Secretion of human parathyroid hormone from rat pituitary cells infected with a recombinant retrovirus encoding preproparathyroid hormone.
In order to study the functions of precursors to secreted proteins, we expressed cloned DNA encoding human preproparathyroid hormone (preproPTH) in rat pituitary cells. We first constructed a recombinant plasmid containing human preproPTH cDNA and retroviral control signals. This recombinant plasmid was transfected into psi-2 cells, a packaging cell line that produces Moloney murine leukemia viral particles containing no retroviral RNA. The transfected psi-2 cells generated helper-free recombinant retrovirus encoding preproPTH, and this recombinant retrovirus was used to infect GH4 rat pituitary cells. Clonal lines of the infected GH4 cells contained copies of the recombinant provirus stably integrated via the long terminal repeats, and the expected RNA transcripts of proviral DNA accumulated in the cytoplasm, although no infectious virus was produced. The infected cells synthesized and processed preproPTH appropriately and secreted PTH in response to thyrotropin-releasing hormone, a secretagogue for GH4 cells. Use of recombinant retrovirus permits the introduction of DNA encoding normal and mutant secreted proteins into a number of cell types specialized for secretion. Analysis of the fate of the resultant proteins will help define the specific molecular interactions involved in transmembrane transport and processing of precursor proteins.
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004247:DNA / Q000378:metabolism; D004262:DNA Restriction Enzymes; D005796:Genes; D006801:Humans; D051379:Mice; D008815:Mice, Inbred Strains; D008979:Moloney murine leukemia virus / Q000235:genetics*; D009693:Nucleic Acid Hybridization; D010281:Parathyroid Hormone / Q000235:genetics* / Q000378:metabolism; D010911:Pituitary Neoplasms; D010957:Plasmids; D011498:Protein Precursors / Q000235:genetics*; D051381:Rats
D010281:Parathyroid Hormone; D011498:Protein Precursors; C019056:preproparathormone; D004247:DNA; D004262:DNA Restriction Enzymes