In order to study the functions of precursors to secreted proteins, we expressed cloned DNA encoding human preproparathyroid hormone (preproPTH) Precursors of secreted proteins contain sequences that direct the proteins along the secretory pathway (1-3). Studies using cell-free extracts have shown that cytoplasmic signal-recognition particles (4) bind to polysomes that are synthesizing these precursors. The signal-recognition particles then bind to receptors on the rough endoplasmic reticulum (5). Little is understood about how' the precursors then cross the membrane of the endoplasmic reticulum and traverse the rest of the secretory pathway.Recombinant DNA technology provides methods for manipulating the sequences of genes encoding secreted proteins and then expressing these genes in cultured cells. This strategy has already been used to show that amino-terminal "signal" sequences and internal "stop transfer" sequences are required for the proper insertion of membrane-bound proteins into those membranes (6-9). To perform analogous experiments to determine how proteins traverse the entire secretory pathway, the genes encoding secreted proteins can be introduced into cells specialized for secretion.As a prelude to a detailed genetic analysis of the secretory pathway, we have expressed cDNA encoding a precursor of the human calcium-regulating parathyroid hormone (PTH), preproPTH (10), in GH4 cells. In the parathyroid gland, the 25-amino-acid amino-terminal "pre" sequence of preproPTH is cleaved to generate proPTH, and then the 6-amino-acid "pro" sequence is cleaved, yielding PTH. GH4 cells are a well-characterized clonal rat pituitary cell line that secrete prolactin and growth hormone (11); this secretion can be stimulated by secretagogues such as thyrotropin-releasing hormone (TRH) (12, 13).Exploiting the retroviral life-cycle, we stably introduced cDNA encoding preproPTH into GH4 cells by infecting GH4 cells with a recombinant retrovirus. Clonal lines derived from the recipient cells synthesized and processed preproPTH appropriately and secreted PTH. TRH stimulated the secretion of PTH as well as prolactin from these cells. MATERIALS AND METHODSPlasmid Construction. The methods for preparation of plasmid DNA, cleavage with restriction enzymes, purification of DNA fragments, ligation with T4 DNA ligase, and transfection of Escherichia coli were performed as described (14, 15).Cells. The rat pituitary cell line GH4C1 (11), a gift of A. H.Tashjian, and NIH 3T3 cells were maintained in Dulbecco's modified Eagle's medium containing 10% calf serum in 95% air/5% CO2. The Cl-C cell line, a clonal NIH 3T3 cell line productively infected with Moloney murine leukemia virus, was a gift of C. Tabin.DNA Transfection and Viruses. DNA transfections were performed with 10 ,ug of plasmid DNA (16,17) by the procedure of Graham and Van der Eb (18) as modified by Parker and Stark (19). Virus infections were performed in the presence of 8 pug of Polybrene per ml for 2.5 hr. Aminopterin was omitted from gpt-selection mediu...
Messenger ribonucleoprotein particles (mRNPs) have been isolated from rabbit reticulocyte polysomes. One of the proteins of the mRNP complex has
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