Faculty Bibliography

A major unresolved issue in the field of secretory granule biogenesis is the extent to which the aggregation of granule content proteins is responsible for the sorting of regulated from constitutively secreted proteins. The aggregation process is postulated to take place in the trans-Golgi network and immature secretory granules as the proteins encounter mildly acidic pH and high calcium concentrations. We have developed in vitro assays that reconstitute the precipitation out of solution of secretory granule content proteins of anterior pituitary gland and adrenal medulla. In the assays, all of the major granule content polypeptides form a precipitate as the pH is titrated below 6.5, and this precipitate can be recovered in the pellet fraction after centrifugation. Addition of calcium is required for the aggregation of chromaffin granule content. In contrast to the proteins secreted by the regulated pathway, the constitutively secreted proteins IgG, albumin, and angiotensinogen, when added to the assays, remain predominantly in the supernatant. Among the individual proteins tested, prolactin is found to aggregate homophilically under these conditions and can drive the co-aggregation of other proteins, such as the chromogranins. Soluble forms of granule membrane proteins, including dopamine beta-hydroxylase and peptidyl glycine alpha-amidating enzyme also co-aggregated with granule content proteins. The results are consistent with the idea that spontaneous aggregation of proteins occurring under ionic conditions similar to those at the sites of granule formation is a property restricted to those proteins packaged in secretory granules. In addition, the association of luminal domains of membrane proteins with content proteins in vitro raises the possibility that analogous interactions between membrane-bound and content proteins also occur during granule formation in intact cells

The mechanism of IgG transport by the placental trophoblast was examined by studying IgG uptake by purified trophoblast maintained in culture. This model retains the ability to bind and endocytose human IgG from human serum. Comparison of the relative IgG uptake by the trophoblast among the four subclasses of both human and mouse IgG indicates that the trophoblast IgG receptor has different affinities from those described for the three known human Fc gamma receptors, FcR gamma I, FcR gamma II, and FcR gamma III. These results suggest the presence of a novel trophoblast Fc gamma receptor. Although Fc gamma RIII has been reported to be present on trophoblasts, immunocytochemical studies failed to detect binding to the cell surface of antibody-specific for Fc gamma RIII, 3G8 MAb. In addition, blocking studies with MAb 3G8 did not interfere with IgG uptake. Scatchard analysis of human IgG uptake revealed a biphasic curve consistent with two distinct mechanisms for the transport of IgG by the trophoblast. The first is a higher affinity system (Ka = 1.7 x 10(7) M-1, 1.7 x 10(4) binding sites/cell) which exhibits IgG subclass and species specificity, and the second is a low affinity system (Ka = 6.9 x 10(3) M-1, 7.5 x 10(7) binding sites/cell)

The mechanisms for segregation of secretory and membrane proteins incorporated into storage granules from those transported constitutively have been thought to be conserved in diverse cell types, including exocrine and endocrine cells. However, GP2, the major protein of pancreatic zymogen granule membranes, in its native glycosyl phosphatidylinositol (GPI)-linked form, is incorporated into secretory granules when expressed in exocrine pancreatic AR42J cells, but not in the endocrine cells such as pituitary AtT20. To determine whether the protein moiety of GP2 contains the cell-type specific information for packaging into granules, a secretory form of GP2 (GP2-GPI-), with the GPI attachment site deleted, was generated and introduced into AR42J and AtT20 cells. Like native GP2, GP2-GPI- localized to the zymogen-like granules of AR42J cells and underwent regulated secretion. In AtT20 cells expressing GP2-GPI-, however, the protein was secreted by the constitutive pathway. Thus, a granule packaging signal is present in the luminal portion of GP2 that is functional only in the exocrine cells. However, this cell-type dependent sorting process is not limited to GP2 or membrane proteins. Amylase, a major content protein of pancreatic acinar and serous salivary gland granules, was also secreted exclusively by the constitutive pathway when expressed in AtT20 cells. The cell-type specific targeting of GP2 to granules correlated with its behavior in an in vitro aggregation assay where it co-aggregated more effectively with content proteins from pancreatic zymogen granules than with those from pituitary granules.(ABSTRACT TRUNCATED AT 250 WORDS)

Exocrine cells are epithelial cells in which secretory granules undergo fusion with the apical plasma membrane upon secretagogue stimulation. Several apical plasma membrane proteins have been found in secretory granules in cells from pancreas and salivary glands raising the possibility that incorporation into secretory granules followed by exocytosis of the granules accounts for their insertion into the apical plasma membrane. To test this hypothesis, we have expressed the influenza hemagglutinin (HA) in pancreatic AR42J cells, which make zymogen-like granules upon incubation with dexamethasone. The influenza virus HA is known to be specifically targeted to the apical plasma membrane of epithelial cells that lack a regulated pathway and is also known to be excluded from secretory granules in virally-infected pituitary AtT20 cells. Localization of the protein by immunofluorescence microscopy revealed that it accumulated at the plasma membrane of the transfected AR42J cells. HA was not observed in the amylase-rich secretory granules. By immunolabeling of ultrathin cryosections of the transfected cells, HA was also found exclusively on the cell surface, with label over secretory granules not exceeding that seen in control, untransfected cells. In addition, in cell fractionation experiments performed on radiolabeled AR42J cell transformants, HA was not detectable in the secretory granule fractions. These results indicate that HA is not efficiently stored in mature secretory granules and is likely to reach the cell surface via constitutive transport pathways

The pancreatic zymogen granule membrane protein GP-2 was introduced into cells of exocrine or endocrine origin by transfection of its cDNA in order to investigate the mechanisms by which proteins are specifically incorporated into the membranes of secretory granules. Permanent transformants expressing GP-2 were isolated from exocrine pancreatic-derived AR42J cells as well as AtT20 cells of anterior pituitary origin and insulinoma-derived Rin5F cells. In AR42J cells, GP-2 was localized by immunofluorescence and immunoelectron microscopy to the endogenous zymogen-like granules as well as to the plasma membrane. In experiments supporting the localization data, incubation of the AR42J transformants with the secretagogue cholecystokinin (CCK8) resulted in enhanced release of a shed form of GP-2 into the medium in parallel with amylase, suggesting that the two proteins were secreted from the same compartment. By contrast, when expressed in AtT20 cells, the protein was found by immunofluorescence microscopy on the plasma membrane as well as in intracellular vesicles that differed in size and location from the endogenous secretory vesicles. By electron microscopy, large (approximately 0.5 micron) multivesicular structures were observed. Single- and double-label immunoelectron microscopy demonstrated that these large organelles labeled with anti-GP-2 antibodies, whereas the smaller adrenocorticotropic hormone (ACTH)-containing secretory vesicles did not. In permanent transformants of Rin5F cells, GP-2 was also excluded from the insulin-containing granules and found in multivesicular bodies similar to those in the AtT20 cells and containing the endosomal/lysosomal marker endolyn-78. Despite the apparent accumulation of GP-2 in lysosome-like structures, it turned over slowly and did not undergo rapid endocytosis from the cell surface. We conclude that GP-2 is targeted to secretory granule membranes by cell type-specific mechanisms that likely involve its interaction with other membrane or content proteins expressed only in the exocrine cells

Human Tamm-Horsfall glycoprotein (T-H) is produced by renal cells of ascending limb of loop of Henle and is largely excreted in urine. N-linked glycans account for close to 30% of the weight of T-H. We studied the biosynthesis of recombinant T-H permanently expressed in HeLa cells. The conversion from the precursor (84 kDa) to the mature form (97 kDa) mainly depends on the processing of glycans from the high-mannose to polyantennary type. The conversion from precursor to mature form is very slow and the glycan structure of precursor appears to be that of a glycoprotein not yet processed by Golgi alpha 1,2 mannosidase. Since T-H has a very high number of disulfide bridges (more than 50 cysteine residues/mol) one may infer that the rate limiting step for the precursor export out of ER is the formation of a correct set of disulfide bonds. Mature T-H isolated from HeLa cells retained one N-linked chain with the high-mannose structure similarly to urinary T-H. This result indicates that the occurrence of one unprocessed high-mannose chain in mature T-H is host-cell independent and very likely related to the T-H primary structure