Comparison of the Effects on Secretion in Chromaffin and PC12 Cells of Rab3 Family Members and Mutants
EVIDENCE THAT INHIBITORY EFFECTS ARE INDEPENDENT OF DIRECT INTERACTION WITH RABPHILIN3*

Sul-Hee ChungDagger §, Gerard Joberty, Eric A. GelinoDagger , Ian G. Macara, and Ronald W. HolzDagger parallel

From the Dagger  Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0632 and the  Center for Cell Signaling, University of Virginia, Charlottesville, Virginia 22908

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The Rab class of low molecular weight GTPases has been implicated in the regulation of vesicular trafficking between membrane compartments in eukaryotic cells. The Rab3 family consisting of four highly homologous isoforms is associated with secretory granules and synaptic vesicles. Many different types of experiments indicate that Rab3a is a negative regulator of exocytosis and that its GTP-bound form interacts with Rabphilin3, a possible effector. Overexpression of Rabphilin3 in chromaffin cells enhances secretion. We have investigated the expression, localization, and effects on secretion of the various members of the Rab3 family in bovine chromaffin and PC12 cells. We found that Rab3a, Rab3b, Rab3c, and Rab3d are expressed to varying degrees in PC12 cells and in a fraction enriched in chromaffin granule membranes from the adrenal medulla. Immunocytochemistry revealed that all members of the family when overexpressed in PC12 cells localize to secretory granules. Binding constants for the interaction of the GTP-bound forms of Rab3a, Rab3b, Rab3c, and Rab3d with Rabphilin3 were comparable (Kd = 10-20 nM). Overexpression of each of the four members of the Rab3 family inhibited secretion. Mutations in Rab3a were identified that strongly impaired the ability of the GTP-bound form to interact with Rabphilin3. The mutated proteins inhibited secretion similarly to wild type Rab3a. Although Rab3a and Rabphilin3 are located on the same secretory granule or secretory vesicle and interact both in vitro and in situ, it is concluded that the inhibition of secretion by overexpression of Rab3a is unrelated to its ability to interact with Rabphilin3.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The Rab class of low molecular weight GTPases has been implicated in the regulation of vesicular trafficking between membrane compartments in eukaryotic cells (see Refs. 1-3 for reviews). Distinct Rab proteins are associated with specific membranes and are necessary for vesicle movement in the secretory (4-6) and endocytosis pathways (7-9). For example, in yeast YPT1 is associated with the endoplasmic reticulum and is necessary for endoplasmic reticulum to Golgi transport; SEC4 is associated with post-Golgi vesicles and is required for vesicle fusion with the plasma membrane. Recent studies of the vacuole-vacuole fusion pathway in yeast implicate a Rab GTPase in a reversible tethering step prior to SNAREmediated docking (10, 11).

We have been interested in Rab3a and related family members that play a role in regulated secretion in mammalian cells. Rab3a is found in cells including neurons and chromaffin cells with a highly differentiated Ca2+-dependent secretory pathway (12-14). We (15) and others (16) had previously demonstrated that Rab3a is a negative regulator of exocytosis in experiments in bovine chromaffin cells and PC12 cells. Examination of various mutants indicated that the GTP-bound rather than GDP-bound Rab3a was the inhibitory form. Experiments in permeabilized chromaffin cells suggest that Rab3a acts upstream of the final fusion events triggered by Ca2+ and may be involved in preparing the granules to undergo exocytosis (15). The inhibition of secretion caused by overexpression of Rab3a is consistent with the enhancement of secretion from hippocampal neurons in Rab3a knockout mice (17) and the enhancement of secretion by injection into chromaffin cells of antisense rab3a oligonucleotide (16).

The precise function of Rab proteins is unknown. It has been postulated that Rab proteins may be important for the proper targeting of donor membranes to the correct acceptor membrane. However, studies in yeast with chimeras of YPT1 and SEC4 indicate that single constructs can substitute for both YPT1 and SEC4 and maintain proper targeting of the vesicles in the secretory pathway (18, 19).

The specific functions of the Rab GTPases are likely to be determined by specific proteins with which they interact (2). For Rab3a, three such proteins have been identified: Rabin3a (20), Rabphilin3 (21), and Rim (Rab3-interacting molecule) (22). All three bind preferentially to Rab3a-GTP. Rabin is predominately cytosolic with a widespread distribution. Rabphilin3 co-localizes with Rab3a to synaptic vesicles and chromaffin granules. Rim is localized to the plasma membrane of nerve terminals, but not to synaptic vesicles.

Our studies have focussed on a possible pathway that modulates regulated secretion through the coordinated interaction of Rab3a and Rabphilin3. Rabphilin3 was initially identified as a 85-kDa protein, which bound Rab3a with a much greater affinity for the GTP- than the GDP-bound forms. Cloning revealed an open reading frame encoding 704 amino acids in bovine brain (21) and a 710 splice variant in bovine chromaffin cells (23). Overexpression of Rabphilin3 in chromaffin cells (23) and insulin-secreting cells (24) enhances stimulated secretion. Both Rab3a and Rabphilin3 are found on synaptic vesicles and chromaffin granules (14, 23, 25-28) where they can directly interact (29). Rabphilin3 has two discrete domains (21, 30), an amino-terminal Rab3a-GTP binding domain (Rp(51-190)) (31) and carboxyl-terminal tandem C2 domains that confer Ca2+-dependent binding to lipid vesicles containing phosphatidylserine and/or phosphatidylinositol 4,5-bisphosphate (32, 33).

The opposite effects on secretion of Rab3a and Rabphilin3 suggest a sequence of events in which the formation of a Rab3a-GTP·Rabphilin3 complex inhibits secretion and the subsequent release of Rabphilin3 from the complex (perhaps because of GTP hydrolysis) enhances secretion (23, 31). Overexpressed Rab3a bound to GTP would inhibit secretion by binding and reducing the amount of activated Rabphilin3 on the chromaffin granule.

Rab3a is one of a subfamily of Rab3 GTPases. The four members of the family have 219-228 residues with 85-90% amino acid identity from amino acids 16 to 191 (34, 35). There is virtually complete identity in the region (residues 51-59) that is homologous to the effector domain (loop 2 or switch I) of Ras (Rab3c is the only member of the family with a non-conservative amino acid in the consensus sequence). There is much less identity (<30%) in the amino and carboxyl termini (residues 4-15 and 192-216) of the Rab3 family.

The Rab3 family members have been found in different tissues. Rab3a and Rab3c are both associated with synaptic vesicles and secretory granules in brain and neuroendocrine cells (13, 14, 25, 36, 37). Rab3b is expressed in epithelial cells (38) and anterior pituitary cells (39). Rab3d is expressed in adipocytes (35) and on zymogen granules in pancreatic acinar cells (40, 41). Rab3a, -b, and -c have been shown to interact with Rabphilin3 (21, 28, 42), although no quantitative comparisons of the binding characteristics of the Rab3 proteins have been reported.

Whereas a variety of different approaches indicate that Rab3a is an inhibitory modulator of exocytosis, there is evidence that Rab3b and Rab3d are positive regulators of secretion (39, 42, 43). To our knowledge, there is no information concerning the direct functional effects on secretion of Rab3c.

The diverse effects of different members of the Rab3 family in different cell types observed using different techniques prompted us to compare the localization and effects on secretion of transiently overexpressed Rab3 family members in chromaffin and PC12 cells. We found that Rab3a, Rab3b, Rab3c, and Rab3d are expressed to varying degrees in PC12 cells and in a fraction enriched in chromaffin granule membranes. Overexpression of each of the four members of the Rab3 family inhibits secretion. Although the binding constants for the interaction of the GTP-bound forms of Rab3a, Rab3b, Rab3c, and Rab3d with Rabphilin3 are comparable, investigation of Rab3a mutants with impaired ability to interact with Rabphilin3 indicates that the inhibition of secretion caused by overexpression of Rab3a is unrelated to its ability to interact with Rabphilin3.

    MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Chromaffin Cell Preparation, Transfection, and Secretion Experiments-- Chromaffin cell preparation, transient transfection and secretion experiments were performed as described previously (15, 23, 44). Ca2+ phosphate precipitation was used for transfections according to Wilson et al. (45) in 12-well plates (22.6-mm well diameter) for secretion experiments and in 35-mm diameter dishes for detection of expressed proteins. Secretion experiments were generally performed 5-6 days after transfection at 27 °C in a physiological salt solution containing 145 mM NaCl, 5.6 mM KCl,, 2.2 mM CaCl2, 0.5 mM MgCl2, 5.6 mM glucose, and 15 mM HEPES (pH 7.4). There were four wells or dishes/group. Human growth hormone was measured with a high sensitivity chemiluminescence assay from Nichols Institute (San Juan Capistrano, CA). Endogenous catecholamine secretion was measured with a fluorescence assay (60). Since only 1-4% of the cells are transfected, catecholamine secretion mainly reflects secretion from nontransfected cells and served as a control in the hGH1 secretion experiments. Secretion was expressed as the percentage of the total cellular human GH (or catecholamine) that was released into the medium. There was usually 0.5-2.0 ng of hGH and 20-40 nmol of catecholamine/22.6-mm diameter well.

Isolation of P2 Fraction and Chromaffin Granule Membranes from Bovine Adrenal Medullae-- Fresh bovine adrenal medullae were homogenized in 0.29 M sucrose, 10 mM Hepes (pH 7.1), 0.5 mM EDTA, and 0.5 mM PMSF. The supernatant from a 10-min 800 × g centrifugation was re-centrifuged at 27,000 × g for 10 min to generate a large granule fraction (P2 fraction). The P2 pellet was resuspended in homogenization buffer, and 8 ml was layered onto 26 ml of a solution containing 1.7 M sucrose, 2 mM Hepes (pH 7.1), and 0.1 mM EDTA. The tubes were centrifuged at 194,000 × g for 60 min in a 50.2 Ti ultracentrifuge rotor (Beckman Instruments, Fullerton, CA). The pellet at the bottom of the tube consisted of highly purified chromaffin granules. The chromaffin granules in the pellet were lysed in 10 mM Hepes (pH 7.1), 0.2 mM EDTA, frozen, and thawed. The chromaffin granule membranes were pelleted by centrifugation at 30,000 × g for 20 min and resuspended in lysis buffer. They were again centrifuged at 30,000 × g for 20 min, and the chromaffin granule membrane pellet was resuspended in 10 mM Hepes (pH 7.1) without EDTA, aliquoted, and stored at -70 °C.

Monolayers of PC12 cells plated in 60-mm dishes were grown for 2 days and subcellular fractionation performed as described previously (46). PC12 cells were harvested into 1 ml of hypotonic lysis buffer (10 mM Tris, pH 7.4, 5 mM KCl, 2 mM MgCl2, and 1 mM PMSF) and incubated on ice for 10 min. The cells were ruptured by passage through a 26-gauge needle and centrifuged at 100,000 × g for 10 min. The supernatant was removed, and the pellet was rinsed twice and resuspended in 1 ml of hypotonic lysis buffer. Trichloroacetic acid was added to a final concentration of 10% and the samples were incubated on ice for 30 min and then centrifuged at 16,000 × g for 15 min. The pellets were washed in acetone and redissolved in 0.1 N NaOH, 0.1% deoxycholate for SDS-PAGE and blotting.

SDS-PAGE, Western Analysis, and Immunocytochemistry-- The expression of transiently transfected plasmids in bovine chromaffin cells and PC12 cells was examined 6 and 2 days, respectively, after transfection. Cells were harvested into sample buffer and subjected to 12% or 15% SDS-PAGE, followed by immunoblotting with anti-HA1 antibody (1:5000 dilution) and protein detected by enhanced chemiluminescence (Amersham Pharmacia Biotech).

Rab3a Binding Assays-- [gamma -32P]Rab3a, Rab3b, Rab3c, and Rab3d binding to GST-Rabphilin3(1-206) was investigated as in previous experiments (46).

Experiments investigating the binding of Rabphilin3 to Rab3a constructs with mutants in the putative effector domain of Rab3a were performed as follows. HA-tagged Rab3a, Rab3a(F51L), Rab3a(T54A), Rab3a(F59S), and Rab5 (as a control) proteins were expressed by transient transfection in COS cells. Cell lysates in 50 mM HEPES, pH 7.4, 1% Triton X-100, 2 mM MgCl2, 150 mM NaCl, 1 mM DTT, and 1 mM PMSF were centrifuged 2 min at 500 × g to remove nuclei, and cell debris and supernatants were spun at 100,000 × g for 45 min. Concentrations of the supernatants were adjusted to 50 mM HEPES, pH 7.4, 0.5% Triton X-100, 1 mM MgCl2, 150 mM NaCl, 1 mM DTT, 5 mM EDTA, and 1 mM PMSF. In half of the supernatants, 1 mM GTPgamma S was added. After 30 min at 4 °C, MgCl2 was added to a final concentration of 10 mM and extracts were incubated for 1 h at 4 °C with 5 mg of GST-Rabphilin(1-206) bound to glutathione-Sepharose beads. Beads were washed twice in 50 mM HEPES, 0.5% Triton X-100, 5 mM MgCl2, 150 mM NaCl, and 1 mM DTT, and then twice in the same buffer minus Triton X-100. Denaturing loading buffer was added to the beads and to the second half of the supernatant from the ultracentrifugation. Samples were submitted to SDS-PAGE and then transferred to nitrocellulose membranes. HA-tagged proteins were detected by incubation with anti-HA1 antibody, followed by enhanced chemiluminescence as described previously (46). The amount of HA-tagged Rab protein bound to Rabphilin was quantitated from the blots and related to the total amount of the protein present in the supernatant.

Immunocytochemistry and confocal microscopy were performed as described previously to determine the localization of transiently expressed hGH and the HA1 epitope in PC12 cell (26, 31, 46). Overlap in the fluorescein isothiocyanate channel (hGH) and the rhodamine channel (HA1-Rab3) was determined by multiplication of the images pixel by pixel in the two channels using NIH Image (1.59). The resulting pixel intensities were scaled to fit an eight-bit scale.

Antibodies and Plasmids-- Human GH and the HA1 epitope (YPYDVPDYA) were detected by confocal microscopy with rabbit anti-human pituitary GH polyclonal antibody (1:1000, National Hormone and Pituitary Program, NIDDKD, National Institutes of Health, Bethesda, MD) and mouse anti-HA1 (monoclonal antibody 12CA5, 1:500-1:2000, Berkeley Antibody Co.) with appropriate secondary anti-rabbit and anti-mouse antibodies tagged with Oregon Green 488 or lissamine rhodamine (Molecular Probes, Eugene, OR). Anti-Rab3a was a rabbit peptide antibody to a unique 13-amino acid stretch near the carboxyl terminus (47). Rabbit polyclonal antibody to human Rab3b (38) was a gift from Dr. K. L. Kirk (University of Alabama, Birmingham, AL). Anti-Rab3c was a rabbit peptide antibody directed against amino acids 195-209 of the bovine sequence and was obtained from Dr. Ahmed Zahraoui (Institut Curie, Paris, France). Anti-Rab3d anti-serum (40) was obtained from Dr. Mark McNiven (Mayo Clinic, Rochester, MN). The specificity of these antibodies has been documented in the literature. We verified that anti-Rab3b detected only Rab3b by SDS-PAGE and immunoblots of COS cells transiently expressing equivalent amounts of Rab3a, Rab3b, Rab3c, or Rab3d. Rat Rab3a, Rab3b, Rab3c, mouse Rab3d, and human Rab8 plasmids were constructed under transcriptional control of the immediate-early enhancer-promoter region of the human cytomegalovirus (CMV) and usually contained the HA1 epitope (YPYDVPDYA) in frame at the amino terminus (48). Rab3a, Rab3b, and Rab3c were amplified from reverse transcribed rat brain mRNA, and sequences were confirmed. Mouse Rab3d was obtained from Drs. H. Ohnishi and J. A. Williams (University of Michigan, Ann Arbor, MI). The plasmid encoding human Rab3b (under control of the CMV promotor) (42) was a gift from Dr. K. L. Kirk. The cDNA encoding Rab8 was obtain from A. Zahraoui (INSERM, U-248, Paris, France). For the immunocytochemistry in Fig. 6, three consecutive HA epitopes were present at the amino terminus of the Rab3 family members. The construction of GFP-Rabphilin was described previously (46).

Data are generally expressed as mean ± standard error of the mean.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Members of the Rab3 Family Are Detected in Chromaffin Granule Membranes and PC12 Cells-- Immunoblots of equal amounts of proteins in a P2 fraction and a highly enriched chromaffin granule membrane fraction prepared from fresh adrenal medulla detected Rab3a, Rab3b, Rab3c, and Rab3d (Fig. 1). The primary antibodies that were used for detection of the various Rab3 proteins were highly specific with virtually no cross-reactivity (see "Materials and Methods"). Significantly more of each of the Rab isoforms were detected in the chromaffin granule membrane fraction than in the P2 fractions consistent with their localization on chromaffin granules.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 1.   Endogenous Rab3a, Rab3b, Rab3c, and Rab3d are detected in a chromaffin granule membrane fraction and in PC12 cells. CC, chromaffin cells; s, soluble fraction; p, particulate fraction; P2, pellet; CG, chromaffin granule membrane fraction.

The expression of the various Rab3s was also investigated in PC12 cells, a clonal cell line from a rat adrenal medullary tumor (Fig. 1). The four different Rab3 GTPases could be detected to varying degrees with Rab3b only weakly detected. Equal proportions of the soluble and particulate fractions were applied to the gels. Rab3a, Rab3b, and Rab3d were mainly particulate. Rab3c was found to greater extent in the soluble fraction.

It had been previously reported that PC12 cells do not express Rab3b (42). The low level of expression was detected with the same anti-Rab3b antibody used in the previous study. The difference in results could reflect a difference in the two PC12 cell lines.

HA-tagged Rab3 Family Members Can Be Transiently Expressed in Bovine Chromaffin Cells and PC12 Cells and Localize to Secretory Granules-- HA-tagged members of the Rab 3 family could be readily expressed in bovine chromaffin cells (Fig. 2A) and PC12 cells (Fig. 2B) by transient transfection.


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 2.   Transiently transfected HA-Rab3a, Rab3b, Rab3c, and Rab3d are expressed in bovine adrenal chromaffin cells and PC12 cells. s, soluble fraction; p, particulate fraction.

Confocal microscopy was performed on PC12 cells transiently cotransfected with a plasmids encoding hGH and one of the HA-Rab3 GTPases. PC12 cells have far fewer secretory granules than bovine chromaffin cells and have a characteristic peripheral localization near the plasma membrane that facilitates resolution of the granules (49). HGH-containing granules tended to be localized adjacent to the plasma membrane (Fig. 3, Control). Often a perinuclear staining was also observed suggestive of Golgi. Transfected HA-Rab3a, -b, -c, and -d all had a punctate, peripheral localization that overlapped with the cotransfected hGH in the cell periphery (right side of each panel in Fig. 3). The transfected Rab3 GTPases were not observed in a perinuclear region.


View larger version (116K):
[in this window]
[in a new window]
 
Fig. 3.   Transfected HA-tagged Rab3a, Rab3b, Rab3c, and Rab3d colocalize with hGH-containing granules in the periphery of PC12 cells. PC12 cells were cotransfected with a plasmid expressing hGH and with plasmids expressing HA-Rab3a, HA-Rab3b, HA-Rab3c, or HA-Rab3d or with a control plasmid, pCMVneo. Two days later, cells were fixed and the expression of hGH and the HA-tagged proteins were detected by confocal microscopy. The left side of each panel shows hGH; the right side shows HA-Rab. Notice the tendency for peripheral localization of hGH-containing granules. The HA-Rab proteins tend to colocalize with hGH granules. All but one of the images were optical sections through the middle of the cells. The middle right panel presents an optical section tangent to the surface of a HA-Rab3b-transfected cell. The calibration bar corresponds to 5 µm.

The colocalization of hGH and the four members of the rab3 family was apparent both in optical sections through the center of cells and in optical sections tangent to the cell surface (e.g. see Rab3b, right panel). The colocalization was confirmed by computer-generated maps of pixels with significant intensities in both channels (dark spots and areas in Fig. 4). The peripheral localization of the secretory granules was not disrupted by the transient expression of any of the Rab3 family members.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 4.   Computer-determined overlap of hGH and HA-Rab immunofluorescence. The dark spots and areas correspond to pixels with significant intensities due to expression of hGH and either HA-Rab3a or HA-Rab3b. The analysis was performed on the images in Fig. 3.

Members of the Rab3 Family of GTPases Interact Similarly with Rabphilin3-- Rabphilin3 interacts with Rab3a-GTP and has been postulated to be part of a Rab3a/Rabphilin3 pathway that regulates exocytosis. The abilities of the Rab3-GTP isoforms to bind to the Rab3a binding domain of Rabphilin3 were compared (Fig. 5). Various concentrations of Rab3-[gamma -32P]GTP were incubated with GST-Rabphilin3(1-206) bound to glutathione-Sepharose. The binding was saturable with Kd values of 10-20 nM for all the Rab3 isoforms (Table I). The amount of GST-Rabphilin3(1-206) in the assay was 1.34 nmol. The Bmax values of the isoforms were similar with a range of 0.8-1.5 nmol, indicating that the stoichiometry of the binding was approximately 1:1. The binding studies suggest that Rab3a, Rab3b, Rab3c, and Rab3d interact similarly with Rabphilin3.


View larger version (18K):
[in this window]
[in a new window]
 
Fig. 5.   Rab3a, Rab3b, Rab3c, and Rab3d bind to Rabphilin3 in a similar manner. Rabphilin3 (1.34 pmol) was present in each assay (100 µl). Data points are the means of duplicates with the range in all cases less than 5%. Lines were plotted with a non-linear curve fitting algorithm (Levenberg-Marquardt) for a simple, one-site binding model (B = Bmax/(Kd + S)), where Bmax = total amount of bound Rab3 (pmol), Kd = dissociation constant, and S = free concentration of Rab3. See Table I for binding parameters.

                              
View this table:
[in this window]
[in a new window]
 
Table I
Rab3a, -b, -c, and -d bind to Rabphilin3 with similar binding constants
Rabphilin3 (1.34 pmol) was present in each assay. See Fig. 5 legend for experimental details and binding curves.

Colocalization of HA-tagged Rab3 Family Members with Cotransfected GFP Rabphilin3 in PC12 Cells-- The similar ability of the members of the Rab3 family to interact with Rabphilin3 in vitro suggested that members of the Rab3 family would similarly co-localize with Rabphilin3 in situ. Confocal microscopy of transfected cells indicates that this prediction is correct (Fig. 6). PC12 cells were transfected with plasmids encoding GFP-Rabphilin3 and one of either HA-Rab3a, -b, -c, or -d. Cells were subsequently treated with nerve growth factor to cause process formation. Cells were fixed and processed for HA1 immunocytochemistry. Rabphilin3 was detected by the fluorescence of GFP. Both the GFP-Rabphilin3 and all of the Rab3 GTPases tended to accumulate in the processes. A pixel-by-pixel analysis demonstrated substantial overlap of GFP-Rabphilin3 and each of the Rab3 proteins.


View larger version (67K):
[in this window]
[in a new window]
 
Fig. 6.   Colocalization of HA-Rab3 proteins with GFP-Rabphilin3 in nerve growth factor-treated PC12 cells. PC12 cells were transfected with plasmids expressing GFP-Rabphilin3 and the indicated HA-Rab3. The localization of the HA-tagged proteins was determined by HA immunocytochemistry. The localization of GTP-Rabphilin3 was determined by the intrinsic fluorescence of GFP. The pixel-by-pixel colocalization of the HA-Rab3 proteins and GFP-Rabphilin3 is shown in the bottom panel.

Members of the Rab3 Family Inhibit Secretion from Bovine Chromaffin Cells and PC12 Cells-- The effects of overexpression of HA-tagged Rab3 family members on secretion were compared in bovine chromaffin cells and PC12 cells (Fig. 7). Cells were transiently co-transfected with plasmids encoding hGH and one of the HA-Rab3 family members. Two days later (PC12 cells) or 6 days later (bovine chromaffin cells), hGH secretion was stimulated with either elevated K+ (PC12 cells) or the nicotinic agonist, DMPP (bovine chromaffin cells). All of the Rab3 family members inhibited secretion (Fig. 7). In both chromaffin and PC12 cells, overexpression of Rab3a, Rab3b, Rab3c, and Rab3d inhibited stimulated secretion by 50% or more. These effects were specific since transfection with a plasmid encoding HA-Rab8 resulted in expression of the GTPase in chromaffin cells (data not shown) but did not alter secretion (Fig. 7A).


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 7.   The Rab3a family of GTPases inhibit secretion from bovine chromaffin cells (A) and PC12 cells (B). A, bovine chromaffin cells were transfected with pXGH5 and a control plasmid (pCMVneo, parent plasmid) or one expressing one of the indicated HA-tagged Rab GTPases. Six days later, secretion was stimulated with the nicotinic agonist DMPP (20 µM) for 2 min. Three separate experiments are shown. B, PC12 cells were transfected similarly to bovine chromaffin cells. Three days later cells were stimulated with elevated K+ for 15 min. [3H]NE secretion induced by elevated K+ was 17.5-20% for all groups. There were four wells per group. Two separate experiments are shown. The inhibition of hGH secretion from both chromaffin cells and PC12 cells by all the Rab3 plasmids was statistically significant (p < 0.01 for all Rab3 GTPases except for the inhibition of secretion from PC12 cells by Rab3c where p < 0.05). Ctrl, control.

It has been reported that ionomycin (calcium ionophore)-induced secretion is enhanced from PC12 cell lines stably expressing human Rab3b (42). Ionomycin-induced hGH secretion from PC12 cells was inhibited 53% (mean of 47% and 58% inhibition in two experiments) by transient expression of HA-Rab3b. Because stable transfectants may express less protein than transient transfectants, the effects of reducing the amount of the HA-Rab3b-encoding plasmid during transfection on the subsequent inhibition of secretion was investigated. Decreasing the amount of plasmid encoding rab3b by 10-fold (keeping the total DNA in the transfection constant with control DNA) did not cause an enhancement of secretion (data not shown). The Rab3b construct was derived from the rat cDNA sequence. Transient transfection with the human rab3b construct used in the earlier study similarly inhibited elevated K+ (secretion relative to CMV.neo control, 0.491 ± 0.039, n = 3 experiments). The experiments do not reveal an ability of Rab3b to enhance secretion in PC12 or chromaffin cells.

Inhibition of Secretion Caused by Mutations in Rab3a That Reduce or Eliminate Rabphilin3 Binding-- A number of point mutations in Rab3a were created and investigated for their ability to inhibit binding to Rabphilin3 and to inhibit secretion. The binding of Rabphilin3 to Rab3a(T54A) or Rab3a(F59S) was reduced by 82% or 98%, respectively, compared with binding to wild type Rab3a (Fig. 8). It has been previously demonstrated in PC12 cells that Rab3a(T54A) partitions between soluble and particulate fractions and colocalizes with GFP-Rabphilin3 in immunocytochemistry experiments identically to wild type Rab3a (46). We found that Rab3a(T54A) colocalized to hGH-containing granules in transfected PC12 cells identically to wild type Rab3a, consistent with these findings (data not shown). Rab3a(F59S) also colocalized to hGH-containing granules. The images suggested that there was also a significant amount of cytosolic Rab3a(F59S) (data not shown). Both these mutations were as effective as wild type Rab3a in inhibiting secretion in chromaffin cells and PC12 cells (Fig. 9). Rab3a (F51L) bound Rabphilin3 similarly to wild type Rab3a and also inhibited secretion. The data suggest that, although all members of the Rab3 family bind Rabphilin3, the inhibition of secretion caused by Rab3 does not require this interaction.


View larger version (26K):
[in this window]
[in a new window]
 
Fig. 8.   Binding of different Rab3a effector mutants to GST-Rabphilin(1-206). Rab3a wild type (wt), effector domain mutants Rab3a(F51L), Rab3A(T54A), Rab3A(F59S), or Rab5 were expressed in COS cells, solubilized with Triton X-100 and incubated with GTPgamma S and GST-Rabphilin bound to beads as described under "Materials and Methods." A, immunodetection after SDS-PAGE and immunoblotting using monoclonal anti-HA antibody. Upper panel, Rab proteins bound to GST-Rabphilin; lower panel, expression of HA-tagged Rab in COS cells. B, binding of Rab3a effector mutant proteins to GST-Rabphilin relative to the binding of the wild type protein (100%). Data are the mean of two to four independent experiments.


View larger version (25K):
[in this window]
[in a new window]
 
Fig. 9.   Rab3a mutants defective in Rabphilin3 binding inhibit secretion from chromaffin cells (A) and PC12 cells (B). Cells were transfected and secretion stimulated as described in Fig. 7. Panels A and B each show two experiments. WT, wild type; Ctrl, control.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The similar primary sequences of the Rab3 family, and the virtually identical sequences of the putative effector domains suggested that they would bind similarly to Rabphilin3 and would have similar effects on secretion. All members of the Rab3 family were expressed in PC12 and chromaffin cells, and bound Rabphilin3 similarly. When overexpressed in chromaffin or PC12 cells they all inhibited secretion. However, analysis of the effects of Rab3a mutants indicated that the binding to Rabphilin3 was unnecessary for the inhibition of secretion. These issues are discussed below.

Members of the Rab3 Family Are Enriched into Chromaffin Granule Fraction and Are Expressed in PC12 Cells-- Antibodies that were specific for each of the Rab3 GTPases demonstrated that Rab3a, Rab3b, Rab3c, and Rab3d are enriched in a chromaffin granule membrane fraction (compared with a P2 fraction) from the adrenal medulla and are expressed to varying degrees in PC12 cells. Although it is possible that some of the Rab3 GTPases in the chromaffin granules fraction are contaminants from adrenal cortical, endothelial or fibroblast cells, their expression in PC12 cells, a cell line derived from tumor of rat chromaffin cells, supports the notion that all four isoforms can be expressed in chromaffin cells. There is evidence that Rab3b may also be associated with the plasma membrane in chromaffin cells (50).

Secretory granules in PC12 cells tend to be peripherally localized (49). Transiently expressed, HA-tagged Rab3a, Rab3b, Rab3c, and Rab3d all co-localized with hGH in granules in the periphery of the cells. This observation is consistent with previous work that demonstrated by immunocytochemistry or subcellular fractionation the presence of endogenous or transfected Rab3a, Rab3b, and Rab3c on chromaffin granules from the adrenal medulla or on secretory granules in PC12 cells (14, 26, 42). Rab3d is associated with zymogen granules in pancreatic acinar cells (40, 41) and perhaps with Glut4-containing vesicles in adipocytes (35). Overexpression of none of the isoforms had noticeable effects on the distribution of the hGH-containing granules in PC12 cells detected by immunocytochemistry.

The Similar Inhibition of Secretion by Rab3 Family Members Does Not Involve Binding to Rabphilin3-- Rab3a, Rab3b, Rab3c, and Rab3d all inhibited secretion from PC12 and chromaffin cells. Direct in vitro binding assays demonstrated similar affinities and Bmax values for Rab3a, Rab3b, Rab3c, and Rab3d for binding to recombinant Rabphilin3. Since the overexpression of Rabphilin3 enhances secretion in chromaffin cells and PC12 cells, it was possible that the inhibition of secretion caused by overexpression of each of the members of the Rab3 family could have been caused by binding endogenous Rabphilin3. However, a mutant with reduced ability to bind Rabphilin3 (Rab3aT54L) and a mutant devoid of measurable Rabphilin3 binding (Rab3aF59S) inhibited secretion from chromaffin and PC12 cells similarly to wild type Rab3a. Both of the mutations are in a region (the putative switch 1 region of Rab3a) that that has been recently demonstrated to interact with Rabphilin3 (51). Phenylalanine 59 directly contacts Rabphilin3. The experiments indicate that the inhibition of secretion caused by overexpression of Rab3a does not involve direct interaction with Rabphilin3.

In an earlier study we provided evidence that the enhancement of secretion induced by overexpression of Rabphilin3 does not require binding to Rab3a-GTP (31). Expression of an amino truncated mutant of Rabphilin3 that did not bind Rab3a-GTP enhanced secretion similarly to wild type Rabphilin3. A similar conclusion was drawn from experiments with insulin-secreting cells (24). Thus, although Rab3a and Rabphilin3 are located on the same secretory granule or vesicle and interact both in vitro (21) and in situ (29), the inhibition of secretion by Rab3a and the enhancement of secretion by Rabphilin3 are each independent of the direct interaction of the two proteins.

Although we have been unable to demonstrate a direct function of the binding of Rab3a and Rabphilin3 in the secretory pathway, there are other known consequences to their interaction. The binding of Rabphilin3 to Rab3 stabilizes Rabphilin3 in chromaffin cells (31) and neurons (52) and may play a role in transporting Rabphilin3 to the nerve terminal (28). In addition, the interaction of Rab3a to the amino terminus of Rabphilin3 prevents the interaction of Rabphilin3 with alpha -actinin (53), an actin-binding protein, and with Rabaptin5 (54), a protein important in the endocytic pathway. Thus, the association of Rab3a and Rabphilin3 may regulate the cytoskeleton and/or endocytosis in secretory cells.

Members of the Rab3 Family Have Diverse Effects on Secretion-- Experiments in chromaffin cells, PC12 cells, an insulin-secreting cell line, and hippocampal neurons suggest a consistent role for Rab3a as an inhibitory modulator of exocytosis. Injection of Rab3a antisense oligonucleotides into chromaffin cells (16, 55) enhances exocytosis. Similarly, removal of the Rab3a gene in mice enhances vesicular release in cultured hippocampal neurons (17). Conversely, overexpression of Rab3a inhibits exocytosis in PC12, chromaffin cells (15, 16) and insulin-secreting cells (37). Secretory granules in PC12 cells, chromaffin cells, and insulin-secreting cells are derived from the trans-Golgi network of the protein secretory pathway, whereas synaptic vesicles participate in an exocytotic-endocytotic cycle. The common negative modulation of both pathways by Rab3a is consistent with a common step in exocytosis being modulated, perhaps related to the function of SNARE proteins (56-58).

Experiments investigating the roles of Rab3b and Rab3d in secretion give a less consistent picture. The introduction of antisense oligonucleotides to Rab3b into pituitary cells inhibits exocytosis (39) and overexpression in mice of Rab3d using a transgenic approach enhances exocytosis, suggesting that both Rab3b and Rab3d normally enhance secretion. On the other hand, we determined in this study that overexpression of Rab3b or Rab3d inhibits secretion in PC12 and chromaffin cells. In addition, expression of Rab3dN135I inhibits regulated secretion in AtT-20 cells (59). A comparable mutation in Rab3a greatly increases GTP and GDP exchange rates and is predicted to render the protein predominantly GTP-bound within cells (48). The N135I mutation in Rab3a inhibits secretion in PC12 cells and chromaffin cells (15, 16). Thus, it is likely that the same Rab protein in different cells can have different overall effects on the secretory pathway.

One possible explanation for the different effects of the same Rab3 isoform in different cells is that different cells have different effector pathways. The present study indicates that differences in the expression of one possible effector, Rabphilin3, are unlikely to be the basis for the difference of effects in the different cells types. In addition to the three proteins that bind Rab3-GTP, Rabin, Rabphilin3, and Rim, there are likely to be other effectors for the Rab3 family. Genetic experiments indicate that the single Rab3 homologue in Caenorhabditis elegans enhances but is not essential for synaptic transmission (60). This GTPase may act through AEX-3, which is homologous to segments in DENN, a human protein of unknown function (61).

Effects of Rab3b in Secretion-- A complication in understanding the role of Rab3b in exocytosis is the finding that when Rab3b is expressed in stably transfected PC12 cells, it enhanced ionomycin-induce [3H]norepinephrine secretion (42). We found that overexpression of Rab3b by transient transfection inhibited protein secretion induced by elevated potassium and ionomycin in PC12 cells and by DMPP in chromaffin cells. Since transient transfectants may express more protein than stable transfectants, we attempted without success to enhance secretion by reducing the amount of Rab3b-encoding plasmid used in the transfections. A possible explanation for the different effects is that, in the previous experiments with stably transfected PC12 cells, there was an average 8-fold increase in [3H]norepinephrine stored intracellularly in the various clonal lines. Although there was no change in the number or distribution of large dense core vesicles in the clones, there may be another vesicular compartment that expanded and accounts for the change. It need not be a protein-secreting compartment since [3H]norepinephrine enters the compartment from the cytosol. The large increase in storage in the stable Rab3b-expressing cell lines compared with control or Rab3a-overexpressing cell lines creates uncertainties in the comparisons of secretory responses of the various clones, despite secretion being normalized to the total [3H]norepinephrine content in the cells. In the present study, immunocytochemistry of Rab3b-transfected cells did not reveal a qualitative change in the number of the growth hormone-containing granules or their distribution in the periphery of the cell.

    ACKNOWLEDGEMENTS

We are grateful to Dr. Kevin Kirk (University of Alabama) for sharing with us the anti-Rab3b antibody and plasmid encoding human Rab3b and to Dr. M. McNiven for the anti-Rab3d antibody. We thank Dr. Mary A. Bittner (University of Michigan) for many helpful discussions and Chuliang Yu for skillful technical support.

    FOOTNOTES

* This work was supported by Grants RO1 DK50127 (to R. W. H.) and CA-56300 (to I. G. M.) from the National Institutes of Health, and by a grant from the Student Biomedical Research Program, University of Michigan Medical School (to E. A. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Current address: Cardiovascular Biology Laboratory, Harvard School of Public Health, Boston, MA 02115.

parallel To whom correspondence should be addressed: Dept. of Pharmacology, University of Michigan Medical School, 1301 MSRB III, Ann Arbor, MI 48109-0632.

    ABBREVIATIONS

The abbreviations used are: hGH, human growth hormone; GH, growth hormone; HA, hemagglutinin; PMSF, phenylmethylsulfonyl fluoride; GTPgamma S, guanosine 5'-O-(thiotriphosphate); DMPP, dimethylphenylpiperazinium; PAGE, polyacrylamide gel electrophoresis; DTT, dithiothreitol; CMV, cytomegalovirus; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
  1. Bean, A. J., and Scheller, R. H. (1997) Neuron 19, 751-754[Medline] [Order article via Infotrieve]
  2. Novick, P., and Zerial, M. (1997) Curr. Opin. Cell Biol. 9, 496-504[CrossRef][Medline] [Order article via Infotrieve]
  3. Schimmoller, F., Simon, I., and Pfeffer, S. R. (1998) J. Biol. Chem. 273, 22161-22164[Free Full Text]
  4. Tisdale, E. J., Bourne, J. R., Khosravi-Far, R., Der, C. J., and Balch, W. E. (1992) J. Cell Biol. 119, 749-761[Abstract]
  5. Nuoffer, C., Davidson, H. W., Matteson, J., Meinkoth, J., and Balch, W. E. (1994) J. Cell Biol. 125, 225-237[Abstract]
  6. Elazar, Z., Mayer, T., and Rothman, J. E. (1994) J. Biol. Chem. 269, 794-797[Abstract/Free Full Text]
  7. Bucci, C., Parton, R. G., Mather, I. H., Stunnenberg, H., Simons, K., Hoflack, b., and Zerial, M. (1992) Cell 70, 715-728[Medline] [Order article via Infotrieve]
  8. van der Sluijs, P., Hull, M., Zahraoui, A., Tavitian, A., Goud, B., and Mellman, I. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 6313-6317[Abstract]
  9. Lombardi, D., Soldati, T., Riederer, M. A., Goda, Y., Zerial, M., and Pfeffer, S. R. (1993) EMBO J. 12, 677-682[Abstract]
  10. Mayer, A., and Wickner, W. (1997) J. Cell Biol. 136, 307-317[Abstract/Free Full Text]
  11. Ungermann, C., Sato, K., and Wickner, W. (1998) Nature 396, 543-548[CrossRef][Medline] [Order article via Infotrieve]
  12. Burstein, E., and Macara, I. G. (1989) Mol. Cell. Biol. 9, 4807-4811[Medline] [Order article via Infotrieve]
  13. Sano, K., Kikuchi, A., Matsui, Y., Teranishi, Y., and Takai, Y. (1989) Biochem. Biophys. Res. Commun. 158, 377-385[Medline] [Order article via Infotrieve]
  14. Darchen, F., Zahraoui, A., Hammel, F., Monteils, M. P., Tavitian, A., and Scherman, D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 5692-5696[Abstract]
  15. Holz, R. W., Brondyk, W. H., Senter, R. A., Kuizon, L., and Macara, I. G. (1994) J. Biol. Chem. 269, 10229-10234[Abstract/Free Full Text]
  16. Johannes, L., Lledo, P. M., Roa, M., Vincent, J. D., Henry, J. P., and Darchen, F. (1994) EMBO J. 13, 2029-2037[Abstract]
  17. Geppert, M., Goda, Y., Stevens, C. F., and Sudhof, T. C. (1997) Nature 387, 810-814[CrossRef][Medline] [Order article via Infotrieve]
  18. Brennwald, P., and Novick, P. (1993) Nature 362, 560-563[CrossRef][Medline] [Order article via Infotrieve]
  19. Dunn, B., Stearns, T., and Botstein, D. (1993) Nature 362, 563-565[CrossRef][Medline] [Order article via Infotrieve]
  20. Brondyk, W. H., McKiernan, C., Fortner, K. A., Stabila, P., Holz, R. W., and Macara, I. G. (1995) Mol. Cell. Biol. 15, 1137-1143[Abstract]
  21. Shirataki, H., Kaibuchi, K., Sakoda, T., Kishida, S., Yamaguchi, T., Wada, K., Miyazaki, M., and Takai, Y. (1993) Mol. Cell. Biol. 13, 2061-2068[Abstract]
  22. Wang, Y., Okamoto, M., Schmitz, F., Hofman, K., and Sudhof, T. (1998) Nature 388, 593-598[CrossRef]
  23. Chung, S.-H., Takai, Y., and Holz, R. W. (1995) J. Biol. Chem. 270, 16714-16718[Abstract/Free Full Text]
  24. Arribas, M., Regazzi, R., Garcia, E., Wollheim, C. B., and De Camilli, P. (1997) Eur. J. Cell Biol. 74, 209-216[Medline] [Order article via Infotrieve]
  25. Mollard, G. F. v., Mignery, G. A., Baumert, M., Perin, M. S., Hanson, R. J., Burger, P. M., Jahn, R., and Sudhof, T. C. (1990) Proc. Natl. Acad. Sci U. S. A. 87, 1988-1992[Abstract]
  26. Darchen, F., Senyshyn, J., Brondyk, W. H., Holz, R. W., Macara, I. G., Tougard, C., and Henry, J. P. (1995) J. Cell Sci. 108, 1639-1649[Abstract/Free Full Text]
  27. Mizoguchi, A., Yano, Y., Hamaguchi, H., Yanigida, H., Ide, C., Zahraoui, A., Shirataki, H., Sasaki, T., and Takai, Y. (1994) Biochem. Biophys. Res. Commun. 202, 1235-1243[CrossRef][Medline] [Order article via Infotrieve]
  28. Li, C., Takei, K., Geppert, M., Daniell, L., Stenius, K. R., Chapman, E. R., Jahn, R., De Camilli, P., and Sudhof, T. C. (1994) Neuron 13, 885-898[Medline] [Order article via Infotrieve]
  29. Stahl, B., Chou, J. H., Li, C., Sudhof, T. C., and Jahn, R. (1996) EMBO J. 15, 1799-1809[Abstract]
  30. Shirataki, H., Kaibuchi, K., Yamaguchi, T., Wada, K., Horiuchi, H., and Takai, Y. (1992) J. Biol. Chem. 267, 10946-10949[Abstract/Free Full Text]
  31. Chung, S.-H., Stabila, P., Macara, I. G., and Holz, R. W. (1997) J. Neurochem. 69, 164-173[Medline] [Order article via Infotrieve]
  32. Yamaguchi, T., Shirataki, H., Kishida, S., Miyazaki, M., Nishikawa, J., Wada, K., Numata, S., Kaibuchi, K., and Takai, Y. (1993) J. Biol. Chem. 268, 27164-27170[Abstract/Free Full Text]
  33. Chung, S.-H., Song, W.-J., Kim, K., Bernarski, J. J., Chen, J., Prestwich, G. D., and Holz, R. W. (1998) J. Biol. Chem. 273, 10240-10248[Abstract/Free Full Text]
  34. Matsui, Y., Kikuchi, A., Kondo, J., Hishida, T., Teranishi, Y., and Takai, Y. (1988) J. Biol. Chem. 263, 11071-11074[Abstract/Free Full Text]
  35. Baldini, G., Hohl, T., Lin, H. Y., and Lodish, H. F. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 5049-5052[Abstract]
  36. Mizoguchi, A., Kim, S., Ueda, T., Kikuchi, A., Yorifuji, H., Hirokawa, N., and Takai, Y. (1990) J. Biol. Chem. 265, 11872-11879[Abstract/Free Full Text]
  37. Regazzi, R., Ravazzola, M., Iezzi, M., Lang, J., Zahraoui, A., Andereggen, E., Morel, P., Takai, Y., and Wollheim, C. B. (1996) J. Cell Sci. 109, 2265-2273[Abstract/Free Full Text]
  38. Weber, E., Berta, G., Tousson, A., St John, P., Green, M. W., Gopalokrishnan, U., Jilling, T., Sorscher, E. J., Elton, T. S., and Abrahamson, D. R. (1994) J. Cell Biol. 125, 583-594[Abstract]
  39. Lledo, P.-M., Vernier, P., Vincent, J.-D., Mason, W. T., and Zorec, R. (1993) Nature 364, 540-544[CrossRef][Medline] [Order article via Infotrieve]
  40. Ohnishi, H., Ernst, S. A., Wys, N., McNiven, M., and Williams, J. A. (1996) Am. J. Physiol. 271, G531-G538[Abstract/Free Full Text]
  41. Valentijn, J. A., Sengupta, D., Gumkowski, F. D., Tang, L. H., Konieczko, E. M., and Jamieson, J. D. (1996) Eur. J. Cell Biol. 70, 33-41[Medline] [Order article via Infotrieve]
  42. Weber, E., Jilling, T., and Kirk, K. L. (1996) J. Biol. Chem. 271, 6963-6971[Abstract/Free Full Text]
  43. Ohnishi, H., Samuelson, L. C., Yule, D. I., Ernst, S. A., and Williams, J. A. (1997) J. Clin. Invest. 100, 3044-3052[Abstract/Free Full Text]
  44. Wick, P. W., Senter, R. A., Parsels, L. A., and Holz, R. W. (1993) J. Biol. Chem. 268, 10983-10989[Abstract/Free Full Text]
  45. Wilson, S. P., Liu, F., Wilson, R. E., and Housley, P. R. (1996) Anal. Biochem. 226, 212-220[CrossRef]
  46. McKiernan, C., Stabila, P., and Macara, I. G. (1996) Mol. Cell. Biol. 16, 4985-4995[Abstract]
  47. Burstein, E., and Macara, I. G. (1989) Mol. Cell. Biol. 9, 4807-4811[Medline] [Order article via Infotrieve]
  48. Brondyk, W. H., McKiernan, C. J., Burstein, E. S., and Macara, I. G. (1993) J. Biol. Chem. 268, 9410-9415[Abstract/Free Full Text]
  49. Pozzan, T., Gatti, G., Dozio, N., Vicentini, L. M., and Meldolesi, J. (1984) J. Cell Biol. 99, 628-638[Abstract]
  50. Lin, C. G., Lin, Y. C., Liu, H. W., and Kao, L. S. (1997) Biochem. J. 324, 85-90[Medline] [Order article via Infotrieve]
  51. Ostermeier, C., and Brunger, A. T. (1999) Cell 96, 363-374[Medline] [Order article via Infotrieve]
  52. Geppert, M., Bolshakov, V. Y., Siegelbaum, S. A., Takei, K., De Camilli, P., Hammer, R. E., and Sudhof, T. C. (1994) Nature 369, 493-497[CrossRef][Medline] [Order article via Infotrieve]
  53. Kato, M., Sasaki, T., Ohya, T., Nakanishi, H., Nishioka, H., Imamura, M., and Takai, Y. (1996) J. Biol. Chem. 271, 31775-31778[Abstract/Free Full Text]
  54. Ohya, T., Sasaki, T., Kato, M., and Takai, Y. (1998) J Biol Chem 273, 613-617[Abstract/Free Full Text]
  55. Johannes, L., Lledo, P. M., Chameau, P., Vincent, J. D., Henry, J. P., and Darchen, F. (1998) J. Neurochem. 71, 1127-1133[Medline] [Order article via Infotrieve]
  56. Sogaard, M., Tani, K., Ye, R. R., Geromanos, S., Tempst, P., Kirchhausen, T., Rothman, J. E., and Sollner, T. (1994) Cell 78, 937-948[Medline] [Order article via Infotrieve]
  57. Lian, J. P., Stone, S., Jiang, Y., Lyons, P., and Ferro-Novick, S. (1994) Nature 372, 698-701[CrossRef][Medline] [Order article via Infotrieve]
  58. Johannes, L., Doussau, F., Clabecq, A., Henry, J. P., Darchen, F., and Poulain, B. (1996) J. Cell Sci. 109, 2875-2884[Abstract/Free Full Text]
  59. Baldini, G., Wang, G., Weber, M., Zweyer, M., Bareggi, R., Witkin, J. W., and Martelli, A. M. (1998) J. Cell Biol. 140, 305-313[Abstract/Free Full Text]
  60. Nonet, M. L., Staunton, J. E., Kilgard, M. P., Fergestad, T., Hartwieg, E., Horvitz, H. R., Jorgensen, E. M., and Meyer, B. J. (1997) J. Neurosci. 17, 8061-8073[Abstract/Free Full Text]
  61. Iwasaki, K., Staunton, J., Saifee, O., Nonet, M., and Thomas, J. H. (1997) Neuron 18, 613-622[Medline] [Order article via Infotrieve]


Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.