized
dimeric and oligomeric forms of the receptor to detergent solubilization. Therefore,
subsequent studies utilized BRET
2
to address the role, if any, of agonist in promoting
hLHR dimerization/oligomerization because this approach could be performed in
living cells, thereby obviating the use of any detergent. The saturation curves
observed in cells incubated with hCG were no different than those of untreated cells
(Guan et al.
2009
), suggesting a lack of effect of agonist on dimerization. However,
it could be argued that if there were an effect of agonist on dimerization of the cell
surface receptor, it might not be detectable due to the contribution to the BRET
2
ratio from intracellular receptor dimers. Therefore, the BRET
2
ratio was also examined
in total membrane lysates as well as membranes separated by sucrose gradient
centrifugation. In all cases, and in particular even the fraction containing predomi-
nantly plasma membranes, there was no difference in the BRET
2
ratio in the absence
or presence of hCG (Guan et al.
2009
) . These results suggest that hLHR dimerization
is a constitutive process that is unaffected by agonist occupancy of the receptor. The
question of whether receptor activation correlates with the extent of dimerization
was further addressed in a different manner taking advantage of mutants of the
hLHR that are either constitutively active or signaling impaired. In the latter case,
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