Functional and structural identification of the WT FSHR variant I1, and M3 mutant (D408Y).

(A) Representative Western blot of FSHR protein variant I1 (WT FSHR) and mutant M3 (D408Y). The immunoblot shows the relevant portion of an autoradiogram in which the mature [80 kDA (m)], plasma membrane-expressed, fully glycosylated form of the D408Y mutant is considerably reduced compared with that of the WT FSHR I1 variant. The blot shows the migration of FSHRs from protein extracts of HEK293 cells transiently transfected with the WT or mutant FSHR cDNAs inserted either in the pSG5 or pcDNA3.1 vectors. The first lane from left to right, shows the migration of the WT FSHR I1 variant from HEK293 cells stably expressing the receptor. The immunoblot also shows that the majority of the D408Y mutant FSHR is present as immature, intracellular forms [kDa ≤ 75; (i)]. (B) FSH-stimulated production of cAMP by cells expressing the WT or D408Y FSHRs. Compared to the WT receptor, cAMP production was significantly reduced in the M3 mutant (areas under the FSH-stimulated dose-response cAMP curve (pmol/well) = 8218 ± 275 and 4224 ± 346 (means ± SEM) for the WT and M3 FSHR, respectively (pt test). (C) Structure domains of the FSHR (magenta ribbons) in the context of the membrane lipid bilayer (white spheres). The seven helix transmembrane domain corresponds to the model of the I1 variant in the lipid bilayer, and the ectodomain was taken from the crystal structure PDB:1XDW [6]. Solvent water molecules were not represented for clarity. Also shown are atoms for residues D408 and N680 depicted as spheres and colored by atom names using the code: C-cyan, O-red, N-blue, and H-white. The hinge region links the transmembrane domain and the ectodomain. (D) Sequence alignments showing the location of the point mutations at the TM helix 2 and the Ctail of the FSHR variants I1 and I2, and mutants M1-3.