Regulatory Processes Governing the Cell Surface Expression of LH and FSH Receptors - GPCR Signalling Complexes: Synthesis, Assembly, Trafficking and Specificity - page 118

of the hLHR, attenuate the basal activities of the constitutively active mutants.
Therefore, the D405N and Y546F mutations appear to stabilize the hLHR in a resting
state, as if the receptor were occupied by an inverse agonist. Interestingly, when a
low concentration of wt hLHR is transiently transfected into cells expressing a
higher density of the signaling impaired hLHR(D405N,Y546F) mutant to promote
the potential dimerization of the wt hLHR with the mutant, a small but reproducible
increase in the EC 50 for hCG-stimulated cAMP is observed relative to cells expressing
the same cell surface density of wt hLHR transiently expressed in cells stably
expressing empty vector. Under these experimental conditions, the attenuation of
hCG-stimulated cAMP through the wt hLHR when co-expressed with the signaling
impaired mutant cannot be attributed to a decrease in cell surface expression of the
wt receptor. One possible cause for the decreased signaling through the wt hLHR
when co-expressed with the signaling impaired mutant is if the signaling impaired
mutant sequestered Gs, thus limiting Gs availability to the wt hLHR. However, the
attenuation of signaling through the wt hLHR when introduced into cells expressing
the signaling impaired mutant was still detected when the cells were also transfected
with the subunits composing Gs. That the attenuation could be attributed to allosteric
effects between dimerized hLHR(wt) and hLHR(D405N,Y546) is supported by data
showing saturable and specific BRET 2 saturation curves between the two forms of
hLHR. Furthermore, it was shown that specific BRET 2 could not be observed
between the MC3R and the signaling impaired hLHR, suggesting a lack of dimeriza-
tion between the MC3R and hLHR, and an attenuation of signaling through the
MC3R by the signaling impaired hLHR was also not observed. Taken altogether,
these data suggest the attenuation of hormone-stimulated cAMP through the wt
hLHR when it is co-expressed with the signaling impaired D405N,Y546F mutant is
due to the dimerization between the two forms of hLHR, allowing for allosteric
regulation of signaling of the wt hLHR by the associated signaling impaired hLHR
mutant.
Other reports have suggested that the dimerization of a binding impaired hLHR
mutant with a signaling impaired hLHR mutant can in some cases result in the
partial functional rescue of hormone-stimulated cAMP production (Ji et al. 2002 ;
Lee et al. 2002 ; Jeoung et al. 2007 ; Rivero-Muller et al. 2010 ) . Studies from our
laboratory, however, question whether functional rescue is in fact occurring in these
cases (M. Zhang, R. Guan, and D.L. Segaloff, manuscript submitted). Using carefully
controlled conditions that precisely matched cell surface receptor densities between
experimental groups and control groups, we did not observe functional rescue
between pairs of receptor mutants previously reported to exhibit functional rescue
when co-expressed. Nor did we observe functional rescue between other pairs of
hLHR mutants of our own design. We found, however, that several mutants previously
described as being expressed at the cell surface and to lack binding activity are
misfolded and not expressed at normal levels at the cell surface. Because the
hormone-dependent cAMP response at low levels of cell surface LHR receptor
densities are exquisitely sensitive to changes in cell surface receptor numbers, very
small increases in cell surface receptor expression can manifest as relatively large
increases in hormone-stimulated cAMP responses (Zhang et al. 2005, 2007 ) .