Retrovirus-mediated expression of preprosomatostatin: posttranslational processing, intracellular storage, and secretion in GH3 pituitary cells.
Somatostatin (SRIF) is a 14-amino acid peptide hormone that is synthesized as part of a larger precursor, preproSRIF, consisting of a signal peptide and a proregion of 80-90 amino acids. The mature hormone, which is located at the carboxyl terminus of the precursor, is preceded by a single pair of basic amino acids. We are studying preproSRIF to investigate intracellular sorting, proteolytic processing, and storage of peptide hormone precursors in the secretory pathway. We used a retroviral expression vector to achieve the high levels of precursor synthesis which are necessary for detailed characterization of processing intermediates and mature somatostatin. Recombinant retroviruses containing RNA transcripts encoding anglerfish preproSRIF I were used to infect rat pituitary GH3 cells which secrete growth hormone and prolactin, neither of which are substrates for endoproteolytic cleavage. In these cells preproSRIF was accurately processed to the mature hormone with an efficiency of approximately 75%. Of the newly synthesized mature SRIF, 55% was sorted into the regulated secretory pathway and released in response to the secretagogue 8-Br-cAMP. The remaining 45% of mature SRIF and residual unprocessed precursor was rapidly secreted. In contrast to SRIF, only 5% of newly synthesized endogenous growth hormone was stored intracellularly, whereas 95% was sorted to the constitutive pathway and secreted rapidly with kinetics identical to proSRIF. Our results show that proSRIF processing is not necessarily dependent on a specific protease found only in SRIF-producing cells and suggest that proteolytic cleavage is not restricted to cells that process endogenous hormones. Moreover, these results demonstrate that GH3 cells have the capacity to discriminate between endogenous and foreign hormones and target the foreign molecule significantly more efficiently to the regulated secretory pathway.
Stoller
T J
TJ
Department of Anatomy, Albert Einstein College of Medicine, Bronx, New York 10461.
Shields
D
D
D000818:Animals; D001692:Biological Transport; D002451:Cell Compartmentation; D002460:Cell Line; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D013006:Growth Hormone / Q000378:metabolism; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats; D012190:Retroviridae / Q000235:genetics*; D013004:Somatostatin / Q000235:genetics / Q000378:metabolism*
D011498:Protein Precursors; D013004:Somatostatin; D013006:Growth Hormone