Identification of surrogate ligands for orphan G protein-coupled receptors

We prepared fusion proteins with an alpha subunit of G protein Gi (Gi1alpha) of 26 orphan G protein-coupled receptors (GPCRs) and with Gsalpha of 10 orphan GPCRs, most of which had been identified from the human genome previously [FEBS Lett 520 (2002) 97]. Ligands for these fusion proteins were screened from a library consisting of approximately 1000 authentic compounds by measuring their effect on [35S]GTPgammaS binding to membrane preparations of insect Sf9 cells expressing these fusion proteins. Eleven compounds were found to act as surrogate agonists for a GPCR-Gsalpha and four GPCR-Gialpha fusion proteins, a compound as an inverse agonist for two GPCR-Gsalpha fusion proteins, and a compound as an endogenous agonist for a GPCR-Gialpha fusion protein.     

Synergistic roles of bone morphogenetic protein 15 and growth differentiation factor 9 in ovarian function

Knockout mouse technology has been used over the last decade to define the essential roles of ovarian-expressed genes and uncover genetic interactions. In particular, we have used this technology to study the function of multiple members of the transforming growth factor-beta superfamily including inhibins, activins, and growth differentiation factor 9 (GDF-9 or Gdf9). Knockout mice lacking GDF-9 are infertile due to a block in folliculogenesis at the primary follicle stage. In addition, recombinant GDF-9 regulates multiple cumulus granulosa cell functions in the periovulatory period including hyaluronic acid synthesis and cumulus expansion. We have also cloned an oocyte-specific homolog of GDF-9 from mice and humans, which is termed bone morphogenetic protein 15 (BMP-15 or Bmp15). To define the function of BMP-15 in mice, we generated embryonic stem cells and knockout mice, which have a null mutation in this X-linked gene. Male chimeric and Bmp15 null mice are normal and fertile. In contrast to Bmp15 null males and Gdf9 knockout females, Bmp15 null females (Bmp15(-/-)) are subfertile and usually have minimal ovarian histopathological defects, but demonstrate decreased ovulation and fertilization rates. To further decipher possible direct or indirect genetic interactions between GDF-9 and BMP-15, we have generated double mutant mice lacking one or both alleles of these related homologs. Double homozygote females (Bmp15(-/-)Gdf9(-/-)) display oocyte loss and cysts and resemble Gdf9(-/-) mutants. In contrast, Bmp15(-/-)Gdf9(+/-) female mice have more severe fertility defects than Bmp15(-/-) females, which appear to be due to abnormalities in ovarian folliculogenesis, cumulus cell physiology, and fertilization. Thus, the dosage of intact Bmp15 and Gdf9 alleles directly influences the destiny of the oocyte during folliculogenesis and in the periovulatory period. These studies have important implications for human fertility control and the maintenance of fertility and normal ovarian physiology.     

A Highlights from MBoC Selection: Synergistic interaction between the fibroblast growth factor and bone morphogenetic protein signaling pathways in lens cells

Fibroblast growth factors (FGFs) play a central role in two processes essential for lens transparency—fiber cell differentiation and gap junction–mediated intercellular communication (GJIC). Using serum-free primary cultures of chick lens epithelial cells (DCDMLs), we investigated how the FGF and bone morphogenetic protein (BMP) signaling pathways positively cooperate to regulate lens development and function. We found that culturing DCDMLs for 6 d with the BMP blocker noggin inhibits the canonical FGF-to-ERK pathway upstream of FRS2 activation and also prevents FGF from stimulating FRS2- and ERK-independent gene expression, indicating that BMP signaling is required at the level of FGF receptors. Other experiments revealed a second type of BMP/FGF interaction by which FGF promotes expression of BMP target genes as well as of BMP4. Together these studies reveal a novel mode of cooperation between the FGF and BMP pathways in which BMP keeps lens cells in an optimally FGF-responsive state and, reciprocally, FGF enhances BMP-mediated gene expression. This interaction provides a mechanistic explanation for why disruption of either FGF or BMP signaling in the lens leads to defects in lens development and function.     

Targeting of bone morphogenetic protein growth factor complexes to fibrillin

Both latent transforming growth factor-beta (TGF-beta)-binding proteins fibrillins are components of microfibril networks, and both interact with members of the TGF-beta family of growth factors. Interactions between latent TGF-beta-binding protein-1 and TGF-beta and between fibrillin-1 and bone morphogenetic protein-7 (BMP-7) are mediated by the prodomain of growth factor complexes. To extend this information, investigations were performed to test whether stable complexes are formed by additional selected TGF-beta family members. Using velocity sedimentation in sucrose gradients as an assay, complex formation was demonstrated for BMP-7 and growth and differentiation factor-8 (GDF-8), which are known to exist in prodomain/growth factor complexes. Comparison of these results with complex formation by BMP-2, BMP-4 (full-length and shortened propeptides), BMP-10, and GDF-5 allowed us to conclude that all, except for BMP-2 and the short BMP-4 propeptides, formed complexes with their growth factors. Using surface plasmon resonance, binding affinities between fibrillin and all propeptides were determined. Binding studies revealed that the N-terminal end of fibrillin-1 serves as a universal high affinity docking site for the propeptides of BMP-2, -4, -7, and -10 and GDF-5, but not GDF-8, and located the BMP/GDF binding site within the N-terminal domain in fibrillin-1. Rotary shadowing electron microscopy of molecules of BMP-7 complex bound to fibrillin-1 confirmed these findings and also showed that prodomain binding targets the growth factor to fibrillin. Immunolocalization of BMP-4 demonstrated fibrillar staining limited to certain tissues, indicating tissue-specific targeting of BMP-4. These data implicate the fibrillin microfibril network in the extracellular control of BMP signaling and demonstrate differences in how prodomains target their growth factors to the extracellular space.     

Suppression of tumor necrosis factor-mediated apoptosis by nuclear factor kappaB-independent bone morphogenetic protein/Smad signaling

The activation of nuclear factor kappaB (NF-kappa B) plays a pivotal role in the regulation of tumor necrosis factor (TNF)-mediated apoptosis. However, little is known about the regulation of TNF-mediated apoptosis by other signaling pathways or growth factors. Here, unexpectedly, we found that bone morphogenetic protein (BMP)-2 and BMP-4 inhibited TNF-mediated apoptosis by inhibition of caspase-8 activation in C2C12 cells, a pluripotent mesenchymal cell line that has the potential to differentiate into osteoblasts depending on BMP stimulation. Utilizing both a trans-dominant IkappaBalpha inhibitor of NF-kappaB expressed in C2C12 cells and IkappaB kinase beta-deficient embryonic mouse fibroblast, we show that BMP-mediated survival was independent of NF-kappaB activation. Rather, the antiapoptotic activity of BMPs functioned through the Smad signaling pathway. Thus, these findings provide the first report of a BMP/Smad signaling pathway that can inhibit TNF-mediated apoptosis, independent of the prosurvival activity of NF-kappaB. Our results suggest that BMPs not only stimulate osteoblast differentiation but can also promote cell survival during the induction of bone formation, offering new insight into the biological functions of BMPs.     

New intracellular components of bone morphogenetic protein/Smad signaling cascades

Bone morphogenetic proteins (BMPs) regulate many processes in the embryo, including cell type specification, patterning, apoptosis, and epithelial-mesenchymal interaction. They also act in soft and hard tissues in adult life. Their signals are transduced from the plasma membrane to the nucleus through a limited number of Smad proteins. The list of Smad-interacting proteins is however growing and it is clear that these partners determine the outcome of the signal. We summarize the present status in BMP/Smad signaling, with emphasis on recently identified Smad partners and how these proteins may cooperate in the regulation of the expression of BMP target genes.     

Involvement of p70 S6 kinase in bone morphogenetic protein signaling: vascular endothelial growth factor synthesis by bone morphogenetic protein-4 in osteoblasts

In the present study, we investigated the effect of bone morphogenetic protein (BMP)-4 on the synthesis of vascular endothelial growth factor (VEGF) in osteoblast-like MC3T3-E1 cells. BMP-4 significantly stimulated VEGF synthesis time-dependently up to 48 h. The stimulatory effect was dose-dependent in the range between 1 and 100 ng/ml. BMP-4 time-dependently phosphorylated p70 S6 kinase. Rapamycin, an inhibitor of p70 S6 kinase, suppressed the BMP-4-stimulated VEGF synthesis as well as the phosphorylation of p70 S6 kinase. The VEGF synthesis by BMP-4 was suppressed by wortmannin and LY294002, inhibitors of phosphatidylinositol 3-kinase. Both wortmannin and LY294002 inhibited the BMP-4-stimulated phosphorylation of p70 S6 kinase. BMP-4 did not affect the phosphorylation of Akt/protein kinase B. Taken together, our results strongly suggest that p70 S6 kinase takes part in BMP-4-stimulated VEGF synthesis as a positive regulator in osteoblasts and that phosphatidylinositol 3-kinase acts at a point upstream from p70 S6 kinase.     

Golgi apparatus casein kinase phosphorylates bioactive Ser-6 of bone morphogenetic protein 15 and growth and differentiation factor 9

Bone morphogenetic protein-15 (BMP-15) and growth and differentiation factor-9 (GDF-9) are oocyte-secreted factors that play essential roles in human folliculogenesis and ovulation. Their bioactivity is tightly regulated through phosphorylation, likely to occur within the Golgi apparatus of the secretory pathway. Here we show that Golgi apparatus casein kinase (G-CK) catalyzes the phosphorylation of rhBMP-15 and rhGDF-9. rhBMP-15, in particular, is an excellent substrate for G-CK. In each protein a single residue is phosphorylated by G-CK, corresponding to the serine residue at the sixth position of the mature region of both rhBMP-15 and rhGDF-9, whose phosphorylation is required for biological activity.     

Growth differentiation factor 9 regulates expression of the bone morphogenetic protein antagonist gremlin

Growth differentiation factor 9 (GDF9) is an oocyte-expressed member of the transforming growth factor beta (TGF-beta) superfamily and is required for normal ovarian follicle development and female fertility. GDF9 acts as a paracrine factor and affects granulosa cell physiology. Only a few genes regulated by GDF9 are known. Our microarray analysis has identified gremlin as one of the genes up-regulated by GDF9 in cultures of granulosa cells. Gremlin is a known member of the DAN family of bone morphogenetic protein (BMP) antagonists, but its expression and function in the ovary are unknown. We have investigated the regulation of gremlin in mouse granulosa cells by GDF9 as well as other members of the TGF-beta superfamily. GDF9 and BMP4 induce gremlin, but TGF-beta does not. In addition, in cultures of granulosa cells, gremlin negatively regulates BMP4 signaling but not GDF9 activity. The expression of gremlin in the ovary was also examined by in situ hybridization. A distinct change in gremlin mRNA compartmentalization occurs during follicle development and ovulation, indicating a highly regulated expression pattern during folliculogenesis. We propose that gremlin modulates the cross-talk between GDF9 and BMP signaling that is necessary during follicle development because both ligands use components of the same signaling pathway.     

Roles of bone morphogenetic protein type I receptors and Smad proteins in osteoblast and chondroblast differentiation

The biological effects of type I serine/threonine kinase receptors and Smad proteins were examined using an adenovirus-based vector system. Constitutively active forms of bone morphogenetic protein (BMP) type I receptors (BMPR-IA and BMPR-IB; BMPR-I group) and those of activin receptor-like kinase (ALK)-1 and ALK-2 (ALK-1 group) induced alkaline phosphatase activity in C2C12 cells. Receptor-regulated Smads (R-Smads) that act in the BMP pathways, such as Smad1 and Smad5, also induced the alkaline phosphatase activity in C2C12 cells. BMP-6 dramatically enhanced alkaline phosphatase activity induced by Smad1 or Smad5, probably because of the nuclear translocation of R-Smads triggered by the ligand. Inhibitory Smads, i.e., Smad6 and Smad7, repressed the alkaline phosphatase activity induced by BMP-6 or the type I receptors. Chondrogenic differentiation of ATDC5 cells was induced by the receptors of the BMPR-I group but not by those of the ALK-1 group. However, kinase-inactive forms of the receptors of the ALK-1 and BMPR-I groups blocked chondrogenic differentiation. Although R-Smads failed to induce cartilage nodule formation, inhibitory Smads blocked it. Osteoblast differentiation induced by BMPs is thus mediated mainly via the Smad-signaling pathway, whereas chondrogenic differentiation may be transmitted by Smad-dependent and independent pathways.     

Cross-talk between bone morphogenetic protein and transforming growth factor-beta signaling is essential for exendin-4-induced insulin-positive differentiation of AR42J cells

A key goal of cellular engineering is to manipulate progenitor cells to become beta-cells, allowing cell replacement therapy to cure diabetes mellitus. As a paradigm for cell engineering, we have studied the molecular mechanisms by which AR42J cells become beta-cells. Bone morphogenetic proteins (BMPs), implicated in a myriad of developmental pathways, have not been well studied in insulin-positive differentiation. We found that the canonical intracellular mediators of BMP signaling, Smad-1 and Smad-8, were significantly elevated in AR42J cells undergoing insulin-positive differentiation in response to exendin-4 treatment, suggesting a role for BMP signaling in beta-cell formation. Similarly, endogenous BMP-2 ligand and ALK-1 receptor (activin receptor-like kinase-1; known to activate Smads 1 and 8) mRNAs were specifically up-regulated in exendin-4-treated AR42J cells. Surprisingly, Smad-1 and Smad-8 levels were suppressed by the addition of BMP-soluble receptor inhibition of BMP ligand binding to its receptor. Here, insulin-positive differentiation was also ablated. BMP-2 ligand antisense also strongly inhibited Smad-1 and Smad-8 expression, again with the abolition of insulin-positive differentiation. These results demonstrate a previously unrecognized key role for BMP signaling in mediating insulin-positive differentiation through the intracellular Smad signaling pathway. In short, BMP signaling may represent a novel downstream target of exendin-4 (glucagon-like peptide 1) signaling and potentially serve as an upstream regulator of transforming growth factor-beta isoform signaling to differentiate the acinar-like AR42J cells into insulin-secreting cells.     

Identification of specific ligands for orphan olfactory receptors. G protein-dependent agonism and antagonism of odorants

Olfactory receptors are the largest group of orphan G protein-coupled receptors with an infinitely small number of agonists identified out of thousands of odorants. The de-orphaning of olfactory receptor (OR) is complicated by its combinatorial odorant coding and thus requires large scale odorant and receptor screening and establishing receptor-specific odorant profiles. Here, we report on the stable reconstitution of OR-specific signaling in HeLa/Olf cells via G protein alphaolf and adenylyl cyclase type-III to the Ca2+ influx-mediating olfactory cyclic nucleotide-gated CNGA2 channel. We demonstrate the central role of Galphaolf in odorant-specific signaling out of OR. The employment of the non-typical G protein alpha15 dramatically altered the odorant specificities of 3 of 7 receptors that had been characterized previously by different groups. We further show for two OR that an odorant may be an agonist or antagonist, depending on the G protein used. HeLa/Olf cells proved suitable for high-throughput screening in fluorescence-imaging plate reader experiments, resulting in the de-orphaning of two new OR for the odorant (-)citronellal from an expression library of 93 receptors. To demonstrate the G protein dependence of its odorant response pattern, we screened the most sensitive (-)citronellal receptor Olfr43 versus 94 odorants simultaneously in the presence of Galpha15 or Galphaolf. We finally established an EC50-ranking odorant profile for Olfr43 in HeLa/Olf cells. In summary, we conclude that, in heterologous systems, odorants may function as agonists or antagonists, depending on the G protein used. HeLa/Olf cells provide an olfactory model system for functional expression and de-orphaning of OR.