Mating types and pheromone recognition in the Homobasidiomycete schizophyllum commune.

In the homobasidiomycete Schizophyllum commune the mating type genes of the B locus encode pheromones and pheromone receptors in multiple allelic specificities. Interaction of non-self pheromones and receptors leads to induction of B-regulated development easily scored in S. commune by the "flat" phenotype which lacks aerial mycelium formation and shows aberrant hyphal morphology. In contrast, self pheromones are not recognized and B-regulated development is not induced. Natural and mutant alleles of receptors have been analyzed for their specificity in transformation assays, and parts of the receptor involved in ligand discrimination can be described. The biological role of pheromone response in S. commune is assumed to be connected to nuclear migration based on the observation that wild-type cells with a receptor gene of different specificity lead to cells capable of nuclear uptake. Other possible roles for pheromone function are discussed.     

A glucocorticoid/retinoic acid receptor chimera that displays cytoplasmic/nuclear translocation in response to retinoic acid. A real time sensing assay for nuclear receptor ligands

Members of the nuclear receptor superfamily play key roles in a host of physiologic and pathologic processes from embryogenesis to cancer. Some members, including the retinoic acid receptor (RAR), are activated by ligand binding but are unaffected in their subcellular distribution, which is predominantly nuclear. In contrast, several members of the steroid receptor family, including the glucocorticoid receptor, are cytoplasmic and only translocate to the nucleus after ligand binding. We have constructed chimeras between RAR and glucocorticoid receptor that selectively respond to RAR agonists but display cytoplasmic localization in the absence of ligand. These chimeric receptors manifest both nuclear translocation and gene activation functions in response to physiological concentrations of RAR ligands. The ability to achieve regulated subcellular trafficking with a heterologous ligand binding domain has implications both for current models of receptor translocation and for structural-functional conservation of ligand binding domains broadly across the receptor superfamily. When coupled to the green fluorescent protein, chimeric receptors offer a powerful new tool to 1) study mechanisms of steroid receptor translocation, 2) detect dynamic and graded distributions of ligands in complex microenvironments such as embryos, and 3) screen for novel ligands of "orphan" receptors in vivo.     

Ligand Recognition in Multiallelic Pheromone Receptors from the Basidiomycete Schizophyllum commune Studied in Yeast

The homobasidiomycete Schizophyllum commune encodes a multiallelic pheromone receptor system that distinguishes more than 20 nonself from at least 2 self pheromones. The well-investigated pheromone response system of the yeast Saccharomyces cerevisiae was used to link the FUS1::lacZ reporter system to the heterologous pheromone receptors from S. commune. To investigate yeast G-protein binding, the unchanged heterologous receptor was compared to constructs carrying an exchange of the 3rd cytoplasmatic loop for the Ste2 sequence. A better coupling could be achieved with the altered constructs. In order to examine activation by single pheromones, an artificial peptide based on the sequence of a new putative pheromone gene, bap2(1), in the Balpha2 mating-type locus encoding the shortest pheromone found so far in fungal mating types was used. Thus, we have reassembled the pheromone recognition of the basidiomycete S. commune and constructed a system ideal for specificity analysis in the yeast S. cerevisiae.     

Interleukin-4-specific signal transduction events are driven by homotypic interactions of the interleukin-4 receptor alpha subunit.

Interleukin-4 (IL-4) exerts its effects through a heterodimeric receptor complex (IL-4R), which contains the IL-4R(alpha) and gamma(c) subunits. IL-4R(alpha) also functions with other partner subunits in several receptor types, including the IL-13 receptor. To examine the roles of the individual subunits within IL-4R complexes, we employed a chimeric system that recapitulates native IL-4R function as verified by the activation of the kinases, JAK1 and JAK3, and induction of STAT-6. When a mutant gamma(c) subunit in which the four cytoplasmic tyrosines were converted to phenylalanine was paired with the cytoplasmic domain of the IL-4R(alpha) chain, specificity within the JAK-STAT pathway was not altered. Signaling events were examined further in cells expressing the IL-4R(alpha) chimera alone without the gamma(c) chimera. Ligand-induced homodimerization of these receptors activated the IL-4 signaling program despite the absence of gamma(c), including induction of JAK1 and STAT-6, phosphorylation of the insulin-related substrate 1 and cellular proliferation. Thus, homotypic interactions of the IL-4R(alpha) subunit are sufficient for the initiation and determination of IL-4-specific signaling events, and such interactions may be integral to signaling through IL-4R complexes.     

A pheromone-binding protein mediates the bombykol-induced activation of a pheromone receptor in vitro

The enormous capacity of the male silkmoth Bombyx mori in recognizing and discriminating bombykol and bombykal is based on distinct sensory neurons in the antennal sensilla hairs. The hydrophobic pheromonal compounds are supposed to be ferried by soluble pheromone-binding proteins (PBPs) through the sensillum lymph toward the receptors in the dendritic membrane. We have generated stable cell lines expressing the candidate pheromone receptors of B. mori, BmOR-1 or BmOR-3, and assessed their responses to hydrophobic pheromone compounds dissolved by means of dimethyl sulfoxide. BmOR-1-expressing cells were activated by bombykol but also responded to bombykal, whereas cells expressing BmOR-3 responded to bombykal only. In experiments employing the B. mori PBP, no organic solvent was necessary to mediate an activation of BmOR-1 by bombykol, indicating that the PBP solubilizes the hydrophobic compound. Furthermore, the employed PBP selectively mediated a response to bombykol but not to bombykal, supporting a ligand specificity of PBPs. This study provides evidence that both distinct pheromone receptors and PBPs play an important role in insect pheromone recognition.     

Changes in mate recognition through alterations of pheromones and receptors in the multisexual mushroom fungus Schizophyllum commune

Schizophyllum commune has thousands of mating types defined in part by numerous lipopeptide pheromones and their G-protein-coupled receptors. These molecules are encoded within multiple versions of two redundantly functioning B mating-type loci, B alpha and B beta. Compatible combinations of pheromones and receptors, produced by individuals of different B mating types, trigger a pathway of fertilization required for sexual development. Analysis of the B beta 2 mating-type locus revealed a large cluster of genes encoding a single pheromone receptor and eight different pheromones. Phenotypic effects of mutations within these genes indicated that small changes in both types of molecules could significantly alter their specificity of interaction. For example, a conservative amino acid substitution in a pheromone resulted in a gain of function toward one receptor and a loss of function with another. A two-amino-acid deletion from a receptor precluded the mutant pheromone from activating the mutant receptor, yet this receptor was activated by other pheromones. Sequence comparisons provided clues toward understanding how so many variants of these multigenic loci could have evolved through duplication and mutational divergence. A three-step model for the origin of new variants comparable to those found in nature is presented.     

Determinants of ligand binding specificity in the glial cell line-derived neurotrophic factor family receptor alpha S

The glial cell line-derived neurotrophic factor (GDNF) family comprise a subclass of cystine-knot superfamily ligands that interact with a multisubunit receptor complex formed by the c-Ret tyrosine kinase and a cystine-rich glycosyl phosphatidylinositol-anchored binding subunit called GDNF family receptor alpha (GFRalpha). All four GDNF family ligands utilize c-Ret as a common signaling receptor, whereas specificity is conferred by differential binding to four distinct GFRalpha homologues. To understand how the different GFRalphas discriminate ligands, we have constructed a large set of chimeric and truncated receptors and analyzed their ligand binding and signaling capabilities. The major determinant of ligand binding was found in the most conserved region of the molecule, a central domain predicted to contain four conserved alpha helices and two beta strands. Distinct hydrophobic and positively charged residues in this central region were required for binding of GFRalpha1 to GDNF. Interaction of GFRalpha1 and GFRalpha2 with GDNF and neurturin required distinct subsegments within this central domain, which allowed the construction of chimeric receptors that responded equally well to both ligands. C-terminal segments adjacent to the central domain are necessary and have modulatory function in ligand binding. In contrast, the N-terminal domain was dispensable without compromising ligand binding specificity. Ligand-independent interaction with c-Ret also resides in the central domain of GFRalpha1, albeit within a distinct and smaller region than that required for ligand binding. Our results indicate that the central region of this class of receptors constitutes a novel binding domain for cystine-knot superfamily ligands.     

The mating-type locus B alpha 1 of Schizophyllum commune contains a pheromone receptor gene and putative pheromone genes.

Analysis of the multispecific B alpha mating-type locus of Schizophyllum commune provided evidence that pheromones and pheromone receptors govern recognition of self versus non-self and sexual development in this homobasidiomycetous fungus. Four subclones of an 8.2 kb genomic fragment carrying B alpha 1 specificity induced B-regulated sexual morphogenesis when introduced into a strain with one of the eight compatible B alpha specificities that are known to exist in nature. One of these clones, which activated all other B alpha specificities, contains a gene termed bar1. The predicted protein product of bar1, as well as that of bar2, a homologous gene isolated from a B alpha 2 strain, has significant homology to known fungal pheromone receptor proteins in the rhodopsin-like superfamily of G protein-linked receptors. The other three active B alpha 1 clones were subcloned further to identify the minimal active element in each clone. Every active subclone contains a putative pheromone gene ending in a signal for possible isoprenylation. A message of approximately 600 bp was observed for one of these genes, bap1(1). This paper presents the first evidence for a system of multiple pheromones and pheromone receptors as a basis for multispecific mating types in a fungus.     

Activation of family C G-protein-coupled receptors by the tripeptide glutathione

The Family C G-protein-coupled receptors include the metabotropic glutamate receptors, the gamma-aminobutyric acid, type B (GABAB) receptor, the calcium-sensing receptor (CaSR), which participates in the regulation of calcium homeostasis in the body, and a diverse group of sensory receptors that encompass the amino acid-activated fish 5.24 chemosensory receptor, the mammalian T1R taste receptors, and the V2R pheromone receptors. A common feature of Family C receptors is the presence of an amino acid binding site. In this study, a preliminary in silico analysis of the size and shape of the amino acid binding pocket in selected Family C receptors suggested that some members of this family could accommodate larger ligands such as peptides. Subsequent screening and docking experiments identified GSH as a potential ligand or co-ligand at the fish 5.24 receptor and the rat CaSR. These in silico predictions were confirmed using an [3H]GSH radioligand binding assay and a fluorescence-based functional assay performed on wild-type and chimeric receptors. Glutathione was shown to act as an orthosteric agonist at the 5.24 receptor and as a potent enhancer of calcium-induced activation of the CaSR. Within the mammalian receptors, this effect was specific to the CaSR because GSH neither directly activated nor potentiated other Family C receptors including GPRC6A (the putative mammalian homolog of the fish 5.24 receptor), the metabotropic glutamate receptors, or the GABAB receptor. Our findings reveal a potential new role for GSH and suggest that this peptide may act as an endogenous modulator of the CaSR in the parathyroid gland where this receptor is known to control the release of parathyroid hormone, and in other tissues such as the brain and gastrointestinal tract where the role of the calcium receptor appears to subserve other, as yet unknown, physiological functions.     

Localization of functional domains in the Escherichia coli coprogen receptor FhuE and the Pseudomonas putida ferric-pseudobactin 358 receptor PupA

Transport of ferric-siderophores across the outer membrane of gram-negative bacteria is mediated by specific outer membrane receptors. To localize the substrate-binding domain of the ferric-pseudobactin 358 receptor, PupA, of Pseudomonas putida WCS358, we constructed chimeric receptors in which different domains of PupA were replaced by the corresponding domains of the related ferric-pseudobactin receptors PupB and PupX, or the coprogen receptor FhuE of Escherichia coli. None of the chimeric proteins composed of pseudobactin receptor domains facilitated growth on any of the original substrates, or they showed only an extremely low efficiency. However, these receptors enabled cells of Pseudomonas BN8 to grow on media supplemented with uncharacterized siderophore preparations. These siderophore preparations were isolated from the culture supernatant of WCS358 cells carrying plasmids that contain genes of Pseudomonas B10 required for the biosynthesis of pseudobactin B10. Hybrid proteins that contained at least the amino-terminal 516 amino acids of mature FhuE were active as a receptor for coprogen and interacted with the E. coli TonB protein. A chimeric PupA-FhuE protein, containing the amino-terminal 94 amino acids of mature PupA, was also active as a coprogen receptor, but only in the presence of Pseudomonas TonB. It is concluded that the carboxy-terminal domain of ferric-pseudobactin receptors is important, but not sufficient, for ligand interaction, whereas binding of coprogen by the FhuE receptor is not dependent on this domain. Apparently, the ligand-binding sites of different receptors are located in different regions of the proteins. Furthermore, species-specific TonB binding by the PupA receptor is dependent on the amino-terminal domain of the receptor.     

Self-compatible B mutants in coprinus with altered pheromone-receptor specificities

A successful mating in the mushroom Coprinus cinereus brings together a compatible complement of pheromones and G-protein-coupled receptors encoded by multiallelic genes at the B mating-type locus. Rare B gene mutations lead to constitutive activation of B-regulated development without the need for mating. Here we characterize a mutation that arose in the B6 locus and show that it generates a mutant receptor with a single amino acid substitution (R96H) at the intracellular end of transmembrane domain III. Using a heterologous yeast assay and synthetic pheromones we show that the mutation does not make the receptor constitutively active but permits it to respond inappropriately to a normally incompatible pheromone encoded within the same B6 locus. Parallel experiments carried out in Coprinus showed that a F67W substitution in this same pheromone enabled it to activate the normally incompatible wild-type receptor. Together, our experiments show that a single amino acid replacement in either pheromone or receptor can deregulate the specificity of ligand-receptor recognition and confer a self-compatible B phenotype. In addition, we use the yeast assay to demonstrate that different receptors and pheromones found at a single B locus belong to discrete subfamilies within which receptor activation cannot normally occur.     

Molecular dissection of G protein preference using Gsalpha chimeras reveals novel ligand signaling of GPCRs

Although only 16 genes have been identified in mammals, several Galpha subunits can be simultaneously activated by G protein-coupled receptors (GPCRs) to modulate their complicated functions. Current GPCR assays are limited in the evaluation of selective Galpha activation, thus not allowing a comprehensive pathway screening. Because adenylyl cyclases are directly activated by G(s)alpha and the carboxyl termini of the various Galpha proteins determine their receptor coupling specificity, we proposed a set of chimeric G(s)alpha where the COOH-terminal five amino acids are replaced by those of other Galpha proteins and used these to dissect the potential Galpha linked to a given GPCR. Unlike G(q)alpha, G(12)alpha, and G(i)alpha outputs, compounding the signals from several Galpha members, the chimeric G(s)alpha proteins provide a superior molecular approach that reflects the previously uncharacterized pathways of GPCRs under the same cAMP platform. This is, to our knowledge, the first time allowing verification of the whole spectrum of Galpha coupling preference of adenosine A1 receptor, reported to couple to multiple G proteins and modulate many physiological processes. Furthermore, we were able to distinguish the uncharacterized pathways between the two neuromedin U receptors (NMURs), which distribute differently but are stimulated by a common agonist. In contrast to the G(q) signals mainly conducted by NMUR1, NMUR2 routed preferentially to the G(i) pathways. Dissecting the potential Galpha coupling to these GPCRs will promote an understanding of their physiological roles and benefit the pharmaceutical development of agonists/antagonists by exploiting the selective affinity toward a certain Galpha subclass.     

Mutational analysis of ligand-induced activation of the Torpedo acetylcholine receptor.

A number of studies have demonstrated that a major portion of the ligand binding site of the Torpedo nicotinic acetylcholine receptor is near cysteines 192 and 193 of the alpha subunit. The role of conserved tyrosine and aspartate residues within this region in ligand binding and receptor activation was investigated using a combination of site-directed mutagenesis and expression in Xenopus oocytes. Wild-type receptors are half-maximally activated (K1/2) by 20 microM acetylcholine with a Hill coefficient, n, of 1.9. Substitution of alpha Y190 and alpha Y198 with phenylalanines (alpha Y190F, alpha Y198F) or alpha D200 with asparagine (alpha D200N) shifts the K1/2 to 408, 117, and 75 microM, respectively, with no effect on the Hill coefficient. To further study the effects of these mutations on activation, the responses of the receptors to the partial agonists phenyltrimethylammonium (PTMA) and tetramethylammonium (TMA) were examined. Wild-type receptors are half-maximally activated by 73 microM PTMA and 2 mM TMA. In contrast, alpha Y190F, alpha Y198F, and alpha D200N receptors are not activated by PTMA and TMA by concentrations of up to 500 microM or 5 mM, respectively. However, PTMA and TMA do act as competitive antagonists of the mutant receptors, an indication that the binding of these compounds is not abolished by these mutations. Comparison of the the Ki values for TMA and PTMA inhibition with the K 1/2 values for TMA and PTMA activation of wild-type receptors indicates that the affinities of these compounds are similar in wild-type and mutant receptors. Therefore, alpha Y190F, alpha Y198F, and alpha D200N mutations do not significantly alter the affinity of the ligand binding site; rather, these mutations appear to interfere with the coupling of ligand binding to channel opening.     

Molecular similarities in the ligand binding pockets of an odorant receptor and the metabotropic glutamate receptors

The 5.24 odorant receptor is an amino acid sensing receptor that is expressed in the olfactory epithelium of fish. The 5.24 receptor is a G-protein-coupled receptor that shares amino acid sequence identity to mammalian pheromone receptors, the calcium-sensing receptor, the T1R taste receptors, and the metabotropic glutamate receptors (mGluRs). It is most potently activated by the basic amino acids L-lysine and L-arginine. In this study we generated a homology model of the ligand binding domain of the 5.24 receptor based on the crystal structure of mGluR1 and examined the proposed lysine binding pocket using site-directed mutagenesis. Mutants of truncated glycosylated versions of the receptor containing only the extracellular domain were analyzed in a radioligand binding assay, whereas the analogous full-length membrane-bound mutants were studied using a fluorescence-based functional assay. In silico analysis predicted that aspartate 388 interacts with the terminal amino group on the side chain of the docked lysine molecule. This prediction was supported by experimental observations demonstrating that mutation of this residue caused a 26-fold reduction in the affinity for L-lysine but virtually no change in the affinity for the polar amino acid L-glutamine. In addition, mutations in four highly conserved residues (threonine 175, tyrosine 223, and aspartates 195 and 309) predicted to establish interactions with the alpha amino group of the bound lysine ligand greatly reduced or eliminated binding and receptor activation. These results define the essential features of amino acid selectivity within the 5.24 receptor binding pocket and highlight an evolutionarily conserved motif required for ligand recognition in amino acid activated receptors in the G-protein-coupled receptor superfamily.     

Platelet-derived growth factor receptor-beta (PDGFR-beta) activation promotes its association with the low density lipoprotein receptor-related protein (LRP). Evidence for co-receptor function

Activation of the platelet-derived growth factor receptor-beta (PDGFR-beta) leads to tyrosine phosphorylation of the cytoplasmic domain of LRP and alters its association with adaptor and signaling proteins, such as Shc. The mechanism of the PDGF-induced LRP tyrosine phosphorylation is not well understood, especially since PDGF not only activates PDGF receptor but also binds directly to LRP. To gain insight into this mechanism, we used a chimeric receptor in which the ligand binding domain of the PDGFR-beta was replaced with that from the macrophage colony-stimulating factor (M-CSF) receptor, a highly related receptor tyrosine kinase of the same subfamily, but with different ligand specificity. Activation of the chimeric receptor upon the addition of M-CSF readily mediated the tyrosine phosphorylation of LRP. Since M-CSF is not recognized by LRP, these results indicated that growth factor binding to LRP is not necessary for this phosphorylation event. Using a panel of cytoplasmic domain mutants of the chimeric M-CSF/PDGFR-beta, we confirmed that the kinase domain of PDGFR-beta is absolutely required for LRP tyrosine phosphorylation but that PDGFR-beta-mediated activation of phosphatidylinositol 3-kinase, RasGAP, SHP-2, phospholipase C-gamma, and Src are not necessary for LRP tyrosine phosphorylation. To identify the cellular compartment where LRP and the PDGFR-beta may interact, we employed immunofluorescence and immunogold electron microscopy. In WI-38 fibroblasts, these two receptors co-localized in coated pits and endosomal compartments following PDGF stimulation. Further, phosphorylated forms of the PDGFR-beta co-immunoprecipitated with LRP following PDGF treatment. Together, these studies revealed close association between activated PDGFR-beta and LRP, suggesting that LRP functions as a co-receptor capable of modulating the signal transduction pathways initiated by the PDGF receptor from endosomes.     

Pheromones and Pheromone Receptors Are the Primary Determinants of Mating Specificity in the Yeast Saccharomyces Cerevisiae

Saccharomyces cerevisiae has two haploid cell types, a and alpha, each of which produces a unique set of proteins that participate in the mating process. We sought to determine the minimum set of proteins that must be expressed to allow mating and to confer specificity. We show that the capacity to synthesize alpha-factor pheromone and a-factor receptor is sufficient to allow mating by mat alpha 1 mutants, mutants that normally do not express any alpha- or a-specific products. Likewise, the capacity to synthesize a-factor receptor and alpha-factor pheromone is sufficient to allow a ste2 ste6 mutants, which do not produce the normal a cell pheromone and receptor, to mate with wild-type a cells. Thus, the a-factor receptor and alpha-factor pheromone constitute the minimum set of alpha-specific proteins that must be produced to allow mating as an alpha cell. Further evidence that the pheromones and pheromone receptors are important determinants of mating specificity comes from studies with mat alpha 2 mutants, cells that simultaneously express both pheromones and both receptors. We created a series of strains that express different combinations of pheromones and receptors in a mat alpha 2 background. These constructions reveal that mat alpha 2 mutants can be made to mate as either a cells or as alpha cells by causing them to express only the pheromone and receptor set appropriate for a particular cell type. Moreover, these studies show that the inability of mat alpha 2 mutants to respond to either pheromone is a consequence of two phenomena: adaptation to an autocrine response to the pheromones they secrete and interference with response to alpha factor by the a-factor receptor.     

The erythropoietin receptor transmembrane region is necessary for activation by the Friend spleen focus-forming virus gp55 glycoprotein.

The erythropoietin receptor (EPO-R), a member of the cytokine receptor superfamily, can be activated by binding either erythropoietin (EPO) or gp55, the Friend spleen focus-forming virus glycoprotein. The highly specific interaction between gp55 and EPO-R triggers cell proliferation and thereby causes the first stage of Friend virus-induced erythroleukemia. We have generated functional chimeric receptors containing regions of the EPO-R and the interleukin-3 receptor (AIC2A polypeptide), a related cytokine receptor which does not interact with gp55. All chimeric receptors were expressed at similar levels, had similar binding affinities for EPO, and conferred EPO-dependent cell growth. Only those chimeric receptors which contained the EPO-R transmembrane region were activated by gp55. These results demonstrate that the transmembrane region of the EPO-R is critical for activation by gp55. In addition, analysis of a soluble, secreted EPO-R and cysteine point mutants of the EPO-R show that the extracytoplasmic region of the EPO-R specifically interacts with gp55.     

H3 relaxin is a specific ligand for LGR7 and activates the receptor by interacting with both the ectodomain and the exoloop 2

Leucine-rich repeat-containing, G protein-coupled receptors (LGRs) represent a unique subgroup of G protein-coupled receptors with a large ectodomain. Recent studies demonstrated that relaxin activates two orphan LGRs, LGR7 and LGR8, whereas INSL3/Leydig insulin-like peptide specifically activates LGR8. Human relaxin 3 (H3 relaxin) was recently discovered as a novel ligand for relaxin receptors. Here, we demonstrate that H3 relaxin activates LGR7 but not LGR8. Taking advantage of the overlapping specificity of these three ligands for the two related LGRs, chimeric receptors were generated to elucidate the mechanism of ligand activation of LGR7. Chimeric receptor LGR7/8 with the ectodomain from LGR7 but the transmembrane region from LGR8 maintains responsiveness to relaxin but was less responsive to H3 relaxin based on ligand stimulation of cAMP production. The decreased ligand signaling was accompanied by decreases in the ability of H3 relaxin to compete for (33)P-relaxin binding to the chimeric receptor. However, replacement of the exoloop 2, but not exoloop 1 or 3, of LGR7 to the chimeric LGR7/8 restored ligand binding and receptor-mediated cAMP production. These results suggested that activation of LGR7 by H3 relaxin involves specific binding of the ligand to both the ectodomain and the exoloop 2, thus providing a model with which to understand the molecular basis of ligand signaling for this unique subgroup of G protein-coupled receptors.