ABSTRACT
G protein-coupled receptors (GPCRs) in humans are classified into the five main families named Glutamate, Rhodopsin, Adhesion, Frizzled and Secretin according to the GRAFS classification. Previous results show that these mammalian GRAFS families are well represented in the Metazoan lineages, but they have not been shown to be present in Fungi. Here, we systematically mined 79 fungal genomes and provide the first evidence that four of the five main mammalian families of GPCRs, namely Rhodopsin, Adhesion, Glutamate and Frizzled, are present in Fungi and found 142 novel sequences between them. Significantly, we provide strong evidence that the Rhodopsin family emerged from the cAMP receptor family in an event close to the split of Opisthokonts and not in Placozoa, as earlier assumed. The Rhodopsin family then expanded greatly in Metazoans while the cAMP receptor family is found in 3 invertebrate species and lost in the vertebrates. We estimate that the Adhesion and Frizzled families evolved before the split of Unikonts from a common ancestor of all major eukaryotic lineages. Also, the study highlights that the fungal Adhesion receptors do not have N-terminal domains whereas the fungal Glutamate receptors have a broad repertoire of mammalian-like N-terminal domains. Further, mining of the close unicellular relatives of the Metazoan lineage, Salpingoeca rosetta and Capsaspora owczarzaki, obtained a rich group of both the Adhesion and Glutamate families, which in particular provided insight to the early emergence of the N-terminal domains of the Adhesion family. We identified 619 Fungi specific GPCRs across 79 genomes and revealed that Blastocladiomycota and Chytridiomycota phylum have Metazoan-like GPCRs rather than the GPCRs specific for Fungi. Overall, this study provides the first evidence of the presence of four of the five main GRAFS families in Fungi and clarifies the early evolutionary history of the GPCR superfamily.
Subject(s)
Cell Adhesion Molecules/isolation & purification , Frizzled Receptors/isolation & purification , Fungi/genetics , Receptors, G-Protein-Coupled/classification , Receptors, G-Protein-Coupled/genetics , Receptors, Glutamate/isolation & purification , Rhodopsin/isolation & purification , Amino Acid Sequence , Animals , Cell Adhesion Molecules/genetics , Cloning, Molecular , Evolution, Molecular , Frizzled Receptors/genetics , Fungi/physiology , Genes, Fungal , Genome, Fungal , Humans , Mammals/genetics , Molecular Sequence Data , Phylogeny , Receptors, Glutamate/genetics , Rhodopsin/geneticsABSTRACT
Secreted frizzled related protein-1 (sFRP-1) inhibitors have the potential to be used for the treatment of osteoporosis or other bone related disorders, since the level of sFRP-1 affects osteoblast apoptosis and proliferation. From high throughput screening, we have identified a class of iminooxothiazolidines as sFRP-1 inhibitors. Structure-activity relationships were established for various regions of the scaffold along with the biochemical characterization of this class to probe selectivity, binding and ex vivo activity.
Subject(s)
Osteogenesis/physiology , Proteins/isolation & purification , Calcification, Physiologic , Cell Differentiation/physiology , Cells, Cultured , Frizzled Receptors/antagonists & inhibitors , Frizzled Receptors/isolation & purification , Intracellular Signaling Peptides and Proteins , Molecular Structure , Proteins/antagonists & inhibitors , RANK LigandABSTRACT
Baculovirus mediated gene transduction of mammalian cells (BacMam) is an emerging technique for rapid recombinant protein expression in mammalian cells. We constructed two baculovirus transfer vectors that incorporate several mammalian transcriptional regulatory elements necessary for high-level protein expression in mammalian cells. Using these vectors, we show that the BacMam system in combination with the 293 GnTI(-) cell line can be used for production of milligram quantities of soluble glycoproteins. Moreover, for crystallization trials, the purified glycoproteins are sensitive to EndoH treatment resulting in a loss of the bulk of the attached N-linked glycosylation. In addition, we also show that a combination of the BacMam system and 293 GnTI(-) cell line can be used for producing milligram quantities of a GPCR-protein ligand complex suitable for crystallization trials.
Subject(s)
Baculoviridae/metabolism , Membrane Glycoproteins/chemistry , Membrane Glycoproteins/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Transduction, Genetic/methods , Animals , Base Sequence , Cell Line , Chemokine CX3CL1/isolation & purification , Chemokine CX3CL1/metabolism , Crystallography, X-Ray , Frizzled Receptors/chemistry , Frizzled Receptors/isolation & purification , Frizzled Receptors/metabolism , Genetic Vectors/genetics , Humans , Mice , Molecular Sequence Data , Protein Structure, Tertiary , Receptor, Parathyroid Hormone, Type 1/chemistry , Receptor, Parathyroid Hormone, Type 1/isolation & purification , Receptor, Parathyroid Hormone, Type 1/metabolism , Receptors, Chemokine/isolation & purification , Receptors, Chemokine/metabolism , Receptors, G-Protein-Coupled/chemistry , Receptors, G-Protein-Coupled/isolation & purification , Receptors, G-Protein-Coupled/metabolism , Solubility , Transfection , Viral Proteins/isolation & purification , Viral Proteins/metabolismABSTRACT
The Wnt signal acts by binding to Frizzled receptors, with the subsequent activation of two different signal transduction cascades, the canonical and the non-canonical Wnt pathways, involved in cell growth, differentiation, migration and fate. The canonical pathway functions through the translocation of beta-catenin to the nucleus and the activation of TCF/LEF transcription factors; it plays an important role in developmental patterning and cell fate decisions during embryogenesis. The non-canonical Wnt pathway is responsible for the planar cell polarity process in invertebrates, and for the convergent-extension movements during vertebrate gastrulation. The final effect of the non-canonical Wnt pathway is the rearrangement of the cell cytoskeleton, through the activation of the subfamily of Ras-like small GTPases. In a recent report we described for the first time the isolation of a Wnt-related gene, Sd-Frizzled, from the most basal animal phylum, the Porifera. In the present study we report the isolation and phylogenetic characterization of several Wnt pathway-related genes from the sponge Suberites domuncula: Sd-TCF/LEF, Sd-GSK3, a recently discovered molecule with a putative function as a Wnt regulator (Sd-LZIC), the small Rho GTPases Sd-RhoA, Sd-Cdc42, and their effector Sd-mrlc. Also the isolation of a secreted frizzled related protein sFRP from another sponge species (Lubomirskia baicalensis) is reported.
