High- and Small-Molecular-Weight GTP-Binding Proteins in Zymogen Granule Membranes of Rat Pancreatic Acinar Cells

Abstract

We have detected high- and small-molecular-weight GTP-binding proteins (G- and smg-proteins, respectively) in zymogen granule membranes (ZGM) of rat pancreatic acinar cells. G_i-proteins with molecular weights of 40/41 kDa were detected by pertussis-toxin induced ADP ribosylation and by [α^-32P] GTP photoaffinity labelling in ZGM. Smg-proteins were analysed by [α^-32P] GTP binding, ADP ribosylation with Clostridium botulinum ADP ribosyltransferase C3 and immunoblotting following one- and two-dimensional gel electrophoresis. Two-dimensional separation of ZGM proteins revealed the presence of up to 30 differently charged smg-proteins with molecular masses between 18 and 27 kDa in ZGM. A monoclonal antibody, which recognizes both rab3A and rab3B (clone 42.1), identified 2 proteins with isoelectric points (pI) of 4.89 and 4.96 as proteins closely related to rab3. These proteins were mainly present in ZGM and to a slight extent also in plasma membranes (PM), microsomal membranes (MM) and cytosol. An antibody, which recognizes rab3A only (clone 42.2), did not react with pancreatic proteins. An anti-rap IB antibody recognized a 23 kDa protein localized in the ZGM, PM and to a lesser extent in MM. A monoclonal antibody against all 3 known ras proteins (p21^Ha-ras, p21^ĸi-ras, p21^N-ras) identified ras proteins only in PM and not in ZGM or other membranes. C3-induced ADP ribosylation as indication for the presence of rho and/or rac proteins was detected in PM, MM and cytosol but not in ZGM. The presence of multiple GTP-binding proteins in ZGM suggests a possible role of these proteins in the regulation of exocytosis in pancreatic acinar cells.

Rap1A and rap1B ras-family proteins are prominently expressed in the nucleus of squamous carcinomas: nuclear translocation of GTP-bound active form

“…For example, in rat salivary gland (D 'Silva et al, 1997'Silva et al, , 1998, neutrophils (Maridonneau-Parini and de Gunzburg, 1992) and platelets (Lapetina et al, 1989;Berger et al, 1994), rap1 has been localized to secretory granules and is thought to regulate secretory granule formation or exocytosis (Bos et al, 2001). Rap1 proteins have also been localized on the Golgi of rat fibroblasts (Beranger et al, 1991), the zymogen granules, plasma membrane and microsomal membrane of pancreatic acini (Schnefel et al, 1992), and in the endocytic compartment of fibroblasts and skeletal muscle cells (Pizon et al, 1994(Pizon et al, , 1996. More recently, rap1 was identified in the perinuclear region of COS-1 cells (Mochizuki et al, 2001).…”

Heterotrimeric G-protein Gq/11 Localized on Pancreatic Zymogen Granules Is Involved in Calcium-regulated Amylase Secretion

“…However, little is known about the presence or function of heterotrimeric G-proteins on zymogen granules. Although the existence of a pertussis toxin-sensitive G-protein on pancreatic zymogen granules was previously suggested (22) its identity is still uncertain. In the current work, we have identified a heterotrimeric G-protein(s), which localizes to zymogen granules and appears to participate in regulated exocytosis.…”

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

“…[ ␣-32 P]-GTP-blot Overlay Assay Low molecular mass GTP-binding proteins were detected by the radiolabeled GTP-blot overlay method of Bhullar and Haslam (1987), with modification as described by Schneffel et al (1992) and Gromov and Celis (1994). Briefly, SGM, PM, CY, CO, ER, rR, and molecular weight standards were separated by SDS-12% PAGE and transferred to nitrocellulose filters.…”

“…These proteins are found in almost all tissues (Bokoch 1993). Like other members of the ras superfamily, rap1 has been identified in a variety of cell types, which include human epidermoid carcinoma cells, CHO cells, the Golgi of rat fibroblasts, the zymogen granule, PM and microsomal membrane of rat pancreatic acinar cells, the specific granules and PM in neutrophils, and cytosol, ␣-granules, and PM fractions of human platelets (Lapetina et al 1989;Nagata et al 1989;Matsui et al 1990;Beranger et al 1991;Maridonneau-Parini and de Gunzburg 1992;Quinn et al 1992;Schneffel et al 1992;Mori et al 1993;Berger et al 1994;Nagata et al 1995). This indicates that it serves a general function regulated via different effector pathways, depending on the cell type involved (Bokoch 1993).…”

“…For example, it may be involved in controlling cell growth and differentiation (Bokoch 1993). Alternatively, the variable localization of rap1 on specific intracellular organelles, such as the SG (Maridonneau-Parini and de Gunzburg 1992;Schneffel et al 1992;Berger et al 1994;Nagata et al 1995) may be responsible for its playing well-defined biological functions that vary with intracellular location and cell type (Bokoch 1993). In neutrophils, in which rap1 is located primarily on the specific granules, studies suggest that it plays a crucial role in phagocytosis (Maridonneau-Parini and de Gunzburg 1992).…”