We have previously shown that asparagine synthetase (AS) mRNA expression can be dramatically upregulated by asparagine deprivation in tsll cells, mutants of BHK hamster cells which encode a temperaturesensitive AS. The expression of AS mRNA was also induced upon starvation for one of several essential amino acids in HeLa cells. We also showed that regulation of AS mRNA expression by amino acid concentration has both transcriptional and posttranscriptional components. Here we report the analysis of the elements in the human AS promoter region important for its basal activity and activation by amino acid starvation. Our results indicate that a DNA fragment spanning from nucleotides -164 to +44 of the AS promoter is sufficient for uninduced and induced gene expression. Mutations in a region located 15 to 30 bp downstream from the major transcription start site that shows good homology to a sequence in the first exon of c-fos implicated as a negative regulatory element resulted in a significant increase in basal gene expression but did not affect regulation. Interestingly, this region binds single-stranded-DNA-binding proteins that are specific for the AS coding strand. Mutations in either one of two putative binding sites for transcription factor Spl, in a region of approximately 60 bp where many minor RNA start sites are located, or at the major transcription start site decreased promoter activity, but significant induction by amino acid starvation was still observed. Strikingly, mutations centered around nucleotide -68 not only decreased the basal promoter activity but also abolished amino acid regulation. This DNA region contains the sequence 5'-CATGATG-3', which we call the amino acid response element (AARE), that can bind a factor(s) present in HeLa cells nuclear extracts that is not capable of binding to an AS promoter with mutations or deletions of the AARE. This finding is in line with the hypothesis that transcriptional activation of AS gene expression is mediated through the binding of a positive regulatory element. We did not detect changes in the level of binding of this factor to the AARE by using nuclear extracts from HeLa cells grown under starved conditions, suggesting that activation of this factor(s) results from posttranslational modification or complexing with other proteins that do not affect its DNAbinding properties.The signals regulating gene expression in prokaryotes and lower eukaryotes in response to nutrient variation are well known and characterized; it is well established that these organisms are quickly able to adjust to variations in nutrient supply and to intracellular amino acid levels by altering their patterns of gene expression. Much less is known about the response of higher eukaryotes to nutrient deprivation (9).We have previously shown (13,15)

Signaling through FGF receptors, which constitute a family of membrane-spanning tyrosine kinases, can stimulate cell proliferation, induce or inhibit cell di erentiation and plays an important role in development. Recently, mutations in FGF receptors have been shown to be associated with a number of genetically dominant human skeletal disorders. A remarkably conserved mutation (Gly 380?Arg) in the transmembrane region of FGFR-3 has been shown to be responsible for achondroplasia (ACH) but it was not clear whether such mutations result in loss of receptor function or constitutive activation. We have therefore made mutations in the transmembrane regions of murine FGFR-2 and FGFR-3 and studied their e ect on receptor activity. We show here that the ACH mutation in FGFR-3 as well as two similar mutations in FGFR-2 result in constitutive activation of these receptors. This is manifested in their ability to become autophosphorylated in the absence of ligand in L6 cells, transforming activity on NIH3T3 ®broblasts, and the ability to inhibit myogenic di erentiation in the absence of growth factor. Thus the transmembrane region of FGFR-2 and FGFR-3 plays a regulatory role in receptor function and the ACH mutation produces a dominant oversignaling receptor which is no longer regulated by FGF binding. These ®ndings also support the newly identi®ed role of FGF signaling as a negative regulator of bone growth.

FGF signaling is clearly important for proper bone development, and several autosomally dominant forms of genetic bone disorders have been mapped to FGF receptors 1, 2, and 3. We have studied the biological effects of the most commonly mutated cysteine residue in FGFR-2 which is detected in individuals with Crouzon syndrome, an autosomally dominant trait which causes premature fusion of the skull bones (craniosynostosis). This Crouzon mutation replaces the cysteine at position 342 with tyrosine, thus disrupting the formation of the third immunoglobulin (Ig)-like loop in the extracellular portion of the receptor. By transfecting mutated and wild-type receptors into a variety of cell lines, we have shown that the C342Y mutation in FGFR-2 produces a receptor which is constitutively activated and capable of transforming NIH3T3 cells and preventing the differentiation of C2 myoblasts in the absence of ligand. Constitutive activation appears to result from the ability of this receptor to form stable interreceptor dimers which involve disulfide bonds between the remaining free cysteine in the mutant receptor. The altered conformation of the third Ig-like domain in the mutated receptor also results in a drastically reduced ability to bind FGF-1 or FGF-2 and in a reduced level of receptor glycosylation. Thus it appears that Crouzon syndrome results from constitutive activation of FGFR-2 and that uncontrolled FGF signaling produces alterations of intramembranous bone development and premature closing of cranial sutures.