Prolactin and GH form reversible aggregates in the trans-Golgi lumen that become the dense cores of secretory granules. Aggregation is an economical means of sorting, because self-association removes the hormones from other possible pathways. Secretory granules containing different aggregates show different behavior, such as the reduction in stimulated release of granules containing R183H-GH compared with release of those containing wild-type hormone. Aggregates may facilitate localization of membrane proteins necessary for transport and exocytosis of secretory granules, and therefore understanding their properties is important. Three types of self-association have been characterized: dimers of human GH that form with Zn(2+), low-affinity self-association of human prolactin caused by acidic pH and Zn(2+) with macromolecular crowding, and amyloid fibers of prolactin. The best candidate for the form in most granules may be low-affinity self-association because it occurs rapidly at Zn(2+) concentrations that are likely to be in granules and reverses rapidly in neutral pH. Amyloid may form in older granules. Determining differences between aggregates of wild type and those of R183H-GH should help to understand why granules containing the mutant behave differently from those containing wild-type hormone. If reversible aggregation of other hormones, including those that are proteolytically processed, is the crucial act in forming granules, rather than use of a sorting signal, then prohormones should form reversible aggregates in solution in conditions that resemble those of the trans-Golgi lumen, including macromolecular crowding.

Neuroendocrine cells and other secretory cell types are able to store secretory proteins in a concentrated form for extended periods until the release of large quantities of protein is triggered. The proteins are stored in dense core secretory granules. The dense cores of these granules are made up of large, insoluble aggregates that form by self-association. These aggregates solubilise rapidly into monomeric proteins in their native conformations when released from the cells by exocytosis of secretory granules. Formation of aggregates is an early event in secretory granule formation in at least some cell types. The function of secretory granules containing protein aggregates varies, depending upon the contents. This may occur because recognition of an aspect, such as a surface motif, of the aggregate facilitates correct assembly of the membrane proteins necessary for transport and exocytosis of the granules.Understanding the principles necessary for aggregation of protein hormones may help in the formulation of proteins for clinical use. Formation of aggregates of human prolactin has been investigated both in cells and in solution. In cells, the aggregation of human prolactin requires a mildly acidic pH, and is slowed in the presence of a membrane-permeable chelator of zinc. In solution, the aggregation of human prolactin at mildly acidic pH and physiological concentrations of Zn2+ resembles that which occurs in cells if the reaction is performed with macromolecular crowding, which will mimic the conditions in cells. The factors causing protein aggregation and the extent to which aggregation plays a role in secretory granule formation are likely to vary with the protein and cell type. Further understanding of the principles involved in forming these aggregates that readily disassociate may enhance the ability to formulate protein preparations. Knowledge of the exact residues involved in the protein : protein interfaces in the aggregates of secretory granule proteins may lead to the ability to use small molecules to interfere with self-association and to regulate the storage of secretory granule proteins.

Women with ovarian cancer have a low survival rate and develop resistance to chemotherapy, so new approaches to treatment are needed. We unexpectedly found administration of a replication-deficient adenovirus containing human growth hormone sequences (AdXGH) was beneficial in a mouse model of human ovarian cancer. Intraperitoneal injections of AdXGH prolonged median survival from a mean of 31 ± 1.2 to 40 ± 1.4 days in immunodeficient SCID mice given SKOV3.ip1 human ovarian cancer cells in the peritoneal cavity. Adenovirus containing human prolactin or del32-71growth hormone sequences had no effect. Repeated injection of growth hormone or implantation of tablets with sustained growth hormone release did not increase survival. Control mice had overlapping tumors throughout the peritoneal cavity and liver and frequent lung metastases 24 days after tumor cell injection. Mice that received two injections of AdXGH had no lung metastases. Mice that received four injections had no lung or liver metastases and peritoneal fibrosis. They did not survive longer than mice that received two injections, but they had enlarged livers with hepatocellular changes, indicating that a limitation of increasing the dose is liver toxicity.

This paper presents the pharmacokinetic of human growth hormone (hGH) implantable tablets tested on a human ovarian cancer mouse model. In order to obtain a sustained release device which permits to administer a high dose of the hormone that keeps its integrity and stability, three different formulations of hGH–poly (d,l-lactic-co-glycolic acid) (PLGA) were elaborated by direct compression method varying hormone load, PLGA content and compactation time. In vitro studies showed that drug release was mainly controlled by hormone load. Pharmacokinetic studies were conducted by using immunodeficient female mice. Four days before the insertion of hGH implantable tablets in the peritoneal cavity, every mouse received 5 × 106 human ovarian cancer cells (SKOV3.ip1). Hormone serum levels were monitored through bleeding from eye orbital vessels. The population pharmacokinetic model used was based on the in series tank model and model parameters were estimated using the maximum likelihood method. The null hypothesis test about differences between formulations leads us to the conclusion that the three formulations showed the same kinetic behavior except for the hGH load. The hormone release was extended all over 2 weeks but no increase or decrease in survival time was observed. These results suggest that hGH serum levels do not facilitate tumoral cells proliferation, an expected effect of hGH and this could explain why survival times of mice treated with implantable tablets are not shorter than those treated with the control ones.

There are several steps that must occur for secretory granules to form: (1) Secretory proteins that make up the dense cores of the granules must be concentrated; (2) membrane proteins necessary for granule function must accumulate in the correct location; and (3) inappropriate membrane proteins and excess membrane must be removed. Reversible aggregation of secretory granule proteins provides a mechanism for concentrating and sorting these proteins. There is specificity in the way secretory granule proteins are treated in cells that make granules. The specificity has been shown in some cases to occur after the aggregation process, so that granules containing different aggregates function differently. An explanation could be that a property of the aggregate, such as a surface motif, might influence the accumulation of membrane proteins necessary for granule function. Such a conclusion implies that the aggregates are not amorphous but have structure. Use of NMR spectroscopy to investigate changes in the environment of amino acid residues in secretory granule proteins as they form oligomers by using 15N relaxation times might provide a means to determine which residues are specifically involved in aggregation.

A prominent feature of neuroendocrine cells is the large dense-core vesicles that contain concentrated stores of protein hormones. Keeping stores of hormones in these vesicles, commonly known as secretory granules, means that large amounts of protein hormones can be rapidly released when needed, so the delay required to synthesize, fold, process, and transport hormones through the secretory pathway is avoided. The release of hormones is triggered by an increase in the concentration of cytosolic Ca2+, which causes the membranes of the secretory granule to fuse with the plasma membrane, releasing the contents into the extracellular space. All vesicular traffic in cells occurs by membrane fusion; the membrane fusion of these other vesicles differs from the membrane fusion that leads to regulated protein hormone release from secretory granules, because other vesicles fuse constitutively and the fusion of these vesicles is not stimulated by increases in Ca2+ in the cytosol. In the last decade of the 20th century, definite progress was made in understanding the mechanisms by which the release of protein hormones occurs; much of the progress came about because most membrane fusion processes have characteristics in common, regardless of whether they occur in yeast or human.

The subcellular structure of peptide hormone-producing cells includes the nucleus, cytoplasm, mitochondria, lysosomes, and plasma membrane, which are present in all cells. The parts of the secretory pathway present in all cells, the endoplasmic reticulum and Golgi complex, are more predominant in peptide hormone-secreting cells, and in these cells there are specialized subcellular components called secretory granules that store hormones until they are needed.

Human prolactin is a 199-residue (23 kDa) protein closely related to growth hormone and placental lactogen with properties and functions resembling both a hormone and a cytokine. As a traditional hormone, prolactin is produced by lactotrophic cells in the pituitary and secreted into the bloodstream where it acts distally to regulate reproduction and promote lactation. Pituitary cells store prolactin in secretory granules organized around large prolactin aggregates, which are produced within the trans layer of the Golgi complex. Extrapituitary prolactin is synthesized by a wide variety of cells but is not stored in secretory granules. Extrapituitary prolactin displays immunomodulatory activities and acts as a growth factor for cancers of the breast, prostate and tissues of the female reproductive system. We have determined the tertiary structure of human prolactin using three-dimensional (3D) and four-dimensional (4D) heteronuclear NMR spectroscopy. As expected, prolactin adopts an “up-up-down-down” four-helical bundle topology and resembles other members of the family of hematopoietic cytokines. Prolactin displays three discrete structural differences from growth hormone: (1) a structured N-terminal loop in contact with the first helix, (2) a missing mini-helix in the loop between the first and second helices, and (3) a shorter loop between the second and third helices lacking the perpendicular mini-helix observed in growth hormone. Residues necessary for functional binding to the prolactin receptor are clustered on the prolactin surface in a position similar to growth hormone. The backbone dynamics of prolactin were investigated by analysis of 15N NMR relaxation phenomena and demonstrated a rigid four-helical bundle with relatively mobile interconnecting loops. Comparison of global macromolecular tumbling at 0.1 mM and 1.0 mM prolactin revealed reversible oligomerization, which was correlated to dynamic light scattering experiments. The existence of a reversible oligomerization reaction in solution provides insight into previous results describing the in vitro and in vivo aggregation properties of human prolactin.

Estimates of the frequency of GH deficiency range from 1:4,000 to 1:10,000. Most cases are sporadic and presumed to be secondary to a wide variety of aetiologies. However, in families with consanguinity, or when a second case occurs in the same family, a genetic cause may be suspected. Given that the patient is isolated GH deficient four distinct familial types of isolated GH deficiencies (IGHD) are well-differentiated on the basis of inheritance, hormonal deficiencies as well as molecular analyses. Two forms are autosomal recessively (IGHD type IA and IB), one is autosomal dominantly (IGHD type II) and one X-linked inherited. In this review, we focus on the secretory pathway of peptide hormones in general and on the possible mechanisms causing IGHD type II in detail. Most interestingly, in IGHD type II the apparently same phenotype of IGHD is caused by distinct GH-1 gene alterations leading to different blockades within the secretory pathway. Furthermore, this type of IGHD, in addition to some other specific GH-1 gene mutations, provides the most important opportunity to shed light on cell-biological mechanisms far beyond its pure description at the DNA/RNA level.

There are three steps in the formation of secretory granules: aggregation of proteins to form the dense cores of granules, accumulation of appropriate membrane proteins necessary for function of the granules, and removal of extraneous membrane and inappropriate proteins by small vesicles. Formation of protein aggregates may be the initial step in this process, which is not well understood. Assays of aggregation of human prolactin and growth hormone in neuroendocrine cells indicate that acidic intracellular compartments are necessary, and Zn2+ and Cu2+ may facilitate aggregation through low affinity binding sites. There is more than one way to make proteins aggregate in solution; precipitates of human prolactin formed in "crowded" conditions most closely resemble what is likely to occur in cells. Understanding the properties of aggregates formed in cells may be important, as there are several examples of granules with different contents that function differently; human R183H-growth hormone, a mutant that causes autosomal dominant isolated growth hormone deficiency, also appears to be an example. Recognition of surface motifs on aggregates of proteins may be important to localize correctly membrane proteins necessary for function, an explanation for the means by which granule content may influence function.

Estimates of the frequency of growth hormone deficiency (GHD) range from 1:4,000 to 1:10,000. Most cases are sporadic and presumed to be secondary to one of a wide variety of causes. However, in families with consanguinity, or when a second case occurs in the same family, a genetic cause may be suspected. Four distinct familial types of isolated GHD (IGHD) have been well differentiated on the basis of inheritance, hormonal deficiencies, and molecular analyses. Two forms are inherited autosomal recessively (IGHD type IA and IB); one is inherited autosomal dominantly (IGHD type II); and one is inherited in an X-linked manner. This review focuses on the secretory pathway of GH and the possible mechanisms causing IGHD type II. In IGHD type II, the apparently same phenotype results from several distinct GH-1 gene alterations leading to different blockades within the secretory pathway. This type of IGHD, in addition to some other specific GH-1 gene mutations, provides the most important opportunity for shedding light on cellular biological mechanisms beyond its description at the DNA/RNA level.

Human R183H-GH causes autosomal dominant GH deficiency type II. Because we show here that the mutant hormone is fully bioactive, we have sought to locate an impairment in its progress through the secretory pathway as assessed by pulse chase experiments. Newly synthesized wild-type and R183H-GH were stable when expressed transiently in AtT20 cells, and both formed equivalent amounts of Lubrol-insoluble aggregates within 40 min after synthesis. There was no evidence for intermolecular disulfide bond formation in aggregates of wild-type hormone or the R183H mutant. Both wild-type and R183H-GH were packaged into secretory granules, assessed by the ability of 1 mM BaCl2 to stimulate release and by immunocytochemistry. The mutant differed from wild-type hormone in its retention in the cells after packaging into secretory granules; 50% more R183H-GH than wild-type aggregates were retained in AtT20 cells 120 min after synthesis, and stimulated release of R183H-GH or a mixture of R183H-GH and wild-type that had been retained in the cell was reduced. The longer retention of R183H-GH aggregates indicates that a single point mutation in a protein contained in secretory granules affects the rate of secretory granule release.

Aggregation of hormones is an important step in the formation of secretory granules that results in concentration of hormones. In transfected AtT20 cells, but not COS cells, Lubrol-insoluble aggregates of human prolactin (PRL) accumulated within 30 min after synthesis. Aggregation in AtT20 cells was reduced by incubation with 30 microM chloroquine, which neutralizes intracellular compartments, and was slowed by incubation with diethyldithiocarbamate, which chelates Cu(2+) and Zn(2+). H27A-PRL aggregated in AtT20 cells as well as wild-type PRL, indicating that a high affinity Zn(2+)-binding site is not necessary. In solution, purified recombinant human PRL was precipitated by 20 microM Cu(2+) or Zn(2+). In solution without polyethylene glycol there was no precipitation with acidic pH alone, precipitation with Zn(2+) was most effective at neutral pH, and the ratio of Zn(2+) to PRL was greater than 1 in the precipitate. In solution with polyethylene glycol, precipitation occurred with acidic pH, precipitation with Zn(2+) occurred effectively at acidic pH, and the ratio of Zn(2+) to PRL was less than 1. The aggregates obtained in polyethylene glycol are therefore better models for aggregates in cells. Unlike human PRL, aggregation of rat PRL has been shown to occur at neutral pH in cells and in solution, and therefore these two similar proteins form aggregates that are the cores of secretory granules in ways that are not completely identical.

Many membrane receptors have truncated soluble forms that circulate in the blood, and a protein in serum with characteristics of the extracellular domain of the human prolactin receptor has recently been identified. Because the extracellular domain of the prolactin receptor binds human growth hormone, does the prolactin-binding protein in serum affect the amount of bound circulating growth hormone? A more general question is whether this serum protein has a minor or a major effect on the biological activities of growth hormone and prolactin.

Soluble protein hormones are concentrated and stored in secretory granules The cisternal maturation model for transport of proteins through the Golgi complex allows a major role for formation of reversible aggregates as a means of both concentrating and sorting hormones, since soluble proteins will be removed in small vesicles, leaving behind the aggregated hormones. The storage of secretory granule proteins, however, is more selective than would be expected if passive aggregation were the only process involved. Aggregation of hormones in the secretory pathway may not be completely passive, but may be controlled by the cells. In addition to aggregation, other layers of sorting must exist, because there is selective retention of proteins after aggregation or packaging into granules.

Familial isolated GH deficiency type II is an autosomal dominant form of short stature, associated in some families with mutations that result in missplicing to produce del32-71-GH, a protein that cannot fold normally. The mechanism by which this mutant suppresses the secretion of wild-type GH encoded by the normal allele is not known. Coexpression of del32-71-GH with wild-type human GH in transient transfections of the neuroendocrine cell lines GH4C1 and AtT20 suppressed accumulation of wild-type GH. The suppression of wild-type GH accumulation by del32-71-GH was a posttranslational effect on wild-type GH caused by decreased stability, rather than decreased synthesis, of wild-type GH. Coexpression of del32-71-GH with human PRL did not suppress accumulation of PRL, indicating that there was not a general suppression of secretory pathway function. Accumulation of del32-71-GH protein was not necessary for the suppression of wild-type GH, because del32-71-GH did not accumulate in the neuroendocrine cell lines in which suppression of accumulation of wild-type GH was observed. Del32-71-GH did accumulate in transfected COS and CHO cells, but did not suppress the accumulation of wild-type GH in these cells. These studies suggest that del32-71-GH may cause GH deficiency in somatotropes of heterozygotes expressing both wild-type and del32-71-GH by decreasing the intracellular stability of wild-type GH.