We identified and characterized elements which confer tissue specificity and cyclic AMP (cAMP) responsiveness to the human glycoprotein a-subunit gene. An enhancer containing an 18-base-pair repeat conferred cAMP responsiveness in a non-tissue-specific fashion. DNase I protection assays revealed DNA-binding factors that bound to this element in both placental and nonplacental cells. It also enhanced the a-subunit promoter in a tissue-specific manner but had a negligible effect on a heterologous promoter. A unique element found upstream of this enhancer had no independent activity but, in combination with the cAMP-responsive enhancet-, distinctly increased the tissue-specific activity of both the a-subunit promoter and a heterologous promoter. A factor that bound to this upstream element was found in placental but not nonplacental cells. We conclude that this novel element acts, perhaps through a specific trans-acting factor, in concert with a cAMP-responsive enhancer to confer tissue specificity to the a-subunit gene.The a-subunit gene of human glycoprotein hormones is expressed coordinately with each of four separate ,B-subunit genes. Their combined products comprise the four glycoprotein hormones: chorionic gonadotropin, luteinizing hormone, follicle-stimulating hormone, and thyroid-stimulating hormone (14,36). These hormones direct the synthesis of several classes of hormones and control aspects of reproductive and metabolic function. They are produced in three separate cell types: placental trophoblasts produce chorionic gonadotropin, pituitary gonadotropes produce folliclestimulating hormone and luteinizing hormone and pituitary thyrotropes produce thyroid-stimulating hormone (36). In each of these cell types, production of the a subunit is regulated differently in coordination with the a subunit with which it becomes associated (16,33,37). For example, thyroid-stimulating hormone expression is induced by thyrotropin-releasing hormone and inhibited by thyroid hormones, while luteinizing hormone and follicle-stimulating hormone are induced by gonadotropin-releasing hormone and inhibited by estrogens (12). Thus, the a-subunit gene must contain regulatory sequences which direct its expression in several different tissues and mediate its differential hormonal responsiveness.Some information is available concerning the mechanisms which control these complex responses. Nuclear transcription assays have denmonstrated that thyroid hormones have direct effects on the transcription of both the a(-and 1B-subunit genes in pituitary thyrotropes (40). In addition, cyclic AMP (cAMP) increases the transcription rate of the axand ,B-chorionic gonadotropin genes in placental cells (21), and the cAMP responsiveness of the ox-subunit gene has been shown by gene transfer experiments to reside in its 5'-flanking sequences (8,21,41 mally involved in transcription in order to modify their activity.In the current studies we analyzed two elements important for the expression of the human a-subunit getne. The 5'-flanking region conta...

SummaryThymic epithelial ceils play a crucial role in the selection of developing thymocytes. Thymocyteepithelial cell interactions involve a number of adhesion molecules, including members of the integrin and immunoglobulin superfamilies. We found that human thymic epithelial cells synthesize an endogenous lectin, galectin-1, which binds to oligosaccharide ligands on the surface of thymocytes and T lymphoblastoid cells. Binding of T lymphoblastoid cells to thymic epithelial cells was inhibited by antibody to galectin-1 on the epithelial cells, and by two antibodies, T305 and 2Bll, that recognize carbohydrate epitopes on the T cell surface glycoproteins CD43 and CD45, respectively. T lymphoblastoid ceils and thymocytes bound recombinant galectin-1, as demonstrated by flow cytometric analysis, and lectin binding was completely inhibited in the presence of lactose. The degree of galectin-1 binding to thymocytes correlated with the maturation stage of the cells, as immature thymocytes bound more galectin-1 than did mature thymocytes. Preferential binding of galectin-1 to immature thymocytes may result from regulated expression of preferred oligosaccharide ligands on those cells, since we found that the epitope recognized by the T305 antibody, the core 2 O-glycan structure on CD43, was expressed on cortical, but not medullary cells. The level of expression of the UDP-GIcNAc:Gal31,3GalNAc-R 31, 6GlcNAc transferase (core 2 31, 6 GlcNAc transferase, or C2GnT), which creates the core 20-glycan structure, correlated with the glycosylation change between cortical and medullary cells. Expression of mRNA encoding the C2GnT was high in subcapsular and cortical thymocytes and low in medullary thymocytes, as demonstrated by in situ hybridization. These results suggest that galectin-1 participates in thymocyte--thymic epithelial cell interactions, and that this interaction may be regulated by expression of relevant oligosaccharide ligands on the thymocyte cell surface.

A temperature-sensitive mutant K12 derived from a Chinese hamster fibroblast has been shown to overproduce three specific proteins of Mr 94,000, 78,000, and 58,000 when incubated at the nonpermissive temperature (40.50C). We previously identified these proteins as glucose-regulated proteins similar to those observed in chicken embryo fibroblasts when the cells are starved of glucose. In this report, we show that the Mr 78,000 proteins isolated from the hamster K12 cell line and from chicken embryo fibroblasts have identical electrophoretic mobilities in two-dimensional isoelectric focusing gels and nearly identical peptide maps. However, these proteins are different from heat-shock proteins previously described for animal cells. We have constructed a library ofcDNA clones by using the RNA extracted from the hamster K12 cells incubated at 40.50C. Clones that hybridize preferentially with cDNA made from RNA at 40.50C were selected. By using the hybrid-selection technique, followed by in vitro translation, a cDNA clone containing a 2550-nucleotide insert coding for the hamster Mr 78,000 protein has been identified.It has been observed in various animal cell lines that, when the glucose in the growth medium is depleted, the synthesis of a specific set ofproteins is enhanced (1-4). Two of these proteins observed in chicken embryo fibroblasts have Mrs of 90,000-95,000 and 73,000-79,000 and were originally thought to be transformation-specific proteins (5, 6). Subsequently, it was demonstrated that increased contents of these proteins were actually due to rapid depletion of glucose from the growth medium by the transformed cells (1). The accumulation of three specific proteins (Mr 94,000, 78,000, and 58,000) related to the glucose-regulated proteins in a temperature-sensitive hamster mutant cell line, K12, has been reported (4). This cell line is particularly useful for the study of the coordinated expression ofthese proteins because, when the cells are shifted to the nonpermissive temperature (40.50C), the increase in synthesis of the glucose-regulated proteins is 5-fold to 10-fold greater than that obtained by glucose starvation. This accumulation is primarily due to new transcription and synthesis (7). Thus, the hamster K12 cells provide a convenient system for the molecular cloning of the genes coding for these proteins.In this report we describe experiments which characterize the relationship of the Mr 78,000 glucose-regulated proteins produced by hamster and chicken embryo fibroblasts. Our results indicate that this protein is highly conserved in the two species. Contrary to an earlier speculation (8), we demonstrate that the glucose-regulated proteins are different from heatshock proteins observed in animal cells. We also present preliminary evidence for the identification ofa cDNA clone coding for the Mr 78,000 glucose-regulated protein.MATERIALS AND METHODS Cell Lines and Culture Conditions. Conditions for culturing the hamster cell lines and labeling the cellular proteins have been described (4). Fo...

Membrane vesicles enriched in acetylcholine receptor were prepared from the electroplax tissue of Torpedo californica. The receptor was reduced with dithiothreitol to expose a sulfhydryl group near the ligand binidng site and then treated in one of the following ways: (1) affinity alkylated treated in one of the following ways: (1) affinity alkylated with bromoacetylcholine, a receptor activator, (2) affinity alkylated with maleimidobenzyltrimethylammonium, a receptor inhibitor, or (3) reoxidized to the native state with dithiobis(2-nitrobenzoate). The affinity labels blocked half of the binding sites for alpha-bungarotoxin. The toxin sites not protected by the affinity labels were protected by carbamylcholine based on studies of toxin binding kinetics. The functional response of native and affinity-alkylated receptors was measured by a sodium ion flux procedure. In the absence of added cholinergic activators, only slow ion flux was observed. In the presence of carbamylcholine, a receptor activator, both native and modified membranes showed the increased sodium flux associated with functional receptors. The concentration of carbamylcholine required for a 50% maximal response was higher in the affinity-labeled membranes. Preincubation of the membranes with carbamylcholine blocked the increased ion flux, indicating that desensitization could be induced. The results provide evidence for the existence of two functional sites on the acetylcholine receptor. Each site corresponds to a bungarotoxin binding site and can be independently activated and desensitized.

The tissue inhibitors of metalloproteinases (TIMPs) comprise a family of proteins, of which two members have so far been described in humans. We have cloned and sequenced a third human TIMP (hTIMP-3) from phorbol ester-differentiated THP-1 cells stimulated with bacterial lipopolysaccharide. The open reading frame encodes a 211-amino-acid precursor including a 23-residue secretion signal. The mature polypeptide has a calculated molecular weight of 21.6 kD and includes an N-linked glycosylation site near the carboxyl terminus. The protein is quite basic, having a predicted isoelectric point of 9.04. We have mapped the single gene encoding human TIMP-3 to chromosome 22. By Northern analysis, transcripts for TIMP-3 were identified in a broad cross-section of tissues examined from both embryonic and adult origin. In all tissues except the placenta, the predominant transcript was 5.0 kb in size, with minor bands around 2.4 and 2.6 kb comprising no more than about 10% of the signal. In the placenta, the smaller bands accounted for close to 50% of the signal. Human TIMP-3 shows slightly closer amino acid sequence similarity to TIMP-2 (44.3%) than to TIMP-1 (38.4%), but is most closely related to a recently reported chicken TIMP, chIMP-3 (80.8% amino acid; 77.7% nucleic acid similarity.