ARTICLE |
Correspondence to: Nisha J. D'Silva, Dept. of Oral Biology, Box 357132, U. of Washington, Seattle, WA 98195.
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Summary |
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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 [-32P]-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. (J Histochem Cytochem 45:965-973, 1997)
Key Words: rap1, small GTP-binding protein, rat parotid gland, widefield microscopy with image deconvolution
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Introduction |
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There are two groups of signal-transducing GTP-binding proteins, heterotrimers and monomers. The heterotrimeric GTP-binding or G-proteins transduce signals between cell-surface receptors and intracellular effectors (-, ß-, and
-subunits that have molecular weights of 39-46, 37, and 8 kD, respectively (
-subunit that is a GTPase, i.e., it binds and hydrolyzes GTP and is primarily responsible for the diversity of heterotrimeric G-proteins. The monomeric or small GTP-binding proteins (smgs) differ from the heterotrimeric G-proteins in that they have smaller molecular masses (18-30 kD), consist of a single subunit, have specific GTPase-activating proteins that enhance intrinsic GTPase activity, and may serve different regulatory functions (
The smg rap has close homology to ras but often differs in cellular and intracellular localization (-granules, and PM fractions of human platelets (
activity (
In this study we identified the smg rap1 on SGM, PM, and CY fractions of the rat parotid gland by radiolabeled GTP-blot overlay analysis, immunoblot analysis, and immunohistochemistry. Rap1 was found to be a tightly bound protein of the SGM, and its location on the cytosolic face of the SG suggests that it may play an important role in exocytosis.
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Materials and Methods |
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Materials
Materials were obtained as follows: renografin-60 from ER Squibb (New Brunswick, NJ); N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), diaminobenzidene tetrahydrochloride (DAB), rabbit anti-human -amylase antibody, 4',6-diamidino-2-phenylindole (DAPI), and hyaluronidase from Sigma Chemicals (St Louis, MO); crude collagenase CLS2 from Worthington Laboratories (Freehold, NJ); di-thiothreitol (DTT), Tween-20, SDS-PAGE chemicals and low molecular weight protein standards from Bio-Rad (Richmond, CA); SDS-PAGE and Western blot apparatus including mini-cell, precast 12% Tris-glycine minigels, blotting transfer module, and polyvinylidene fluoride (PVDF) filters from Novex (Encinitas, CA); nitrocellulose filters from Schleicher & Schuell, 0.2 µm (Keene, NH); [
-32P]-GTP from Dupont-New England Nuclear Research Products (Boston, MA); enhanced chemiluminescent (ECL) Western blotting detection system and Hyperfilm MP autoradiography film from Amersham (Arlington Heights, IL); affinity-purified horseradish peroxidase (HRP)-conjugated donkey anti-rabbit IgG and normal rabbit serum from Jackson ImmunoResearch Laboratories (West Grove, PA); rabbit anti-rap1 affinity-purified polyclonal antibody and its neutralizing peptide from Santa Cruz Biotechnology (Santa Cruz, CA); Vectastain ABC kit, affinity-purified fluorescein-conjugated goat anti-rabbit IgG, affinity-purified Texas red-conjugated goat anti-rabbit IgG, and Vectashield from Vector Laboratories (Burlingame, CA). Recombinant rap1b (rR) was a generous gift from Dr. Thomas H. Fischer, (University of North Carolina). Rabbit antiserum against purified parotid secretory granule membrane was kindly provided by Drs. A. Castle and J.D. Castle (University of Virginia).
Preparation of Rat Parotid Plasma and Secretory Granule Membranes
Briefly, the parotid glands were removed from fasted male Sprague-Dawley rats weighing 100-120 g. The glands were minced and homogenized and a 250 x g supernatant and pellet were obtained as described by
Preparation of Purified Rough Endoplasmic Reticulum
Endoplasmic reticular membranes (ER) were prepared from whole rat parotid glands as described previously by
Preparation of Rat Parotid Acinar Cells
Parotid acini were prepared as described by
Preparation of Cytosol
For the CY preparation, rat parotid acini were prepared as described above, homogenized and centrifuged at 250 x g for 5 min. Cytosol was prepared from the supernatant by centrifugation at 100,000 x g for 1 hr at 4C.
-32P]-GTP-blot Overlay Assay
Low molecular mass GTP-binding proteins were detected by the radiolabeled GTP-blot overlay method of -32P]-GTP/ml, with or without added unlabeled GTP (10 µM) for 60 min at room temperature, and washed repeatedly in cold buffer without DTT. The filters were air-dried and exposed to autoradiographic film with and without intensifying screens for 25 hr to 13 days. The data were analyzed by laser densitometry.
Immunoblot Detection of Rap1
Secretory granule membrane, PM, CY, CO, ER and molecular weight standards were separated by SDS-12% PAGE and transferred to PVDF filters. The filters were incubated with rabbit anti-rap 1 affinity-purified polyclonal antibody (1 µg/ml) or with antibody preincubated with a 20-fold excess of its neutralizing peptide. Antibody binding was detected with the ECL detection system, using HRP-linked donkey anti-rabbit IgG secondary antibody (1:15,000).
Immunohistochemistry
A parotid gland was harvested and placed in Carnoy's fixative (acetic acid 10%, methanol 60%, chloroform 30%) for 3 hr, transferred to decreasing grades of alcohol to replace the choloroform with water, dehydrated to remove the water, which is not miscible with paraffin, and paraffin-embedded. Rap1 was detected on 5-µm tissue sections using rabbit anti-rap1 affinity-purified polyclonal antibody (5 µg/ml) with the avidin-biotin-peroxidase immunoassay using the Vecta-stain ABC kit and the DAB colorimetric detection system.
For widefield microscopy with image deconvolution studies (
Controls for the immunoperoxidase studies included omission of the primary antibody or substitution of the primary antibody with rabbit IgG. For the widefield deconvolution microscopy studies, the controls included substitution of the second primary antibody (against SGM) with buffer or IgG.
Tightly Bound vs Loosely Adherent Protein/Epitope Orientation
To determine whether rap 1 is tightly bound to the SGM or whether it becomes associated with it during isolation procedures, SGMs were treated with 0.1 M sodium carbonate as described by
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Results |
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Detection of Monomeric GTP-binding Proteins by [-32P]-GTP Overlay Assay
Initial experiments were designed to determine the presence of smg proteins, particularly rap1, in cell fractions isolated from rat parotid gland. When separated by one-dimensional gel electrophoresis and immobilized on a nitrocellulose filter, recombinant rap1, peptides of rat parotid SGM, CY, PM and ER-enriched fractions were found to bind [-32P]-GTP (Figure 1A). Four smg proteins with molecular weights of 24.5, 26.5, 27.5, and 29 kD were present on the PM fraction (as determined by laser densitometry). At least three smgs with molecular weights of 24.5, 27, and 28 kD were present on the SGM: the GTP binding did not resolve into discrete bands for finite molecular mass determination of a possible fourth band present between the 24.5- and 27-kD bands. Those membrane-bound smg proteins, having an apparent common molecular mass, were most abundant in the SGM fraction, where the levels were severalfold that found in the PM-enriched fraction. The 26.5- and 29-kD smg signals present in the CY were low compared to those present in the PM. In addition, a
21-kD smg was also detected in the CY but not in the other subcellular fractions. For those peptides that bind GTP specifically, micromolar concentrations of unlabeled GTP successfully competed for radiolabeled GTP (Figure 1B).
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Endoplasmic reticulum, found to be the major organelle contaminating the SGM fraction (-32P]-GTP-binding proteins, two of which had molecular masses of 26.5 and 29 kD. The third GTP-binding protein had a slightly higher molecular weight than the 26.5-kD signal but could not be resolved for finite molecular weight determination. These signals were in common with those present in the PM, suggesting that these fractions have smgs in common. [
-32P]-GTP binding to ER fractions, however, was severalfold less than that found in the SGM and PM fractions, based on analysis of similar amounts of resolved proteins. The lower abundance of the 26.5-kD protein in the ER relative to membrane fractions indicates that the SGM population of smg-proteins cannot result solely from ER contamination.
In contrast to -32P]-GTP on a GTP overlay. This may have been due to a slight difference in the GTP-binding buffer, e.g., the presence of DTT in our buffer.
-32P]-GTP binding to GTP-binding proteins on a GTP overlay. However, despite the presence of DTT, CY rap1 transferred to a nitrocellulose filter was not detected by GTP. In addition,
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Immunoblot Detection of Rap1 in Subcellular Fractions
The various subcellular fractions prepared were probed on an immunoblot for rap1. Rap1 was detected as an 24-kD signal and was found to be present in highest concentration in the SGM fraction; rap1 was also detected in the PM, but to a lesser extent (Figure 2A). Comparatively weaker signals were obtained with the ER-enriched fraction, CO and CY. To verify the specificity of this signal, the antibody was incubated with a 20-fold excess (20 µg/ml) of its neutralizing peptide for 1 hr before rap1 detection. In the presence of peptide, the 24-kD rap1 signal was removed in most fractions and markedly reduced in the recombinant rap1 fraction (Figure 2B). The latter would probably have been removed with longer peptide neutralization or a higher concentration of the peptide. The other nonspecific signals were also removed and appeared to be proteins that have immunological epitopes in common with rap 1. The
55-kD nonspecific signal observed in the SGM, CO, and CY fractions was consistent with the molecular weight for amylase, present in the secretory granule contents. However, this was ruled out because rap1 antibody purified on an amylase column crossreacted with this signal (data not shown). The
28-kD nonspecific signal also observed in the SGM fraction may be an immunoglobulin breakdown product because the rap1 antibody was found to crossreact with mouse IgG at
28 kD (data not shown). The peptide with a molecular weight lower than that of rap551 is probably a breakdown product of the latter, since it is also seen in the purified rR lane.
To show that the 24.5-kD GTP-binding protein identified in the SGM and PM fractions by [
-32P]-GTP binding analysis (Figure 1A) was, in fact, rap1 identified by immunoblot in Figure 2A, the filter used for the GTP overlay assay was subsequently immunoblotted with the rap1 antibody. Data presented in Figure 3 showed exact correlation between the
24.5-kD [
-32P]-GTP-binding protein detected in the PM and SGM (Figure 1A) and the protein labeled with the rap1 antibody.
Immunohistochemistry
Immunohistochemical studies revealed strongest staining with the rap1 antibody in the apical region of the acinar cell where the SGs are located (Figure 4A). This staining was removed when the tissue sections were incubated with antibody that had been preincubated with a 10-fold excess of its neutralizing peptide (Figure 4B). Negative controls, which included omission of the primary antibody or substitution with rabbit IgG, were negative.
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To rule out the possibility that labeling of the tissue sections in the apical region of the cell may have been due to the reactivity of the antibody with the nonspecific 55-kD signal detected by immunoblot in the CO, we utilized widefield deconvolution microscopy, a technique that yields high-resolution images (
Negative controls, which included substituting the second primary antibody, i.e., the SGM antibody, with buffer or IgG, were negative for Texas red staining.
Tightly Bound vs Loosely Adherent to the Secretory Granule Membrane
Because proteins may become loosely adherent to an organelle as a function of tissue homogenization and subcellular fraction preparation (
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Epitope Orientation
In other studies, intact SGs were treated with 100 µg/ml trypsin to determine whether rap1 was present on the cytosolic vs the inner surface of the SG. If rap1 is present on the cytosolic surface, it should be sensitive to protease digestion whereas a protein on the intragranular surface should not. Data presented in Figure 7B suggest that rap1 is present on the cytosolic surface of the SG, because it was sensitive to protease treatment. Amylase, a protein in the CO, was not removed (Figure 7A) thereby indicating that the SGs were intact when treated with trypsin.
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Discussion |
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The identification and orientation of smgs on specific cellular organelles suggest their function. For example, rab3A's (smg25A) localization on SGs in endocrine cells suggests a role in exocytosis (-32P]-GTP-blot overlay, immunoblot analysis, and immunohistochemistry, i.e., immunoperoxidase and widefield deconvolution microscopy. We found, by all of these methods, that rap1 is present on the SGM and PM. Two-dimensional gel electrophoretic analysis of the SGM, PM, and CY fractions may help to ascertain if they do indeed have identical smgs.
The significance of the present findings lies in our ability to provide definitive data showing that rap1 is localized to the SGM. Studies using immunoblot analysis are subject to question, because rap1 could have translocated to the SG from another location. Alternatively or additionally, immunoblot localization of rap 1 on the SGM could have been the consequence of organelle contamination produced during isolation of the granules. Even with stringent investigation of contaminating organelles by enzyme marker analysis, it is difficult to make definitive conclusions about localization. Hence, in our studies, we supported our immunoblot data with immunohistochemical data, i.e., immunoperoxidase and widefield deconvolution microscopy. Although the immunoperoxidase data showed maximal staining in the apical region of acinar cells, where SGs are located, it was compromised by the limited resolution of light microscopy, and therefore was unable to distinguish between the SGM vs the CO. This was considered critical because rap1 crossreacts on immunoblots with a 55-kD signal in the CO. This was addressed by using widefield deconvolution microscopy, which provided a very clear picture of the localization of rap1 on the SGM and PM. Orientation was facilitated with the DNA fluorescent stain, DAPI. We specifically show that rap1 co-localizes with a parotid granule-specific antibody (
In other studies, the finding that rap 1 present on the SGM-enriched fraction is a tightly bound protein rather than a loosely adherent contaminant protein is consistent with and supports the immunoperoxidase and widefield deconvolution microscopy studies. The orientation of rap1 on the SG cytoplasmic face is of significance in postulating a function for rap1 because exocytosis involves the fusion of the cytoplasmic face of the SGM with the cytoplasmic face of the PM with subsequent release of CO. Therefore the localization and high concentration of rap1 on the SGM and its cytosolic localization suggest that it may play a role in the regulation of secretion.
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Acknowledgments |
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Supported by the National Institute of Dental Research of the National Institutes of Health, grants DE 07023 and DE 10733.
Received for publication January 27, 1997; accepted February 6, 1997.
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