Regulated phosphorylation of secretory granule membrane proteins of the rat parotid gland

1990 ◽  
Vol 259 (1) ◽  
pp. G70-G77 ◽  
Author(s):  
C. R. Marino ◽  
J. D. Castle ◽  
F. S. Gorelick
Keyword(s):  
Membrane Proteins ◽  
Parotid Gland ◽  
Secretory Granule ◽  
Integral Membrane Proteins ◽  
Dependent Manner ◽  
Amylase Release ◽  
Rat Parotid Gland ◽  
Granule Membrane ◽  
Rat Parotid ◽  
Granule Membrane Proteins

An antiserum raised against purified rat parotid secretory granule membrane proteins has been used to identify organelle-specific protein phosphorylation events following stimulation of intact cells from the rat parotid gland. After lobules were prelabeled with [32P]orthophosphate and exposed to secretagogues, phosphoproteins were immunoprecipitated with the granule membrane protein antiserum, separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and visualized by autoradiography. Parallel studies of stimulated amylase release were performed. Isoproterenol treatment of parotid lobules resulted in an increase in the phosphate content of immunoprecipitable 60- and 72-kDa proteins that correlated with amylase release in a time-dependent manner. Forskolin addition mimicked these effects, but only the isoproterenol effects were reversed by propranolol treatment. To confirm the specificity of the antiserum to the secretory granule membrane fraction, subcellular isolation techniques were employed following in situ phosphorylation. The 60- and 72-kDa phosphoproteins were immunoprecipitated from both a particulate fraction and a purified secretory granule fraction. Furthermore, the extraction properties of both species suggest that they are integral membrane proteins. These findings support the possibility that stimulus-regulated secretion may involve phosphorylation of integral membrane proteins of the exocrine secretory granule.

scholarly journals Immunolocalization of Rap1 in the Rat Parotid Gland: Detection on Secretory Granule Membranes

1997 ◽  
Vol 45 (7) ◽  
pp. 965-973 ◽  
Author(s):  
Nisha J. D'Silva ◽  
Dennis H. DiJulio ◽  
Carol M. Belton ◽  
Kerry L. Jacobson ◽  
E.L. Watson
Keyword(s):  
Parotid Gland ◽  
Secretory Granule ◽  
Immunoblot Analysis ◽  
Image Deconvolution ◽  
High Concentration ◽  
Cytoplasmic Face ◽  
Rat Parotid Gland ◽  
Granule Membrane ◽  
Rat Parotid ◽  
Immunohistochemical Techniques

The objective of this study was to localize rap1 in the rat parotid gland. Rap1 is a small GTP-binding protein that has been linked to phagocytosis in neutrophils and various functions in platelets. In this study, we used [α-32 P]-GTP-blot overlay analysis, immunoblot analysis, and immunohistochemistry to identify rap1 in rat parotid gland. The immunohistochemical techniques included immunoperoxidase and widefield microscopy with image deconvolution. Rap1 was identified in the secretory granule membrane (SGM), plasma membrane (PM), and cytosolic (CY) fractions, with the largest signal being in the SGM fraction. The tightly bound vs loosely adherent nature of SGM-associated rap1 was determined using sodium carbonate, and its orientation on whole granules was assessed by trypsin digestion. Rap1 was found to be a tightly bound protein rather than a loosely adherent contaminant protein of the SGM. Its orientation on the cytosolic face of the secretory granule (SG) is of significance in postulating a function for rap1 because exocytosis involves the fusion of the cytoplasmic face of the SG with the cytoplasmic face of the PM, with subsequent release of granule contents (CO). Therefore, the localization and high concentration of rap1 on the SGM and its cytosolic orientation suggest that it may play a role in the regulation of secretion.

scholarly journals Mistargeting of secretory cargo in retromer-deficient cells

2020 ◽  
pp. dmm.046417
Author(s):  
Sarah D. Neuman ◽  
Erica L. Terry ◽  
Jane E. Selegue ◽  
Amy T. Cavanagh ◽  
Arash Bashirullah
Keyword(s):  
Membrane Proteins ◽  
Salivary Glands ◽  
Secretory Granule ◽  
Complex Function ◽  
Amyloid Β ◽  
Secretory Cells ◽  
Regulated Exocytosis ◽  
Retromer Complex ◽  
Granule Membrane ◽  
Granule Membrane Proteins

Intracellular trafficking is a basic and essential cellular function required for delivery of proteins to the appropriate subcellular destination; this process is especially demanding in professional secretory cells, which synthesize and secrete massive quantities of cargo proteins via regulated exocytosis. The Drosophila larval salivary glands are professional secretory cells that synthesize and secrete mucin proteins at the onset of metamorphosis. Using the larval salivary glands as a model system, we have identified a role for the highly conserved retromer complex in trafficking of secretory granule membrane proteins. We demonstrate that retromer-dependent trafficking via endosomal tubules is induced at the onset of secretory granule biogenesis, and that recycling via endosomal tubules is required for delivery of essential secretory granule membrane proteins to nascent granules. Without retromer function, nascent granules do not contain the proper membrane proteins; as a result, cargo from these defective granules is mistargeted to Rab7-positive endosomes, where it progressively accumulates to generate dramatically enlarged endosomes. Retromer complex dysfunction is strongly associated with neurodegenerative diseases, including Alzheimer's disease, characterized by accumulation of amyloid β (Aβ). We show that ectopically expressed amyloid precursor protein (APP) undergoes regulated exocytosis in salivary glands and accumulates within enlarged endosomes in retromer-deficient cells. These results highlight recycling of secretory granule membrane proteins as a critical step during secretory granule maturation and provide new insights into our understanding of retromer complex function in secretory cells. These findings also suggest that missorting of secretory cargo, including APP, may contribute to the progressive nature of neurodegenerative disease.

Effect of substance P C-terminal heptapeptide on amylase release in rat parotid gland

1980 ◽  
Vol 25 (8-9) ◽  
pp. 597-600 ◽  
Author(s):  
Akifumi Togari ◽  
Takeshi Kato ◽  
Toshiharu Nagatsu
Keyword(s):  
Substance P ◽  
Parotid Gland ◽  
Amylase Release ◽  
Rat Parotid Gland ◽  
Rat Parotid

scholarly journals Effects of clonidine on amylase release from rat parotid gland

1989 ◽  
Vol 49 ◽  
pp. 174
Author(s):  
Kazuhiro Yamada ◽  
Akifumi Togari ◽  
Shosei Matsumoto
Keyword(s):  
Parotid Gland ◽  
Amylase Release ◽  
Rat Parotid Gland ◽  
Rat Parotid
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