Trans-Activation of Mutant Follicle-Stimulating Hormone Receptors Selectively Generates Only One of Two Hormone Signals

Abstract

Previously, we reported that a liganded LH receptor (LHR) is capable of activating itself (cis-activation) and other nonliganded LHRs to induce cAMP (trans-activation). Trans-activation of the LHR raises two crucial questions. Is trans-activation unique to LHR or common to other G protein-coupled receptors? Does trans-activation stimulate phospholipase Cbeta as it does adenylyl cyclase? To address these questions, two types of novel FSH receptors (FSHRs) were constructed, one defective in hormone binding and the other defective in signal generation. The FSHR, a G protein-coupled receptor, comprises two major domains, the N-terminal extracellular exodomain that binds the hormone and the membrane-associated endodomain that generates the hormone signals. For signal defective receptors, the exodomain was attached to glycosyl phosphatidylinositol (ExoGPI) or the transmembrane domain of CD8 immune receptor (ExoCD). ExoGPI and ExoCD can trans-activate another nonliganded FSH. Surprisingly, the trans-activation generates a signal to activate either adenylyl cyclase or phospholipase Cbeta, but not both. These results indicate that trans-activation in these mutant receptors is selective and limited in signal generation, thus providing new approaches to investigating the generation of different hormone signals and a novel means to selectively generate a particular hormone signal. Our data also suggest that the FSHR's exodomain could not trans-activate LHR.

Rescue of defective G protein–coupled receptor function in vivo by intermolecular cooperation

“…As expected, LHR LH− showed no specific ligand binding and LHR cAMP− no signaling, but when the two receptor mutants were coexpressed, the cAMP response to hCG stimulation was partially restored (Fig. 1 C-E), confirming earlier reports with similar receptor mutants (35) or even by more defective mutants, such as the exo-domain of the FSHR linked to a cell membrane phospholipid which is capable of activating a binding-deficient mutant (36).…”

“…We selected two LHR mutant receptors for their inability to bind the ligand (LH or hCG) or to transduce signaling after ligand binding. The first mutant receptor (LHR LH− ) harbored an inactivating Cys 22 to Ala 22 mutation in the ligand binding extracellular domain (35,36). The second mutant (LHR cAMP− ) contained a deletion of TM helices 6 and 7 in exon 11 (amino acids deleted from Val 553 to Ala 689 ), and it was chosen because of involvement of the deleted region in G protein coupling and second messenger generation (37).…”

“…Moreover, the LHR cAMP− mutant was able to bind hCG in testis homogenates without any phenotypic signs of LHR activa- tion, and the LHR LH− mutant was not able to bind hCG. Previous cell culture studies on expression of complementary receptor mutants have shown that the observed intermolecular cooperation was not due to rescue of one mutant, trapped in the endoplasmic reticulum (ER), by the other to the cell membrane, but to direct interaction of two complementary mutants at the cell membrane (33,35), as was also the case with the intermolecular cooperation of FSHRs (36). Furthermore, overexpression of the mutant receptors alone in HEK-293 cells could not demonstrate any cAMP signaling activity, indicating that the mutant receptors do not attain spurious activation as monomers even when overexpressed.…”

“…Earlier cell culture studies have revealed quantitative difference in the cAMP and phosphoinositide responses of gonadotropin receptors upon their cis-and trans-activation (33,36,46), but more pronounced qualitative differences in the responses to the two modes of receptor activation are possible. The only difference we found between the WT and LHR LH−/cAMP− mice was the 2-fold elevation of LH in the latter, suggesting that the trans-activated receptor complex is slightly less sensitive or less responsive to stimulation than the WT receptor.…”

Crosstalk in G protein-coupled receptors: Changes at the transmembrane homodimer interface determine activation

“…In the family C heterodimeric GABA B receptor, the N terminus of GB1 (but not GB2) binds GABA (13), whereas cytoplasmic loops of GB2 couple to G protein (14-16). Transactivation has also been reported for a family C metabotropic glutamate receptor and for the family A leutinizing hormone (LH), follicle-stimulating hormone (FSH), and thyroid-stimulating hormone (TSH) receptors (17)(18)(19)(20).An understanding of the structural basis of crosstalk between receptors in a dimer requires identification of the dimerization interface and its changes, but information about these interfaces is still rather limited. Based on the spatial arrangement of rhodopsin arrays visualized by atomic force microscopy (AFM) of mouse retinal disk membranes in the inactive state, Liang et al (21) built a molecular model that features a symmetric homodimer interface involving both transmembrane segment (TM) 4 and TM5 (Fig.…”