The role of tyrosine residues in fibroblast growth factor receptor 1 signaling in PC12 cells. Systematic site-directed mutagenesis in the endodomain.

To assess the contribution of the intracellular domain tyrosine residues to the signaling capacity of fibroblast growth factor receptor 1 (FGFR1), stably transfected chimeras bearing the ectodomain of the platelet-derived growth factor receptor (PDGFR) and the endodomain of FGFR1 were systematically altered by a tyrosine to phenylalanine bloc and individual conversions. The 15 tyrosine residues of the endodomain of this construct (PFR1) were divided into four linear segments (labeled A, B, C, and D) that contained 4, 4, 2, and 5 tyrosine residues, respectively. When stimulated by platelet-derived growth factor, derivatives in which the A, B, or A + B blocs of tyrosines were mutated were about two-thirds as active as the unmodified chimera at 48 h but achieved full activity by 96 h in a neurite outgrowth assay in transfected PC12 cells. Elimination of only the two activation loop tyrosines (C bloc) also inactivated the receptor. All derivatives in which 4 (or 5) of the D bloc tyrosines were mutated were inactive in producing differentiation but showed low levels of kinase activity in in vitro assays. Derivatives in which 1, 2, or 3 tyrosines of the D bloc in different combinations were systematically changed demonstrated that 2 residues (Tyr(677) and Tyr(701), using hFGFR1 numbering) were essential for bioactivity, but the remaining 3 residues, including Tyr(766), the previously identified site for phospholipase C gamma (PLC gamma) activation, were not. Differentiation activity was paralleled by the activation (phosphorylation) of FRS2, SOS, and ERK1/2. PLC gamma activity was dependent on the presence of Tyr(766) but also required Tyr(677) and/or Tyr(701). Although fully active chimeras did not require PLC gamma, the responses of chimeras showing reduced activation of FRS2 were significantly enhanced by this activity. These results establish that PFR1 does not utilize any tyrosine residues, phosphorylated or not, to activate FRS2. However, it does require Tyr(677) and/or Tyr(701), which may function to stabilize the active conformation directly or indirectly.

Discoidin domain receptor 1 (DDR1) signaling in PC12 cells: activation of juxtamembrane domains in PDGFR/DDR/TrkA chimeric receptors.

The discoidin domain receptor (DDR1) is characterized by a discoidin I motif in the extracellular domain, an unusually long cytoplasmic juxtamembrane (JM) region, and a kinase domain that is 45% identical to that of the NGF receptor, TrkA. DDR1 also has a major splice form, which has a 37 amino acid insert in the JM region with a consensus Shc PTB site that is lacking in the shorter receptor. One class of ligands for the DDR receptors has recently been identified as being derived from the collagen family, but neither native PC12 cells, which express modest amounts of DDR1, nor transfected PC12 cells, which express much larger amounts of DDR1, respond to this ligand. A chimeric receptor, containing the extracellular domain of hPDGFRbeta fused to the transmembrane and intracellular regions of DDR1, also fails to mediate neuronal-like differentiation in stably transfected PC12 cells and is only weakly autophosphorylated. However, chimeric receptors, which are composed of combinations of intracellular regions from DDR1 and TrkA (with the extracellular domain of hPDGFRbeta), in some cases provided ligand (PDGF) -inducible receptor responses. Those with the TrkA kinase domain and the DDR1 JM regions were able to produce differentiation to varying degrees, whereas the opposite combination did not. Analysis of the signaling responses of the two chimeras with DDR1 JM sequences (with and without the insert) indicated that the shorter sequence bound and activated FRS2 whereas the insert-containing form activated Shc instead. Both activated PLCgamma through the carboxyl-terminal tyrosine of the TrkA domain (Y785 in TrkA residue numbering). Mutation of this site (Y-->F) eliminated PLCgamma activation (indicating there are no other cryptic binding sites for PLCgamma in the DDR1 sequences) and markedly reduced the differentiative activity of the receptor. This is in contrast to TrkA (or PDGFRbeta/TrkA chimeras), where ablation of this pathway has no notable effect on PC12 cell morphogenic responses. Thus, the activation of FRS2 and Shc (leading to MAPK activation) is weaker in the DDR1/TrkA chimeras than in TrkA alone, and the PLCgamma contribution becomes essential for full response. Nonetheless, both DDR1 JM regions contain potentially usable signaling sites, albeit they apparently are not activated directly in DDR1 (or DDR1 chimeras) in a ligand-dependent fashion. These findings suggest that the DDR1 receptors do have signaling capacity but may require additional components or altered conditions to fully activate their kinase domains and/or sustain the activation of the JM sites.

Comparison of the intracellular signaling responses by three chimeric fibroblast growth factor receptors in PC12 cells.

Stably transfected PC12 cell lines expressing similar amounts of chimeric receptors composed of the extracellular domain of the human platelet-derived growth factor (PDGF)beta 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 Cgamma, 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 dominant-negative 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, PLCgamma; 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.

Effect of transmembrane and kinase domain mutations on fibroblast growth factor receptor 3 chimera signaling in PC12 cells. A model for the control of receptor tyrosine kinase activation.

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 beta 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 Mr 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 Mr (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.

FGF signal transduction in PC12 cells: comparison of the responses induced by endogenous and chimeric receptors.

Rat phaeochromocytoma (PC12) cells respond to many growth factors and produce different phenotypes, including neurite outgrowth. Receptor tyrosine kinases (RTK), which activate multiple signalling pathways in response to ligand binding, initiate many of these. One such family of receptors, the fibroblast growth factor receptor (FGFR), has four different members and expresses at least three of these in PC12 cells. A chimeric tyrosine kinase receptor, consisting of the extracellular domain of human plasma-derived growth factor receptor-beta (hPDGFR-beta) and the transmembrane and intracellular region of FGFR1 (designated PFR1), was constructed and was stably transfected into cloned PC12 cell lines. This chimera, which can be activated without stimulating endogenous RTK including other FGFR, induces neurite outgrowth in a PDGF-dependent manner. By altering the protocol for preparing the retroviral vectors, cells with a wide range of expression levels can be obtained. The amount of these chimeric receptors seems to correlate with the time and the intensity of response as observed in neurite outgrowth assays. Analysis of proteins implicated in FGFR1 signalling indicates that upon stimulation, a tyrosine phosphorylated protein designated FRS2 associates with SOS, Grb2 and the receptor. The chimeric receptor appears entirely similar to that observed for the stimulation of native PC12 cells with FGF2. These results support the view that FRS2 is the dominant FGFR1 signalling entity in PC12 cells.

Chimeras of the native form or achondroplasia mutant (G375C) of human fibroblast growth factor receptor 3 induce ligand-dependent differentiation of PC12 cells.

Mutations in the gene for human fibroblast growth factor receptor 3 (hFGFR3) cause a variety of skeletal dysplasias, including the most common genetic form of dwarfism, achondroplasia (ACH). Evidence indicates that these phenotypes are not due to simple haploinsufficiency of FGFR3 but are more likely related to a role in negatively regulating skeletal growth. The effects of one of these mutations on FGFR3 signaling were examined by constructing chimeric receptors composed of the extracellular domain of human platelet-derived growth factor receptor beta (hPDGFR beta) and the transmembrane and intracellular domains of hFGFR3 or of an ACH (G375C) mutant. Following stable transfection in PC12 cells, which lack platelet-derived growth factor (PDGF) receptors, all clonal cell lines, with either type of chimera, showed strong neurite outgrowth in the presence of PDGF but not in its absence. Antiphosphotyrosine immunoblots showed ligand-dependent autophosphorylation, and both receptor types stimulated strong phosphorylation of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase, an event associated with the differentiative response of these cells. In addition, ligand-dependent phosphorylation of phospholipase Cgamma and Shc was also observed. All of these responses were comparable to those observed from ligand activation, such as by nerve growth factor, of the native PC12 cells used to prepare the stable transfectants. The cells with the chimera bearing the ACH mutation were more rapidly responsive to ligand with less sustained MAPK activation, indicative of a preactivated or primed condition and consistent with the view that these mutations weaken ligand control of FGFR3 function. However, the full effect of the mutation likely depends in part on structural features of the extracellular domain. Although FGFR3 has been suggested to act as a negative regulator of long-bone growth in chrondrocytes, it produces differentiative signals similar to those of FGFR1, to which only positive effects have been ascribed, in PC12 cells. Therefore, its regulatory effects on bone growth likely result from cellular contexts and not the induction of a unique FGFR3 signaling pathway.

A cooperative model for receptor recognition and cell adhesion: evidence from the molecular packing in the 1.6-A crystal structure of the pheromone Er-1 from the ciliated protozoan Euplotes raikovi.

The crystal structure of the pheromone Er-1 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-1 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.

A diphtheria toxin/fibroblast growth factor 6 mitotoxin selectively kills fibroblast growth factor receptor-expressing cell lines.

The fibroblast growth factors (FGFs) constitute a family of nine polypeptides implicated in a number of physiological and pathological processes. They bind to at least three types of cell surface molecules, including low and high affinity receptor families. The role of FGFs and their receptors in human tumorigenesis has been suspected but not formally proven. FGF6 is an oncogene encoding a precursor protein of 208 amino acids that has been shown to bind to FGF receptors. Its normal function has not been identified, but its restricted pattern of expression suggests a role in muscle development or function. We have constructed, produced, and purified a diphtheria toxin/FGF6 mitotoxin that selectively kills FGF receptor-expressing cells. Interestingly, at least two cell lines that normally respond to FGF6 have been found resistant to DT/FGF6, suggesting that FGF6 acts on these cells through a transduction pathway that does not involve FGF receptor.

Single step purification of biologically active recombinant rat basic fibroblast growth factor by immobilized metal affinity chromatography.

The construction and use of a plasmid which allows the expression and single step purification of recombinant rat basic fibroblast growth factor (bFGF) is described. A cDNA encoding rat bFGF was subcloned into the expression plasmid pQE-9 (Qiagen) in such a way that the bFGF which is produced from the resulting construct contains 6 histidine residues near the amino terminus. The resulting plasmid, pQE-9-bFGF, was expressed in the E. coli strain M15[pREP4] and the 6 x His-bFGF was purified to homogeneity from the soluble fraction of the bacterial cell lysate in a single step by affinity chromatography on a nickel chelate resin. About 5 mg of 6 x His-bFGF was obtained from the soluble fraction from one liter of bacterial cell culture. Testing of the 6 x His-bFGF in a PC12 cell differentiation assay showed that its activity was comparable to the activities for native bFGF and recombinant bFGF purified by multistep methods.

Staurosporine causes epidermal growth factor to induce differentiation in PC12 cells via receptor up-regulation.

Although they all utilize tyrosine kinase receptors and activate signaling pathways characterized by a similar set of phosphoproteins, epidermal growth factor (EGF) promotes only cell division while fibroblast growth factor (FGF) and nerve growth factor (NGF) can induce division followed by differentiation in PC12 cells. EGF, in contrast to NGF and FGF, cannot maintain the sustained phosphorylation and activation of mitogen-activated protein (MAP) kinase kinase and MAP kinases, which may account for the difference in phenotypic response. The pretreatment of PC12 cells with staurosporine, a protein kinase inhibitor, causes a substantial increase in both receptor and MAP kinase phosphorylation that results in a differentiative response (neurite proliferation). However, neurites begin to disappear after 3 days, despite the continual presence of EGF, and are largely gone after 5 days, which is not the case with NGF and FGF. Thus, the effect of staurosporine is not permanent. Northern and Western blots indicate that the staurosporine response mainly results from a substantial up-regulation in EGF receptor synthesis, thus providing a much stronger cell surface signal and supporting the view that quantitative rather than qualitative differences distinguish the EGF versus NGF/FGF signaling pathways in these cells.

Activation of phosphatidylinositol 3-kinase by epidermal growth factor, basic fibroblast growth factor, and nerve growth factor in PC12 pheochromocytoma cells.

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 NGF-stimulated 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-gamma 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.

The disulfide bond pairing of the pheromones Er-1 and Er-2 of the ciliated protozoan Euplotes raikovi.

The disulfide pairings of the two Euplotes raikovi pheromones Er-1 and Er-2 have been determined by chemical and mass spectrometric analyses. Cystine-linked peptides from thermolytic digestions of the native molecules were purified by reverse-phase high performance liquid chromatography and identified in the known sequences to make the assignments. The same pairing, Cys(I)-Cys(IV), Cys(II)-Cys(VI), and Cys(III)-Cys(V), was found in both pheromones, suggesting that this pattern occurs commonly throughout this family of molecules. This arrangement of disulfides indicates that the three-dimensional structure is defined by three loops, which can vary in size and charge distribution from one pheromone to another.

Primary structure of Euplotes raikovi pheromones: comparison of five sequences of pheromones from cells with variable mating interactions.

The amino acid sequences of five pheromones, Er-2, Er-3, Er-9, Er-11, 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-1), 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-1 (Er-3) and Er-10 and between Er-11 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 of the other cell types, including cells secreting Er-11, despite the fact that Er-20 and Er-11 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.

Differential induction of primary-response (TIS) genes in PC12 pheochromocytoma cells and the unresponsive variant PC12nnr5.

As a measure of the transmembrane signals that they transduce, two neurotrophic agents, nerve growth factor (NGF) and basic fibroblast growth factor (bFGF), and the muscarinic agonist carbachol were compared for their ability to induce TIS (tetradecanoyl phorbol acetate-inducible sequences) transcripts, representing a family of immediate early response genes, in the rat pheochromocytoma cell line PC12 and the morphologically unresponsive variant PC12nnr5. Three genes, TIS1 (also designated NGFIB), TIS8 (also designated NGFIA), and TIS21, induced in these cells by NGF (Kujubu, D.A., Lim, R.W., Varnum, B.C., and Herschman, H.R. (1987) Oncogene 1, 257-262, 1987), are also induced by bFGF and carbachol. In native PC12 cells the level of expression of TIS8 and TIS21 is similar for all three stimuli, as well as for tetradecanoyl phorbol acetate (TPA). In contrast, the induction of TIS1 by NGF and TPA is slight and is only just detectable after stimulation by bFGF, but is strong for carbachol. Thus, although all of these agents can stimulate protein kinase (PK-C), at least one TIS gene can apparently be differentially regulated by these ligands, suggesting that alternative signaling pathways must also exist. In keeping with this view, bFGF, and to a lesser degree NGF, can elicit a TIS gene response in PC12 cells in which PK-C has been down-regulated with TPA. The response to carbachol (and TPA) is effectively blocked under these conditions. Since both NGF and bFGF stimulate neurite outgrowth in such cells, PK-C is apparently not essential, i.e. does not represent the sole mechanism, for signal transduction leading to modulation of gene expression for these factors. Consistent with this model, putative protein kinase inhibitors, K252a and sphingosine, did not inhibit the TIS gene responses to bFGF. However, these agents also failed to block TIS gene responses to carbachol and TPA indicating that they were ineffective as PK-C inhibitors under these conditions. The NGF-induced response was, however, blocked by K252a indicating a unique step in the mechanism of this factor not shared by the other ligands. Sphingosine did not block TIS induction with NGF. The mutant cell line PC12 nnr5 does not respond morphologically to either NGF or bFGF. However, TIS gene responses to bFGF are unaffected, whereas those to NGF are completely abolished. The response to TPA is altered quantitatively but not qualitatively; the induction by carbachol is largely eliminated, apparently as a result of a 90% reduction in muscarinic receptors.(ABSTRACT TRUNCATED AT 400 WORDS)

Crystallization of the Euplotes raikovi mating pheromone Er-1.

A protein mating pheromone Er-1 from the ciliate Euplotes raikovi has been crystallized from (NH4)2SO4 in two forms. Both are suitable for structural studies to at least 2.8 A resolution. Both unit cell sizes are consistent with a tetramer of molecular weight 17,640 in the asymmetric unit.

Specific and common epitopes in mating pheromones of Euplotes raikovi revealed by monoclonal antibodies.

Polypeptide mating pheromones Er-1 and Er-2, purified from the supernatant of Euplotes raikovi cultures of mating type I and mating type II, respectively, were used to immunize mice and obtain monoclonal antibodies. Five hybridoma clones producing antibodies specific to the mating pheromones were selected. They were analyzed for immunospecificity by immunoperoxidase assay, immunoblotting, and for their efficacy in inhibition of mating pheromone activity. Monoclonal antibodies from two hybridoma clones recognized only the mating pheromone used as antigen: those from the other three clones reacted, to comparable extents, with both mating pheromones. On the basis of these results it was assumed that two immunogenic sites exist in Er-1 and Er-2, one specific and the other common to both mating pheromones.

Purification, characterization, and amino acid sequence of the mating pheromone Er-10 of the ciliate Euplotes raikovi.

The mating pheromone Er-10 from mat-10 homozygous Euplotes raikovi was purified by a three-step purification procedure with an overall yield of 62%. It was identified as a protein of molecular weight 8000 having an isoelectic point of 3.9. Its complete primary structure was determined by automated Edman degradation of the whole protein after performic acid oxidation and of peptides generated by cyanogen bromide and Staphylococcus aureus V8 protease. The proposed sequence is Asp1-Leu-Cys-Glu-Gln-Ser-Ala-Leu-Gln-Cys10-Asn-Glu-Gln-Gly-Cys-His -Asn-Phe-Cys- Ser20-Pro-Glu-Asp-Lys-Pro-Gly-Cys-Leu-Gly-Met30-Val-Trp-Asn- Pro-Glu-Leu-Cys- Pro38. The calculated molecular weight of 4191.7, which is in good agreement with the value of m/z 4190.7 obtained by fission fragment ionization mass spectrometry, suggests that the native structure is a dimer with three intrachain disulfide bonds in each subunit. The amino acid sequence is 43% identical with that of the E. raikovi mating pheromone Er-1, with the identities concentrated in the amino-terminal half. The half-cystine locations are conserved, but Er-10 is two residues shorter than Er-1. Prediction of the secondary structure suggests that Er-10 may also contain a helical structure at the amino terminus. These results indicate that the mating pheromones of E. raikovi form a homologous family.

Primary structure of the mating pheromone Er-1 of the ciliate Euplotes raikovi.

The complete amino acid sequence of the mating pheromone Er-1 purified from Euplotes raikovi homozygous for mat-1 was determined by automated Edman degradation of the whole protein and peptides generated by cyanogen bromide, trypsin, Staphylococcus aureus V8 protease, and chymotrypsin. The proposed sequence is: Asp-Ala-Cys-Glu-Gln-Ala-Ala-Ile-Gln-Cys-Val-Glu-Ser-Ala-Cys-Glu-Ser-Leu- Cys-Thr-Glu-Gly-Glu-Asp-Arg-Thr-Gly-Cys-Tyr-Met-Tyr-Ile-Tyr-Ser-Asn-Cys- Pro-Pro-Tyr-Val The calculated molecular weight is 4411.0, which is in agreement with the averaged mass of 4410.2 obtained by fission fragment ionization mass spectrometry. Previously reported values of the native molecular weight, determined by gel filtration, have ranged from 9,000 to 12,000. Thus, the native structure is likely a dimer (or larger aggregate) of identical subunits with the three disulfide bonds present occurring as intrachain links. Secondary structure predictions suggest a helical structure at the amino terminus. A comparison of the Er-1 amino acid sequence with known protein sequences did not reveal any significant similarities.