Distinct missense mutations of the FGFR3 lys650 codon modulate receptor kinase activation and the severity of the skeletal dysplasia phenotype.

The fibroblast growth factor-receptor 3 (FGFR3) Lys650 codon is located within a critical region of the tyrosine kinase-domain activation loop. Two missense mutations in this codon are known to result in strong constitutive activation of the FGFR3 tyrosine kinase and cause three different skeletal dysplasia syndromes-thanatophoric dysplasia type II (TD2) (A1948G [Lys650Glu]) and SADDAN (severe achondroplasia with developmental delay and acanthosis nigricans) syndrome and thanatophoric dysplasia type I (TD1) (both due to A1949T [Lys650Met]). Other mutations within the FGFR3 tyrosine kinase domain (e.g., C1620A or C1620G [both resulting in Asn540Lys]) are known to cause hypochondroplasia, a relatively common but milder skeletal dysplasia. In 90 individuals with suspected clinical diagnoses of hypochondroplasia who do not have Asn540Lys mutations, we screened for mutations, in FGFR3 exon 15, that would disrupt a unique BbsI restriction site that includes the Lys650 codon. We report here the discovery of three novel mutations (G1950T and G1950C [both resulting in Lys650Asn] and A1948C [Lys650Gln]) occurring in six individuals from five families. Several physical and radiological features of these individuals were significantly milder than those in individuals with the Asn540Lys mutations. The Lys650Asn/Gln mutations result in constitutive activation of the FGFR3 tyrosine kinase but to a lesser degree than that observed with the Lys540Glu and Lys650Met mutations. These results demonstrate that different amino acid substitutions at the FGFR3 Lys650 codon can result in several different skeletal dysplasia phenotypes.

A novel skeletal dysplasia with developmental delay and acanthosis nigricans is caused by a Lys650Met mutation in the fibroblast growth factor receptor 3 gene.

We have identified a novel fibroblast growth factor receptor 3 (FGFR3) missense mutation in four unrelated individuals with skeletal dysplasia that approaches the severity observed in thanatophoric dysplasia type I (TD1). However, three of the four individuals developed extensive areas of acanthosis nigricans beginning in early childhood, suffer from severe neurological impairments, and have survived past infancy without prolonged life-support measures. The FGFR3 mutation (A1949T: Lys650Met) occurs at the nucleotide adjacent to the TD type II (TD2) mutation (A1948G: Lys650Glu) and results in a different amino acid substitution at a highly conserved codon in the kinase domain activation loop. Transient transfection studies with FGFR3 mutant constructs show that the Lys650Met mutation causes a dramatic increase in constitutive receptor kinase activity, approximately three times greater than that observed with the Lys650Glu mutation. We refer to the phenotype caused by the Lys650Met mutation as "severe achondroplasia with developmental delay and acanthosis nigricans" (SADDAN) because it differs significantly from the phenotypes of other known FGFR3 mutations.

Activating mutations in the extracellular domain of the fibroblast growth factor receptor 2 function by disruption of the disulfide bond in the third immunoglobulin-like domain.

Multiple human skeletal and craniosynostosis disorders, including Crouzon, Pfeiffer, Jackson-Weiss, and Apert syndromes, result from numerous point mutations in the extracellular region of fibroblast growth factor receptor 2 (FGFR2). Many of these mutations create a free cysteine residue that potentially leads to abnormal disulfide bond formation and receptor activation; however, for noncysteine mutations, the mechanism of receptor activation remains unclear. We examined the effect of two of these mutations, W290G and T341P, on receptor dimerization and activation. These mutations resulted in cellular transformation when expressed as FGFR2/Neu chimeric receptors. Additionally, in full-length FGFR2, the mutations induced receptor dimerization and elevated levels of tyrosine kinase activity. Interestingly, transformation by the chimeric receptors, dimerization, and enhanced kinase activity were all abolished if either the W290G or the T341P mutation was expressed in conjunction with mutations that eliminate the disulfide bond in the third immunoglobulin-like domain (Ig-3). These results demonstrate a requirement for the Ig-3 cysteine residues in the activation of FGFR2 by noncysteine mutations. Molecular modeling also reveals that noncysteine mutations may activate FGFR2 by altering the conformation of the Ig-3 domain near the disulfide bond, preventing the formation of an intramolecular bond. This allows the unbonded cysteine residues to participate in intermolecular disulfide bonding, resulting in constitutive activation of the receptor.

Constitutive activation of fibroblast growth factor receptor 3 by mutations responsible for the lethal skeletal dysplasia thanatophoric dysplasia type I.

Thanatophoric dysplasia type I (TDI) is a neonatal lethal skeletal dysplasia caused by several mutations in the extracellular domain of fibroblast growth factor receptor 3. These mutations occur either in the Ig2-Ig3 linker domain or in the extracellular juxtamembrane domain, and all involve mutation of the wild-type residue to Cys. In all cases, the presence of the mutant Cys residue allows the receptor to dimerize abnormally, resulting in ligand-independent activation. This is also manifested by increased biological signaling, increased tyrosine phosphorylation, and in vitro kinase activity associated with dimeric receptors. These results suggest that TDI is caused by Cys-mediated intermolecular disulfide bonding, leading to constitutive receptor activation as a result of these mutations. Mutations causing TDI are discussed with respect to activating mutations in other receptors that are implicated in human disease.

Enhanced signaling and morphological transformation by a membrane-localized derivative of the fibroblast growth factor receptor 3 kinase domain.

Fibroblast growth factor (FGF) receptors (FGFRs) are membrane-spanning tyrosine kinase receptors that mediate regulatory signals for cell proliferation and differentiation in response to FGFs. We have previously determined that the Lys650-->Glu mutation in the activation loop of the kinase domain of FGFR3, which is responsible for the lethal skeletal dysplasia thanatophoric dyplasia type II (TDII), greatly enhances the ligand-independent kinase activity of the receptor. Here, we demonstrate that expression of this construct induces a c-fos promoter construct approximately 10-fold but does not lead to proliferation or morphological transformation of NIH 3T3 cells. In contrast, the isolated kinase domain of activated FGFR3, targeted to the plasma membrane by a myristylation signal, is able to stimulate c-fos expression by 40-fold, induce proliferation of quiescent cells, and morphologically transform fibroblasts. This result suggests that the extracellular and transmembrane domains of FGFRs exert a negative regulatory influence on the activity of the kinase domain. Targeting of the activated kinase domain to either the cytoplasm or the nucleus does not significantly affect biological signaling, suggesting that signals from FGFR3 resulting in mitogenesis originate exclusively from the plasma membrane. Furthermore, our novel observation that expression of a highly activated FGFR3 kinase domain is able to morphologically transform fibroblasts suggests that dysregulation of FGFR3 has the potential to play a role in human neoplasia.

Transmembrane domain sequence requirements for activation of the p185c-neu receptor tyrosine kinase.

The receptor tyrosine kinase p185c-neu can be constitutively activated by the transmembrane domain mutation Val664-->Glu, found in the oncogenic mutant p185neu. This mutation is predicted to allow intermolecular hydrogen bonding and receptor dimerization. Understanding the activation of p185c-neu has assumed greater relevance with the recent observation that achondroplasia, the most common genetic form of human dwarfism, is caused by a similar transmembrane domain mutation that activates fibroblast growth factor receptor (FGFR) 3. We have isolated novel transforming derivatives of p185c-neu using a large pool of degenerate oligonucleotides encoding variants of the transmembrane domain. Several of the transforming isolates identified were unusual in that they lacked a Glu at residue 664, and others were unique in that they contained multiple Glu residues within the transmembrane domain. The Glu residues in the transforming isolates often exhibited a spacing of seven residues or occurred in positions likely to represent the helical interface. However, the distinction between the sequences of the transforming clones and the nontransforming clones did not suggest clear rules for predicting which specific sequences would result in receptor activation and transformation. To investigate these requirements further, entirely novel transmembrane sequences were constructed based on tandem repeats of simple heptad sequences. Activation was achieved by transmembrane sequences such as [VVVEVVA]n or [VVVEVVV]n, whereas activation was not achieved by a transmembrane domain consisting only of Val residues. In the context of these transmembrane domains, Glu or Gln were equally activating, while Lys, Ser, and Asp were not. Using transmembrane domains with two Glu residues, the spacing between these was systematically varied from two to eight residues, with only the heptad spacing resulting in receptor activation. These results are discussed in the context of activating mutations in the transmembrane domain of FGFR3 that are responsible for the human developmental syndromes achondroplasia and acanthosis nigricans with Crouzon Syndrome.

FGFR activation in skeletal disorders: too much of a good thing.

During the past two years, a growing number of mutations have been identified in three of the four members of the fibroblast growth factor receptor (FGFR) family as causing autosomal dominant disorders of skeletal and cranial development. These mutations map to the extracellular domain, the transmembrane domain, or the tyrosine kinase domain of these receptors. Recent studies demonstrate that a common mechanism, constitutive activation of receptors signaling, underlies most, if not all, of these disorders. This suggests a normal role for FGFRs in the negative regulation of bone growth.

Profound ligand-independent kinase activation of fibroblast growth factor receptor 3 by the activation loop mutation responsible for a lethal skeletal dysplasia, thanatophoric dysplasia type II.

Thanatophoric dysplasia type II (TDII) is a neonatal lethal skeletal dysplasia caused by a recurrent Lys-650-->Glu mutation within the highly conserved activation loop of the kinase domain of fibroblast growth factor receptor 3 (FGFR3). We demonstrate here that this mutation results in profound constitutive activation of the FGFR3 tyrosine kinase, approximately 100-fold above that of wild-type FGFR3. The mechanism of FGFR3 activation in TDII was probed by constructing various point mutations in the activation loop. Substitutions at position 650 indicated that not only Glu but also Asp and, to a lesser extent, Gln and Leu result in pronounced constitutive activation of FGFR3. Additional mutagenesis within the beta10-beta11 loop region (amino acids Tyr-647 to Leu-656) demonstrated that amino acid 650 is the only residue which can activate the receptor when changed to a Glu, indicating a specificity of position as well as charge for mutations which can give rise to kinase activation. Furthermore, when predicted sites of autophosphorylation at Tyr-647 and Tyr-648 were mutated to Phe, either singly or in combination, constitutive kinase activity was still observed in response to the Lys-650-->Glu mutation, although the effect of these mutations on downstream signalling was not investigated. Our data suggest that the molecular effect of the TDII activation loop mutation is to mimic the conformational changes that activate the tyrosine kinase domain, which are normally initiated by ligand binding and autophosphorylation. These results have broad implications for understanding the molecular basis of other human developmental syndromes that involve mutations in members of the FGFR family. Moreover, these findings are relevant to the study of kinase regulation and the design of activating mutations in related tyrosine kinases.

Constitutive receptor activation by Crouzon syndrome mutations in fibroblast growth factor receptor (FGFR)2 and FGFR2/Neu chimeras.

Crouzon syndrome is an autosomal dominant condition primarily characterized by craniosynostosis. This syndrome has been associated with a variety of amino acid point mutations in the extracellular domain of fibroblast growth factor receptor 2 (FGFR2). FGFR2/Neu chimeras were generated by substituting the extracellular domain of Neu with that of FGFR2 containing the following Crouzon mutations: Tyr-340-->His; Cys-342-->Tyr; Cys-342-->Arg; Cys-342-->Ser; Ser-354-->Cys: and delta17 (deletion of amino acids 345-361). Each of the mutant chimeric FGFR2/Neu constructs stimulated focus formation in NIH 3T3 cells, indicating that Crouzon mutations can stimulate signal transduction through a heterologous receptor tyrosine kinase. In vitro kinase assay results indicate that FGFR2 receptors containing Crouzon mutations have increased tyrosine kinase activity and, when analyzed under nonreducing conditions, exhibited disulfide-bonded dimers. Thus the human developmental abnormality Crouzon syndrome arises from constitutive activation of FGFR2 due to aberrant intermolecular disulfide-bonding. These results together with our earlier observation that achondroplasia results from constitutive activation of the related receptor FGFR3, leads to the prediction that other malformation syndromes attributed to FGFRs, such as Pfeiffer syndrome and Thanatophoric dysplasia, also arise from constitutive receptor activation.

Constitutive activation of fibroblast growth factor receptor 3 by the transmembrane domain point mutation found in achondroplasia.

Achondroplasia, the most common genetic form of dwarfism, is an autosomal dominant disorder whose underlying mechanism is a defect in the maturation of the cartilage growth plate of long bones. Achondroplasia has recently been shown to result from a Gly to Arg substitution in the transmembrane domain of the fibroblast growth factor receptor 3 (FGFR3), although the molecular consequences of this mutation have not been investigated. By substituting the transmembrane domain of the Neu receptor tyrosine kinase with the transmembrane domains of wild-type and mutant FGFR3, the Arg380 mutation in FGFR3 is shown to activate both the kinase and transforming activities of this chimeric receptor. Residues with side chains capable of participating in hydrogen bond formation, including Glu, Asp, and to a lesser extent, Gln, His and Lys, were able to substitute for the activating Arg380 mutation. The Arg380 point mutation also causes ligand-independent stimulation of the tyrosine kinase activity of FGFR3 itself, and greatly increased constitutive levels of phosphotyrosine on the receptor. These results suggest that the molecular basis of achondroplasia is unregulated signal transduction through FGFR3, which may result in inappropriate cartilage growth plate differentiation and thus abnormal long bone development. Achondroplasia may be one of the number of cogenital disorders where constitutive activation of a member of the FGFR family leads to development abnormalities.

Cellular transformation by a transmembrane peptide: structural requirements for the bovine papillomavirus E5 oncoprotein.

The E5 oncoprotein of bovine papillomavirus, only 44 amino acids long, occurs as a disulfide-bonded transmembrane dimer. This remarkable oncoprotein stimulates signal transduction through activation of the platelet-derived growth factor (PDGF) receptor, and E5 exhibits limited amino acid sequence similarity with PDGF. Results presented here suggest that a key feature of the hydrophobic transmembrane domain is an amino acid side chain that participates in interhelical hydrogen bond formation. These data are reminiscent of the activated neu oncogene, in which a point mutation in the transmembrane domain leads to ligand-independent dimerization and activation of a receptor tyrosine kinase. Significantly, the transmembrane domain of E5 can be largely replaced by the transmembrane domain from the activated neu receptor tyrosine kinase. Extensive mutagenesis defines the minimal structural features required for transformation by the E5 oncoprotein as, first, the ability to dimerize and, second, presentation of a negatively charged residue at the extracellular side of the membrane. The biological activity of E5 mutants that lack most amino acid residues similar to PDGF suggests that E5 and PDGF activate the PDGF receptor by distinct mechanisms.

The v-sis protein retains biological activity as a type II membrane protein when anchored by various signal-anchor domains, including the hydrophobic domain of the bovine papilloma virus E5 oncoprotein.

Membrane-anchored forms of the v-sis oncoprotein have been previously described which are oriented as type I transmembrane proteins and which efficiently induce autocrine transformation. Several examples of naturally occurring membrane-anchored growth factors have been identified, but all exhibit a type I orientation. In this work, we wished to construct and characterize membrane-anchored growth factors with a type II orientation. These experiments were designed to determine whether type II membrane-anchored growth factors would in fact exhibit biological activity. Additionally, we wished to determine whether the hydrophobic domain of the E5 oncoprotein of bovine papilloma virus (BPV) can function as a signal-anchor domain to direct type II membrane insertion. Type II derivatives of the v-sis oncoprotein were constructed, with the NH2 terminus intracellular and the COOH terminus extracellular, by substituting the NH2 terminal signal sequence with the signal-anchor domain of a known type II membrane protein. The signal-anchor domains of neuraminidase (NA), asialoglycoprotein receptor (ASGPR) and transferrin receptor (TR) all yielded biologically active type II derivatives of the v-sis oncoprotein. Although transforming all of the type II signal/anchor-sis proteins exhibited a very short half-life. The short half-life exhibited by the signal/anchor-sis constructs suggests that, in some cases, cellular transformation may result from the synthesis of growth factors so labile that they activate undetectable autocrine loops. The E5 oncoprotein encoded by BPV exhibits amino acid sequence similarity with PDGF, activates the PDGF beta-receptor, and thus resembles a miniature membrane-anchored growth factor with a putative type II orientation. The hydrophobic domain of the E5 oncoprotein, when substituted in place of the signal sequence of v-sis, was indistinguishable compared with the signal-anchor domains of NA, TR, and ASGPR, demonstrating its ability to function as a signal-anchor domain. NIH 3T3 cells transformed by the signal/anchor-sis constructs exhibited morphological reversion upon treatment with suramin, indicating a requirement for ligand/receptor interactions in a suramin-sensitive compartment, most likely the cell surface. In contrast, NIH 3T3 cells transformed by the E5 oncoprotein did not exhibit morphological reversion in response to suramin.

Immediate-early transcriptional regulation and rapid mRNA turnover of a putative serine/threonine protein kinase.

Serine/threonine protein kinases are important regulators of diverse cellular processes including metabolism, proliferation, and differentiation. We have previously identified the cDNA for a 49-kDa serine/threonine kinase, designated sgk, which is transcriptionally responsive to glucocorticoid hormones and serum in epithelial cells. We report here that sgk expression is also rapidly induced by dexamethasone or serum in Rat2 fibroblasts. Nuclear run-on and Northern blot analysis revealed that the induction of sgk mRNA is an immediate-early transcriptional response to serum stimulation of quiescent fibroblasts, which occurs just after the peak in c-jun transcription. In contrast to the glucocorticoid-stimulated sgk expression in Rat2 fibroblasts, the transcriptional induction of sgk by serum was transient and sgk transcripts decayed with a particularly rapid half-life of 20 min. The rapid turnover of sgk, in combination with its immediate-early transcriptional response to serum, suggests a novel mechanism for responding to mitogenic signals during G0 to S transition and entry into the cell cycle.

Characterization of sgk, a novel member of the serine/threonine protein kinase gene family which is transcriptionally induced by glucocorticoids and serum.

A novel member of the serine/threonine protein kinase gene family, designated sgk, for serum and glucocorticoid-regulated kinase, was identified in a differential screen for glucocorticoid-inducible transcripts expressed in the Con8.hd6 rat mammary tumor cell line. sgk encodes a protein of 49 kDa which has significant sequence homology (45 to 55% identity) throughout its catalytic domain with rac protein kinase, the protein kinase C family, ribosomal protein S6 kinase, and cyclic AMP-dependent protein kinase. sgk mRNA is expressed in most adult rat tissues, with the highest levels in the thymus, ovary, and lung, as well as in several rodent and human cell lines. sgk mRNA was stimulated by glucocorticoids and by serum within 30 min, and both inductions were independent of de novo protein synthesis. The transcriptional regulation by glucocorticoids is a primary response, since the promoter of sgk contains a glucocorticoid response element consensus sequence 1.0 kb upstream of the start of transcription which is able to stimulate chloramphenicol acetyltransferase reporter gene activity in a dexamethasone-dependent manner. Antibodies that specifically recognize sgk-encoded protein on an immunoblot were generated. This protein was shown to increase in abundance with glucocorticoid treatment in a manner which paralleled the mRNA accumulation. This is the first report of a presumed serine/threonine protein kinase that is highly regulated at the transcriptional level by glucocorticoid hormones and suggests a novel interplay between glucocorticoid receptor signalling and a protein kinase of the second messenger family.

Glucocorticoids coordinately disrupt a transforming growth factor alpha autocrine loop and suppress the growth of 13762NF-derived Con8 rat mammary adenocarcinoma cells.

We have demonstrated previously that the synthetic glucocorticoid dexamethasone suppresses the growth of Con8 rat mammary tumor cells, which are derived from the 13762NF transplantable, hormone-responsive rat mammary adenocarcinoma. Dexamethasone inhibited [3H]thymidine incorporation into Con8 cells at high cell density under both serum and serum-free conditions. Fractionation in nonreducing sodium dodecyl sulfate-polyacrylamide gels of proteins secreted from dexamethasone-treated and untreated Con8 mammary tumor cells revealed two size classes of glucocorticoid inhibited mitogenic activities; a larger M(r) 27,000-33,000 and a smaller M(r) 5,000-12,000 activity. Both size classes of mitogens restimulated the growth of glucocorticoid-suppressed Con8 cells suggesting that they can act in an autocrine fashion. The smaller mitogen was identified as transforming growth factor alpha (TGF-alpha) since this activity competed with 125I-epidermal growth factor (EGF) for EGF receptor binding and was selectively immunodepleted with monoclonal TGF-alpha antibodies but not with EGF antibodies. Western blots and radioreceptor assay of Con8-secreted proteins revealed that glucocorticoids inhibited the production of a M(r) 5500 immunoreactive TGF-alpha protein by 10-fold. Consistent with a steroid effect on the level of TGF-alpha production, rather than on its activity, the specific mitogenic activities of the TGF-alpha s secreted by dexamethasone-treated and untreated Con8 cells were identical to that of recombinant human TGF-alpha. Treatment of intact cells with suramin, which dissociates ligand-receptor complexes, revealed that the EGF receptor-mediated mitogenic response is functional in both glucocorticoid-treated and untreated cells. Taken together, our results demonstrate that glucocorticoids suppress Con8 mammary tumor cell growth and disrupt a potential TGF-alpha autocrine loop which results in a dramatic reduction in the level of extracellular TGF-alpha.

Overexpression of transforming growth factor alpha overrides the glucocorticoid-mediated suppression of Con8 mammary tumor cell growth in vitro and in vivo.

In a preceding paper (D. B. Alexander et al., Cancer Res., 53: 1808-1815, 1993), we demonstrated that the in vitro glucocorticoid inhibition of Con8 mammary tumor cell growth is accompanied by the disruption of a transforming growth factor alpha (TGF-alpha) autocrine loop. This growth suppression response functions in vivo since proliferation of Con8-derived tumors was inhibited in rats treated with the synthetic glucocorticoid, dexamethasone. The effect of dexamethasone on Con8-derived tumor growth was reversible in that tumors rapidly grew at the site of inoculation after discontinuing injections of dexamethasone. To test the in vivo relationship between the glucocorticoid growth suppression response and the TGF-alpha autocrine loop, Con8 cells were transfected with a TGF-alpha expression vector and single cell-derived neomycin-resistant subclones were recovered. [3H]Thymidine incorporation of cultured monolayers of transfected Con8 mammary cells and measurement of tumor diameters in rats revealed that dexamethasone failed to suppress the in vitro proliferation or in vivo tumor growth of Con8-derived cells producing high constitutive levels of secreted TGF-alpha. In contrast, both the in vivo and in vitro growth of Con8 cells transfected with vector controls were fully suppressible by glucocorticoids. Consistent with our in vitro observations, these results demonstrate that the regulation of TGF-alpha production plays a key role in the in vivo glucocorticoid suppression of Con8-derived mammary tumor growth.

Glucocorticoid growth suppression response in 13762NF adenocarcinoma-derived Con8 rat mammary tumor cells is mediated by dominant trans-acting factors.

The in vitro and in vivo growth of Con8 cells, a single cell-derived subclone of the 13762NF-transplantable rat mammary adenocarcinoma, is strongly suppressed by glucocorticoid hormones. Hybrids were formed between glucocorticoid-suppressible Con8.hD6 mammary tumor cells (Con8 transfected with the histidinol dehydrogenase selectable marker) and either glucocorticoid-resistant 8RUV7 mammary tumor cells (derived from Con8) or MCT-HTC rat hepatoma cells. Both of the glucocorticoid-resistant 8RUV7 and MCT-HTC fusion partners express functional glucocorticoid receptors, since hormone-responsive genes such as plasminogen activator inhibitor are fully dexamethasone inducible. Karyotypic analyses revealed that the hybrid cell populations possessed the appropriate number of chromosomes for a fusion between the glucocorticoid-suppressible and either of the two resistant cell types. Moreover, Northern blots showed that the intertissue hybrids expressed transcripts for both the milk fat globule membrane protein gene originating from the parental Con8.hD6 mammary tumor cells as well as mouse mammary tumor virus glycoprotein sequences which had been transfected into the MCT-HTC hepatoma cells as a molecular tag. Analysis of DNA content and [3H]thymidine incorporation demonstrated that growth of both the intratissue (Con8.hD6 x 8RUV7) and intertissue (Con8.hD6 x MCT-HTC) hybrids was glucocorticoid suppressible, even though the absolute rates of proliferation differed depending on the parental cells. Analysis of conditioned medium isolated from glucocorticoid-treated and untreated Con8.hD6 cells indicated that the growth suppression response is not mediated through the elaboration of an extracellular growth inhibitor. Taken together, our results demonstrate that the glucocorticoid-suppressible phenotype of Con8 rat mammary tumor cells is dominant, suggesting the existence of intracellular regulatory factors under glucocorticoid control that may function as trans-acting suppressors of tumor cell growth.

Noncoordinate expression of Drosophila glue genes: Sgs-4 is expressed at many stages and in two different tissues.

The glue genes of Drosophila melanogaster comprise a family of genes expressed at high levels in the salivary glands of late third instar larvae in response to the insect hormone ecdysone. We present evidence that, in contrast to the other glue genes, Sgs-4 is turned on throughout Drosophila development and is not expressed exclusively in the larval salivary glands. Larvae transformed with an Sgs-4/Adh (alcohol dehydrogenase) hybrid gene exhibit Sgs-4-directed Adh expression in the larval proventriculus as well as in the salivary glands as early as the first instar. Sgs-4-specific RNA can be detected at very low levels during all stages of development. During late third instar, levels of Sgs-4 RNA in the salivary glands increase several-thousand-fold, thereby accounting for the large amounts of Sgs-4 protein present in the glue produced by the salivary glands. This pattern of expression is unique to the Sgs-4 gene. While expression of several of the other glue genes can be detected in embryos and early larvae, they appear to be expressed neither throughout development nor in the larval proventriculus. Appearance of the glue gene RNAs in mid third instar salivary glands is noncoordinate, even for the chromosomally clustered genes Sgs-3, Sgs-7, and Sgs-8.

Suppression of rat mammary tumor cell growth in vitro by glucocorticoids requires serum proteins. Characterization of wild type and glucocorticoid-resistant epithelial tumor cells.

CON8 is a single-cell derived subclone of the 13762NF transplantable, hormone-responsive rat mammary tumor that proliferates rapidly in serum-free medium. Addition of either glucocorticoids or calf serum alone caused a slight stimulation of CON8 proliferation. However, glucocorticoids required the presence of specific serum proteins to strongly suppress CON8 cell growth. Furthermore, the anchorage-independent growth of CON8 cells was significantly reduced in the presence of glucocorticoids and serum. We have designated this serum activity GMGSF, for glucocorticoid modulating growth suppression factor. Inhibition of cell growth was limited to steroids with strong glucocorticoid biological activity, while exposure to the glucocorticoid antagonist RU38486 prevented this response. Half-maximal growth inhibition and half-maximal expression of a glucocorticoid-inducible gene product (2 nM) occurred slightly below the half-maximal receptor binding of [3H]dexamethasone (10nM). We have also selected a variant mammary epithelial tumor cell line, derived from CON8, denoted 8RUV7, whose proliferation and soft agar colony formation failed to be suppressed by glucocorticoids in the presence of serum. These glucocorticoid-resistant variant cells possess functional glucocorticoid receptors, competently produce the glucocorticoid-responsive gene product plasminogen activator inhibitor, and along with CON8 cells express milk fat globule protein antigens on their cell surface, indicative of their mammary epithelial cell character. We are using this variant line to genetically dissect the molecular mechanism of the glucocorticoid/GMGSF growth suppression pathway in mammary epithelial tumor cells.