The formation of polyglutamine-containing aggregates and inclusions are hallmarks of pathogenesis in Huntington's disease that can be recapitulated in model systems. Although the contribution of inclusions to pathogenesis is unclear, cell-based assays can be used to screen for chemical compounds that affect aggregation and may provide therapeutic benefit. We have developed inducible PC12 cell-culture models to screen for loss of visible aggregates. To test the validity of this approach, compounds that inhibit aggregation in the PC12 cell-based screen were tested in a Drosophila model of polyglutamine-repeat disease. The disruption of aggregation in PC12 cells strongly correlates with suppression of neuronal degeneration in Drosophila. Thus, the engineered PC12 cells coupled with the Drosophila model provide a rapid and effective method to screen and validate compounds.

The crystal structure of the pheromone Er-I from the unicellular eukaryotic organism Euplotes raikovi was determined at 1.6 A resolution and refined to a crystallographic R factor of 19.9%. In the tightly packed crystal, two extensive intermolecular helix-helix interactions arrange the Er-i molecules into layers. Since the putative receptor of the pheromone is a membrane-bound protein, whose extracellular C-terminal domain is identical in amino acid sequence to the soluble pheromone, the interactions found in the crystal may mimic the pheromone-receptor interactions as they occur on a cell surface. Based on this, we propose a model for the interaction between soluble pheromone molecules and their receptors. In this model, strong pheromone-receptor binding emerges as a consequence of the cooperative utilization of several weak interactions. The model offers an explanation for the results of binding studies and may also explain the adhesion between cells that occurs during mating.Pheromones from the ciliated protozoan Euplotes raikovi are proteins of 37-40 amino acids that function both as growth factors and as signaling molecules in cellular adhesion during mating (1-3). Over a dozen different cell types of E. raikovi have been identified based on their ability to form mating pairs (1). In the presence of only one cell type, the homologous secreted Er (Er-x = E. raikovi pheromone of type x) molecules stimulate growth of the cells by binding to cell-surface receptors in an autocrine fashion. In the presence of two different cell types or just one cell type to which heterologous pheromone has been added, Er molecules also stimulate cell adhesion between mating pair partners presumably by binding to the same cell-surface receptors (2, 3) in a paracrine manner. The putative receptors of the pheromones are membranebound proteins whose extracellular C-terminal domain is identical in amino acid sequence to the soluble pheromones (4). They arise by alternate splicing of the transcripts of the same gene that carries the information for the soluble pheromone (4).Different cell types can also be distinguished by the amino acid sequence of their pheromones. Sequences from nine different cell types have been determined (5-8), yielding seven unique sequences with pairwise sequence identities ranging from about 25% to 95%. Only seven residues are conserved among all sequences; these are the N-terminal aspartic acid and six cysteines that are involved in formation of three disulfide bridges (9).The NMR structures of three of the pheromones (Er-1, Er-2, and Er-10) have been determined and compared to each other (10-13). They revealed the three-helical bundle fold of the proteins and the orientations for about two-thirds of the side chains. The crystal structure reported here offers a detailed picture of the monomer and reveals two types of interactions between molecules that provide the basis for a model for receptor recognition, cell adhesion, and signaling. (14) is 1.53 A3/Da and the solvent content of the crystals is ...

Epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), and nerve growth factor (NGF), which stimulate the phosphorylation of proteins on tyrosine in PC12 cells, initiate these modifications through ligand-specific cell surface receptors that contain the causative tyrosine kinases. One apparent substrate for these enzymes is phosphatidylinositol 3-kinase (PI 3-kinase), an enzyme that phosphorylates the D-3 position of the inositol ring and associates with several protein tyrosine kinases, as indicated by the fact that it is immunoprecipitated from EGF-, bFGF-, and NGFstimulated PC12 cells by an anti-phosphotyrosine antibody. All three growth factors increase immunoprecipitable PI 3-kinase activity after 2 min of addition at concentrations able to stimulate either mitogenic or neurotrophic responses in PC12 cells. The level of stimulation of PI 3-kinase activity by EGF, bFGF, and NGF is 15-to 20-fold, 2-to 3-fold, and 8-to 10-fold, respectively. Moreover, tyrosine phosphorylation of PI 3-kinase was detected in EGF-, bFGF-, and NGF-stimulated PC12 cells, and the amount of the phosphorylation correlated with the level of stimulation of enzyme activity. In contrast, phosphatidylinositol 4-kinase, which produces the inositol phospholipids cleaved by phospholipase C-y to yield diacylglycerol and inositol-1,4,5-trisphosphate, is not affected by these growth factors. The pattern of stimulation of PI 3-kinase does not correlate with the induction of neurite outgrowth but rather with the mitotic responses, suggesting that PI 3-kinase and its products may be more important for signaling in cell division than in trophic processes. However, the levels of phosphatidylinositol 3-phosphate do not coincide with the stimulation of [3H]thymidine incorporation by these growth factors, rendering its role in mitotic functions, at least in PC12 cells, also uncertain.Nerve growth factor (NGF) and basic fibroblast growth factor (bFGF) induce reversible differentiation accompanied by neurite proliferation in the rat pheochromocytoma PC12 cell line, whereas epidermal growth factor (EGF) induces only mitogenic responses. As the receptors for all three growth factors contain tyrosine kinases, differences in protein tyrosine phosphorylation may be responsible for differences in intracellular signaling and phenotypic response generated by these receptors. Only few substrates for these induced tyrosine phosphorylations that are likely to be involved mechanistically have been identified in PC12 cells. Those include phospholipase C-y and several other kinases (1-6).Recently, several kinase-containing growth factor receptors have been demonstrated to physically associate with and activate an inositol phospholipid kinase, termed type I or phosphatidylinositol 3-kinase (PI 3-kinase). This enzyme phosphorylates phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PI-4-P), and phosphatidylinositol 4,5-bisphosphate (PI-4,5-P) at the D-3 position of the myoinositol ring. PI 3-kinase is present in anti-phosphotyrosine immun...

Stably transfected PC12 cell lines expressing similar amounts of chimeric receptors composed of the extracellular domain of the human platelet-derived growth factor (PDGF)␤ receptor and the transmembrane and intracellular domains of the fibroblast growth factor receptors (FGFRs) 1, 3, and 4 undergo ligand-induced differentiation. The FGFR1 chimera (PFR1) is the most potent of the three, and PFR4 requires more frequent (every 24 hr) addition of ligand to maintain the response. Both PFR1 and -3 also show significant ligand-independent autophosphorylation but PFR4 does not. All of the chimeras activated phospholipase C␥, Shc, FGFR substrate (FRS)2, and the mitogen-activated protein kinases, ERK1 and 2. PFR4 was moderately weaker in stimulating these effects as well; PFR1 and -3 were comparable. None of the chimeras induced Sos association or were coprecipitated with Shc. Cotransfection of a dominantnegative Shc derivative, with tyrosine at 239, 240, and 317 replaced with phenylalanine, in the PFR-expressing cells was without effect on PDGF-induced neurite outgrowth. The same derivative substantially inhibited the response of these cells to NGF. These results indicate that FGFR1, 3, and 4 (i) are capable of signaling in a similar fashion; (ii) primarily use FRS2 and, perhaps, PLC␥; and (iii) do not utilize Shc. The results also suggest that the principal difference between FGFR1, 3, and 4 is in the strength of the tyrosine kinase activity and that qualitative differences in signaling capacity are likely to be less important.

The effect of six point mutations causing various human skeletal dysplasias, occurring in the transmembrane (TM) and kinase domains (KD) of fibroblast growth factor receptor 3, were introduced into a chimera composed of the extracellular domain of human platelet-derived growth factor ␤ and the TM and intracellular domains of hFGFR3. Stable transfectants in rat PC12 cells showed distinct differences in the two classes of mutations. The cells containing TM mutants displayed normal expression and activation but higher responsiveness to lower doses of ligand. The KD mutants showed significantly altered expression patterns. Normal amounts of a lower M r receptor (p130) reflecting incomplete glycosylation, but only greatly decreased amounts of the mature (p170) form, were observed. However, the latter material showed normal ligand-dependent activation. In contrast, the p130 form, which is regularly observed in the expression of both native and chimeric receptors, exhibits strong ligand-independent tyrosine phosphorylation, particularly with the K650E mutation. Expression of two of the KD mutants (K650M and K650E), under control of an inducible metallothionein promoter, indicated that this receptor was sufficiently autoactivated to produce at least partial differentiation and, in the case of the K650E mutation, to induce ligand-independent neurite outgrowth. A model is presented that suggests that the low M r (p130) KD mutants can, under the right conditions, signal intracellularly, but when they are fully glycosylated and move to the cell surface they adopt a normal, inhibited conformation, in the form of ligand-independent dimers, that neutralizes the effects of the mutations. When ligands bind, these dimeric receptors are activated in a normal manner. This model suggests that unliganded dimers may be a common intermediate in receptor tyrosine kinase signaling.The fibroblast growth factor receptor (FGFR) 1 tyrosine kinase family consists of four (FGFR1-4) members that are implicated in many developmental and regenerative processes, including cell proliferation, differentiation, and angiogenesis (1-3). Signaling by these receptors is mediated by the high affinity binding of the fibroblast growth factors (FGF), a diverse family consisting of at least 10 characterized members, aided by heparan sulfate proteoglycan interactions (4). Further diversity in expression and ligand specificity is achieved by the generation of subforms of the FGFRs mediated by alternative mRNA splicing events (4, 5). The ligand-receptor complexes activate the receptor kinases inducing tyrosine phosphorylation and a cascade of signaling events mediated by target effector proteins including Ras/MAPK and phospholipase C␥ (PLC␥) (6 -8).It is now well known that mutations in three of the four members of the FGFR family (FGFR1-3) cause a variety of autosomal dominant human skeletal dysplasias (for review see Refs. 9 and 10). Among these is the most common genetic form of short-limbed dwarfism, achondroplasia (ACH) (11,12) caused by a point mutation in...

Euplotes raikovi of the wild-type strain 13 was found to be heterozygous (mat-l/mat-2) for the genetic locus mat, which is supposed to control the mating-type specificity of freely released mating pheromones ("euplomones"), and capable of yielding the two types of corresponding homozygotes (mat-i/mat-i, mat-2/mat-2). The results of euplomone purification, performed in parallel on Euplotes of the three different genotypes, showed that the heterozygous cells corelease two mat-specific euplomones (namely, euplomone r 1 associated with mat-i and euplomone r 2 associated with mat-2), while the homozygous cells release either one of the two types according to their genotype. The two euplomones coreleased by the mat heterozygous cells were resolved as separate molecular species present in different relative amounts (euplomone r 1, -70%; euplomone r 2, -30%). A closely similar degree of eccentricity in the euplomone r 1/euplomone r 2 production was again found between the two homozygous cell types. It was concluded that the alleles mat-i and mat-2 exhibit a relationship of nondominance: the heterozygote apparently behaves as the simple combination of the two corresponding homozygotes. It was inferred that the observed quantitative variations in the production of the different euplomones may be the result of a differential mat gene-type amplification occurring during the development of the cell somatic macronucleus.Cell union in conjugation in most species of Euplotes is regulated by multiple mating-type systems via mat gene-typespecific mating substances that may diffuse into the extracellular environment as mating pheromones (1), referred to also as gamones (2). Two mating-type expression patterns are known: the patella pattern (3) and the vannus pattern (4), according to the species of Euplotes in which they were first described. In the former, the mat heterozygous cell is distinctive from the two corresponding types of homozygote as it interacts with both of them. In the latter, the mat heterozygous cell is not distinctive from the dominant homozygote as it does not interact with it. Based only on results of mating-type interactions, the differences between these two patterns have been explained by assuming that the mat alleles exhibit codominance in the patella pattern whereby the heterozygote would produce two types of pheromone (3,5,6), while obeying a serial dominance relationship in the vannus pattern (4).Here we provide genetic and biochemical evidence verifying the assumption that a mat heterozygous mating type of the patella pattern actually behaves as a simple combination of the two corresponding homozygous mating types. The system used is Euplotes raikovi, which is particularly amenable for a biochemical analysis of pheromones in parallel with a traditional genetic study of mating types: most wildtype strains, representing different mating types, have proven to be constitutive pheromone excreters and capable of yielding viable offspring from both inter-and intraclonal conjugation (7). In particular,...

No abstract

The amino acid sequences of five pheromones, Er-2, Er-3, Er-9, Er-li, and Er-20, secreted by cells of different mating types of the ciliated protozoa Euplotes raikovi, have been determined by automated Edman analyses of the whole proteins and germane fragments. In each case, the molecular mass was determined by plasma desorption or laser desorption mass spectrometry and was in excellent agreement with the calculated values. Where available, the determined sequences were also in accord with the corresponding segments of the precursor molecules predicted from relevant nucleic acid sequences. Of the five, two were found to be identical (Er-2 and Er-9) and one (Er-3) was identical to a pheromone previously sequenced (Er-i), even though mating pair formation was found to take place (although to a limited extent) when cells secreting those pheromones were combined in a mixture.Comparison of the five unique sequences suggested a closer relationship between Er-i (Er-3) and Er-10 and between Er-il and Er-20 (44% and 56% identity, respectively) than was generally observed among the other members. This pairing was also supported by hydrophobicity analyses. Interestingly, Er-20 cannot, as a rule, induce cell union in any ofthe other cell types, including cells secreting Er-il, despite the fact that and Er-ll are the most similar of the five unique sequences. Thus sequence identity and secondary structure profiles are not a good indicator of biological relatedness as manifested in heterologous receptor interaction.