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

Abstract

WashingtonS U M M A R Y 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 [ ␣-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.

CalDAG-GEFI and protein kinase C represent alternative pathways leading to activation of integrin αIIbβ3 in platelets

“…Studies in other cell types demonstrated that Rap1 is localized to the surface of secretory granules and that it translocates to the plasma membrane on cellular stimulation, suggesting that it may play a role in granule secretion. [27][28][29] Furthermore, CalDAG-GEFII and CalDAG-GEFIII have been implicated in granule release in mast cells and endocrine tissue, respectively. 30,31 CalDAG-GEFmediated granule release in these cells involved activation of the small GTPase RhoA, and microtubule formation and was, at least in part, independent of PKC function.…”

GTP-Binding Proteins and Regulated Exocytosis

“…Rap lA, referred to as smg p21 or Krevl, is 95% homologous to raplB. Like other members of the ras superfamily, rap 1 has been identified in a variety of cell types and has been found to be localized to SGs in parotid acinar cells, the specific granules in neutrophils, and cx granules in platelets (Lapetina et al, 1989;Maridonneau-Parini and de Gunzburg, 1992;D'Silva et al, 1997 G-PROTEINS G-proteins have been found in the cytosol and on intracellular organelles including ER, Golgi, and SGMs (Fig. 1).…”

“…Most recently, widefield deconvolution microscopy, a technique that yields highresolution images (Agard et al, 1989) and is less damaging to tissue than confocal microscopy because it does not require laser luminology, was found to be very beneficial for the localization of rap! (D'Silva et al, 1997 Strategies for Determining the Function of GTP-binding Proteins/SNAREs ed important insights into the process of regulated secretion and support for a regulatory role of GTP-binding proteins in this process. In early studies, GTP analogs such as GTPyS were used in permeabilized cell systems to introduce a way of dissecting the exocytotic process (Baker and Knight, 1978) and to examine the role of GTPbinding proteins (Barrowman et al, 1986).…”

Bromodomain Protein Brd4 Binds to GTPase-Activating SPA-1, Modulating Its Activity and Subcellular Localization

“…The Rap1 protein may have different cellular functions depending on the isoform and its subcellular localization, as well as the proliferative capacity and differentiation state of the particular cell type. For example Rap1 proteins have been localized in secretory granules of rat salivary gland (13), neutrophils (28), and platelets (26); in the Golgi complex of rat fibroblasts (2); in the endocytic compartment of fibroblasts and skeletal muscle cells (35); and in the perinuclear region of COS-1 cells (32). More recently Rap1 has been found to be highly expressed in the nucleus of squa-mous cell carcinomas, making it the second (after Ran) of over 100 small GTP binding proteins to be identified in the nucleus.…”