Abstract
Rat prolactin in the dense cores of secretory granules of the pituitary gland is a Lubrol-insoluble aggregate. In GH(4)C(1) cells, newly synthesized rat prolactin and growth hormone were soluble, but after 30 min about 40% converted to a Lubrol-insoluble form. Transport from the endoplasmic reticulum is necessary for conversion to Lubrol insolubility, since incubating cells with brefeldin A or at 15 degrees C reduced formation of insoluble rat (35)S-prolactin. Formation of Lubrol-insoluble aggregates has protein and cell specificity; newly synthesized human growth hormone expressed in AtT20 cells underwent a 40% conversion to Lubrol insolubility with time, but albumin did not, and human growth hormone expressed in COS cells underwent less than 10% conversion to Lubrol insolubility. del32-46 growth hormone, a naturally occurring form of growth hormone, and P89L growth hormone underwent conversion, although they were secreted more slowly, indicating that there is some tolerance in structural requirements for aggregation. An intracellular compartment with an acidic pH is not necessary for conversion to Lubrol insolubility, because incubation with chloroquine or bafilomycin slowed, but did not prevent, the conversion. GH(4)C(1) cells treated with estradiol, insulin, and epidermal growth factor accumulate more secretory granules and store more prolactin, but not more growth hormone, than untreated cells; Lubrol-insoluble aggregates of prolactin and growth hormone formed to the same extent in hormone-treated or untreated GH(4)C(1) cells, but prolactin was retained longer in hormone-treated cells. These findings indicate that aggregation alone is not sufficient to cause retention of secretory granule proteins, and there is an additional selective process.
Highlights
Other secretory cells have a specialized pathway to the cell surface in which certain secreted proteins are stored in concentrated forms in vesicles called secretory granules, so that the proteins are rapidly available in large amounts when needed
Formation of Lubrol-insoluble aggregates has protein and cell specificity; newly synthesized human growth hormone expressed in AtT20 cells underwent a 40% conversion to Lubrol insolubility with time, but albumin did not, and human growth hormone expressed in COS cells underwent less than 10% conversion to Lubrol insolubility. del32– 46 growth hormone, a naturally occurring form of growth hormone, and P89L growth hormone underwent conversion, they were secreted more slowly, indicating that there is some tolerance in structural requirements for aggregation
In GH4C1 cells treated with hormones, over 90% of newly synthesized 35S-prolactin was soluble when cell membranes were disrupted with Lubrol
Summary
Other secretory cells have a specialized pathway to the cell surface in which certain secreted proteins are stored in concentrated forms in vesicles called secretory granules, so that the proteins are rapidly available in large amounts when needed. Prolactin in solution has a tendency to aggregate at mildly acidic pH [6, 7]; the tendency to aggregate in solution at acidic pH is a property shared by many proteins that are stored in granules (8 –13) Such aggregation in solution has led to one of the simplest models for concentrating secretory granule proteins, which is that secretory proteins are soluble in the secretory pathway until they reach the trans-Golgi region, where they aggregate, facilitated by the decrease in pH that occurs there. Prolactin and growth hormone have several properties that make them useful for understanding the processes that lead to storage in secretory granules They are small (22 kDa), monomeric proteins that are stored in granules and neither glycosylated nor proteolytically processed beyond the cleavage of the signal sequence necessary for transport into the endoplasmic reticulum. We have investigated whether hormone treatment increases the ability of cells to store prolactin by regulating their ability to aggregate prolactin preferentially
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