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...

Transcription of proto-oncogene los is induced by elevated levels of intracellular cAMP. We report that human c-fos promoter recombinants transfected into rat pheochromocytoma cells (PC12) and human choriocarcinoma cells {JEG-3) are induced by stimulation of adenylate cyclase and that this induction is diminished considerably in the mutant PC12 cell line A126-1B2, which is deficient in cAMP-dependent protein kinase II. An element centered at position -60 of the c-los promoter, which encompasses a consensus cAMP response element (CRE), is sufficient to confer cAMP responsiveness to a herpes thymidine kinase/CAT fusion gene. The specific binding of a nuclear protein to the c-los CRE can be competed by the somatostatin and ~-chorionic gonadotropin {a-CG} promoter regions that contain CREs. Gel mobility shift assays with double-stranded oligonucleotides containing either the wild-type or mutated c-los CRE sequence have demonstrated that binding occurs only to the wild-type CRE. The nuclear factor binding to the c-los CRE is likely to be transcription factor CREB (CRE nuclear binding protein), because an affinity-purified 43-kD CREB isolated from PC12 cells binds efficiently in a DNA footprinting assay. Thus, regulation of the c-los gene transcription appears to involve a mechanism common to many genes that respond to cAMP as a second message leading to cell growth and differentiation.

To evaluate whether catabolic levels of glucocorticoids activate the ubiquitin pathway in conjunction with their known proteolytic effect in skeletal muscle, rats were injected daily with corticosterone (CTC; 10 mg/100 g body wt) for 7 days. Two peaks of urinary excretion of 3-methylhistidine (3-MH), a specific marker of myofibrillar proteolysis, were observed at days 1 and 3 (165 and 295% of controls, respectively). Levels of ubiquitin pathway mRNAs in skeletal muscle were assessed around the 3-MH peaks. In the extensor digitorum longus, a first rise of two polyubiquitin (pUb) mRNAs was seen at day 1 (183 and 162% of control for the UbB and UbC transcripts, respectively, P < 0.01). An accumulation of both E2-14k mRNAs (140%, P < 0.02, and 157% of controls, P < 0.01) and proteasome C8 subunit mRNA (222% of control, P < 0.05) was seen at day 2. A second more important peak of induction of pUb mRNA was seen at day 3 (251 and 217% of controls for the UbB and UbC transcripts, respectively, P < 0.001). All transcripts returned to near control levels by day 4. In the soleus, induction of E2-14k mRNA started at day 3 and reached 216 and 208% of controls at day 4 (P < 0.001), whereas an increase of pUb mRNA was observed at days 3 (213 and 241%, P < 0.05) and 4 (211 and 221%, P < 0.001). A rise of proteasome C8 subunit mRNA accumulation was also seen in the soleus at days 3 (217%, P < 0.05) and 4 (157%, P < 0.05). Reduced ubiquitin conjugate levels, possibly due to their rapid degradation through increased proteasome activity, were observed in both muscle types at day 3. The parallel between the catabolic effects of CTC and activation of the ubiquitin pathway in muscles of CTC-treated rats strongly suggests the involvement of this system in glucocorticoid-induced muscular atrophy.

We describe the molecular cloning and characterization of a novel giant human cytoplasmic protein, trabeculin-␣ (M r ‫؍‬ 614,000). Analysis of the deduced amino acid sequence reveals homologies with several putative functional domains, including a pair of ␣-actinin-like actin binding domains; regions of homology to plakins at either end of the giant polypeptide; 29 copies of a spectrinlike motif in the central region of the protein; two potential Ca 2؉ -binding EF-hand motifs; and a Ser-rich region containing a repeated GSRX motif. With similarities to both plakins and spectrins, trabeculin-␣ appears to have evolved as a hybrid of these two families of proteins. The functionality of the actin binding domains located near the N terminus was confirmed with an F-actin binding assay using glutathione S-transferase fusion proteins comprising amino acids 9 -486 of the deduced peptide. Northern and Western blotting and immunofluorescence studies suggest that trabeculin is ubiquitously expressed and is distributed throughout the cytoplasm, though the protein was found to be greatly up-regulated upon differentiation of myoblasts into myotubes. Finally, the presence of cDNAs similar to, yet distinct from, trabeculin-␣ in both human and mouse suggests that trabeculins may form a new subfamily of giant actin-binding/cytoskeletal cross-linking proteins.