Barbara Lindau-Shepard

Molecular, structural, and cellular biology of follitropin and follitropin receptor

Follitropin and the follitropin receptor are essential for normal gamete development in males and females. This review discusses the molecular genetics and structural and cellular biology of the follitropin/follitropin receptor system. Emphasis is placed on the human molecules when possible. The structure and regulation of the genes for the follitropin beta subunit and the follitropin receptor is discussed. Control of systemic and cellular protein levels is explained. The structural biology of each protein is described, including protein structure, motifs, and activity relationships. Finally, the follitropin/follitropin receptor signal transduction system is discussed.

Hormone-induced conformational change of the purified soluble hormone binding domain of follitropin receptor complexed with single chain follitropin.

Human follicle-stimulating hormone receptor (hFSHR) belongs to family I of G protein-coupled receptors. FSHR extracellular domain (ECD) is predicted to have 8–9 αβ or leucine-rich repeat motif elements. The objective of this study was to identify elements of the FSHR ECD involved in ligand binding. Preincubation of recombinant hFSHR ECD with rabbit antisera raised against synthetic peptides of hFSHR ECD primary sequence abolished follitropin binding primarily in the region of amino acids 150–254. Accessibility of hFSHR ECD after hormone binding, captured by monoclonal antibodies against either ECD or FSH, was decreased for the region of amino acids 150–220 but additionally for amino acids 15–100. Thus, when hFSH bound first, accessibility of antibody binding was decreased to a much larger extent than if antibody was bound first. This suggestion of a conformational change upon binding was examined further. Circular dichroism spectra were recorded for purified single chain hFSH, hFSHR ECD, and hFSHR ECD-single chain hFSH complex. A spectral change indicated a small but consistent conformational change in the ECD·FSH complex after hormone binding. Taken together, these data demonstrate that FSH binding requires elements within the leucine-rich repeat motifs that form a central region of hFSHR ECD, and a conformational change occurs upon hormone binding.

Transfection of a manganese-containing superoxide dismutase gene into hamster tracheal epithelial cells ameliorates asbestos-mediated cytotoxicity

To determine if overexpression of manganese-containing SOD (MnSOD) alters cell sensitivity to asbestos, an expression cassette containing murine MnSOD cDNA was cotransfected with pSV2neo, a plasmid conferring resistance to the antibiotic G418, into a diploid cell line of hamster tracheal epithelial (HTE) cells. Pools of G418-resistant transfectants were characterized by Southern and Northern blot analyses and enzyme activity assays. Although increases in MnSOD gene copies in individual cell pools ranged from approximately 7- to 86-fold in comparison to cells transfected with pSV2neo alone, steady-state levels of MnSOD mRNA were increased only by 1.4-to 2.3-fold. Despite modest increases in MnSOD mRNA, significant elevations in MnSOD enzyme activity were observed in pools of G418-resistant cells. MnSOD-transfected cell lines were more resistant to the cytotoxic effects of crocidolite asbestos using a sensitive colony-forming efficiency (CFE) assay. These data show that MnSOD has a direct role in cell defense against asbestos-induced cytotoxicity, an oxidant-dependent process.

Reversible immunoneutralization of human follitropin receptor.

Investigations of the human Follitropin receptor (hFSHR) have failed to identify the tertiary structure that forms the active hormone-receptor interaction site which is essential to develop an immunocontraceptive based upon the receptor. To identify such a domain of hFSHR, an immunoneutralizing monoclonal antibody (mAb) 106-105 (IgG2b) was generated. Flow cytometry tested whether mAb 106-105 recognized native hFSHR. The epitope of mAb 106-105 was mapped by Western blot and by peptide ELISA. Inhibition of hFSH binding and bioactivity was determined by radioreceptor assay and by cAMP production, respectively. MAb 106-105 bound native hFSHR through an epitope including residues 300-315. MAb 106-105 completely blocked hormone binding to receptor and cAMP production by Y1-R cells expressing hFSHR. These effects were completely reversible by increasing the concentration of hFSH. Coincubation of this antibody with peptide D300-F315 blocked antibody activity. These data demonstrate that a discrete linear hFSHR epitope is a target for interference with hormone activity. These results further demonstrate that antibody binding to the extracellular domain (ECD) of hFSHR and subsequent bioactivation can be modulated through a domain specific hindrance, offering a reversible immunoneutralizing target.

Structural determinants in the second intracellular loop of the human follicle-stimulating hormone receptor are involved in Gs protein activation

In the present study, we analyzed the structural determinants present in the second intracellular loop (IL-2) of the human follicle-stimulating hormone (FSH) receptor (R) involved in G(s) protein-mediated signal transduction. Human embryonic kidney 293 (HEK-293) cells, stably expressing wild-type (Wt) human FSHR (HEK-293((+))), were transiently transfected with plasmids containing cDNAs encoding the entire IL-2 or several IL-2 sequences mutated in R467 (a residue located at the center of the conserved ERW motif in the glycoprotein hormone receptors), T470 (a potential site for phosphorylation by protein kinase-A and -C) or L477 (a residue conserved in all glycoprotein hormone receptors). Expression of the IL-2 Wt in HEK-293((+)) cells reduced the maximum FSH-stimulated cAMP production significantly by approximately 40%; similar results were observed with the R467A and R467K IL-2 mutants. The IL-2(R467H), IL-2(T470A), the triple R467A/T470A/L477A IL-2 mutant and the IL-2 of the oxytocin receptor (G(q/11)-coupled) had no effects on Wt FSHR-mediated intracellular signaling whereas the L477A mutation provoked a higher ( approximately 55%) inhibition of FSH-stimulated cAMP than the free, Wt IL-2. These results suggested a specific role of IL-2 residues in FSHR function. Site directed mutagenesis of the FSHR and the expression of resulting mutants in HEK-293 cells were performed in order to corroborate the effects of these substitutions. Expression of FSHR(R467H), FSHR(R467A) and FSHR(T470A) failed to mediate ligand-provoked G(s) protein activation, whereas the R467K mutant behaved as the Wt receptor. Interestingly, the expression of L477A, L477D and L477P FSHR mutants conferred elevated basal cAMP levels to HEK-293 cells. This study indicates that the IL-2 of the human FSHR possesses amino acid residues that are important for both coupling the receptor to the G(s) protein (R467 and T470) and maintaining the receptor molecule in an inactive conformation (L477). It appears that this particular intracellular domain may act as a conformational switch to produce the activation of G proteins as has been reported for the IL-2 of other G protein-coupled receptors.

Expression of human catalase in acatalasemic murine SV-B2 cells confers protection from oxidative damage

Reactive oxygen species have been implicated in aerobic organisms as causative agents in damage to DNA, proteins, and lipids. Catalase is a major enzyme in the defense against such oxidant damage. To determine whether increased catalase expression confers greater resistance to oxidant stress, a eukaryotic expression vector harboring a human catalase cDNA clone was constructed. Acatalasemic murine fibroblasts were then co-transfected with that catalase expression vector and pSV2-neo, and successfully transfected cells were identified by their ability to grow in the presence of geneticin. Clones that contained integrated copies of the catalase expression vector were identified by Polymerase Chain Reaction (PCR) analysis. Stably transfected geneticin-resistant cell lines that overexpressed catalase in potentially positive cell lines were confirmed by catalase enzyme assays. To examine the physiological relevance of catalase overexpression, cells were exposed to oxidant stresses (hydrogen peroxide and hyperoxia), and survival rates were determined. Results demonstrated a significant resistance to oxidative stress in cells overexpressing catalase when compared to controls. These transfected cell lines will provide important models for further evaluation of the role of catalase in protecting cells against the toxic effects of oxygen-derived free radicals and their derivatives.

Single-Molecule Analyses of Fully Functional Fluorescent Protein-Tagged Follitropin Receptor Reveal Homodimerization and Specific Heterodimerization with Lutropin Receptor

ABSTRACT We have previously shown that the carboxyl terminus (cT) of human follicle-stimulating hormone (FSH, follitropin) receptor (FSHR) is clipped before insertion into the plasma membrane. Surprisingly, several different constructs of FSHR fluorescent fusion proteins (FSHR-FPs) failed to traffic to the plasma membrane. Subsequently, we discovered that substituting the extreme cT of luteinizing hormone (LH) receptor (LHR) to create an FSHR-LHRcT chimera has no effect on FSHR functionality. Therefore, we used this approach to create an FSHR-LHRcT-FP fusion. We found this chimeric FSHR-LHRcT-FP was expressed in HEK293 cells at levels similar to reported values for FSHR in human granulosa cells, bound FSH with high affinity, and transduced FSH binding to produce cAMP. Quantitative fluorescence resonance energy transfer (FRET) analysis of FSHR-LHRcT-YFP/FSHR-LHRcT-mCherry pairs revealed an average FRET efficiency of 12.9 ± 5.7. Advanced methods in single-molecule analyses were applied in order to ascertain the oligomerization state of the FSHR-LHRcT. Fluorescence correlation spectroscopy coupled with photon-counting histogram analyses demonstrated that the FSHR-LHRcT-FP fusion protein exists as a freely diffusing homodimer in the plasma membrane. A central question is whether LHR could oligomerize with FSHR, because both receptors are coexpressed in differentiated granulosa cells. Indeed, FRET analysis revealed an average FRET efficiency of 14.4 ± 7.5 when the FSHR-LHR cT-mCherry was coexpressed with LHR-YFP. In contrast, coexpression of a 5-HT2cVSV-YFP with FSHR-LHR cT-mCherry showed only 5.6 ± 3.2 average FRET efficiency, a value indistinguishable from the detection limit using intensity-based FRET methods. These data demonstrate that coexpression of FSHR and LHR can lead to heterodimerization, and we hypothesize that it is possible for this to occur during granulosa cell differentiation.

Human follicle stimulating hormone receptor variants lacking transmembrane domains display altered post-translational conformations.

Variant splicing of gonadotropin receptor mRNA commonly occurs, however expression of receptor protein variants and their trafficking has yet to be studied in detail. To determine receptor variant trafficking and intracellular processing in mammalian cells, the intracellular fate of intentionally truncated variants of human follicle stimulating hormone receptor (hFSH-R) expressed in CHO cells was examined. Monoclonal antibodies (mAbs) were made against the hFSH-Rs extracellular domain (ECD) expressed in insect cells. Four mAbs 106.156, 106.290, 106.318, and 106.263 were chosen as probes. Epitope mapping using synthetic peptides, and truncated hFSH-R variants revealed that mAb 106.156 bound to ECD residues 183-220, while mAbs 106.318, 106.290, 106.263 bound ECD residues 300-331. Immunofluorescence microscopy showed that mAbs 106.318 and 106.156 stained the surface of fixed, intact CHO cells expressing wild type hFSH-R. However, following cell permeabilization all four antibodies stained hFSH-R in Golgi and endoplasmic reticulum. Permeabilized cells expressing truncated variants ECD213 and ECD254 showed staining accumulated in the endoplasmic reticulum/nuclear envelope continuum. ECD335/His was found to accumulate in extended endoplasmic reticulum (ER). The ER location of ECD335/His was confirmed by double labeling experiments with concanavalin A and ECD mAb. Glycosidase digestion followed by Western blot analysis show ECD213 and ECD335/His to be glycosylated, but not ECD254. Both glycosylated truncated hFSH-R variants were sensitive to peptide-N-glycanase F and endoglycosidase H but insensitive to neuraminidase indicating that these variants possess high mannose type oligosaccharides. Thus truncated hFSH-R variants do not reach the medial or trans Golgi where high mannose oligosaccharides are trimmed and sialic acid is added. These data suggest that the conformation the ECD of the wild type receptor is different from the ECD alone expressed in the endoplasmic reticulum. This information suggests that the ECD serves two distinct roles; the first is to bind FSH and the other is likely to contact the endodomain of the receptor, which presumably leads to activation of the endodomain for signal transduction.

A pollicle-stimulating hormone receptor ecto-domain epitope that is a target for receptor immunoneutralization yet does not affect ligand contact and activation

The follicle-stimulating hormone receptor (FSHR) large extracellular domain suggests that interaction of ligand with receptor is likely to be complex. Residues 265–296 of the FSHR are part of a sequence primarily nonhomologous with other glycoprotein hormone receptors. A reasonable hypothesis is that this sequence of the FSHR plays a role in binding FSH. Flow cytometry studies of this region revealed that antibody x179 against peptide R265–S296 bins to humans FSHR expressed by CHO cells and can be competed against by preincubating the cells with hFSH. These results suggested that the region corresponding to residues 265–296 in the extracellular domain of the FSHR is involved in binding to hormone. To test this hypothesis 10 scanning alanine mutants of rFSH at the 265–296 epitope were generated, and the binding characteristics of these mutants were studied. Their affinity constants for 125I-hFSH did not deviate greatly from that of wild-type FSHR, in which some mutants exhibited an approximately two- to threefold reduction in Ka compared to wild-type receptor, and no mutation abolished signal transduction. These results lead to rejection of the hypothesis that this region contains residues critical for conveying hormone specificity and receptor-dependent hormone action.