Rab3 Isoforms Research Articles

Molecular cloning of mouse homologues of Rab3 isoforms in osteoclasts

Molecular cloning of mouse homologues of Rab3 isoforms in osteoclasts

A rab3 homolog in sea urchin functions in cell division

Rabs are monomeric GTP binding proteins belonging to the ras superfamily that function throughout the secretory pathway. Members of the rab3 family function in the final steps of the secretory pathway, vesicle fusion with the plasma membrane. In contrast to mammalian systems with several rab3 isoforms (rab3A-D), a single family member homologue of rab3 is present in the rapidly dividing cleavage stage sea urchin embryo that localizes to numerous vesicles enriched at the cell cortex. We hypothesized that whereas the contents of these rab3-positive vesicles may contribute to the embryonic extracellular matrix, the membrane and its constituent proteins may be important for other aspects of cell division. We tested the function of rab3 in cell division by the microinjection of either antibodies or competing effector domain peptides to interfere with its function. We found that perturbing rab3 function results in cessation of cell division, whereas cells injected with either heat-inactivated antibodies or control scrambled peptides develop as normal. Moreover, neither endocytosis nor general membrane topology are affected by rab3 perturbation. Thus, we conclude that rab3-associated vesicles and/or their contents are critical for cell division.–Conner, S. D., Wessel, G. M. A rab3 homolog in sea urchin functions in cell division. FASEB J. 14, 1559–1566 (2000)

A rab3 homolog in sea urchin functions in cell division.

Rabs are monomeric GTP binding proteins belonging to the ras superfamily that function throughout the secretory pathway. Members of the rab3 family function in the final steps of the secretory pathway, vesicle fusion with the plasma membrane. In contrast to mammalian systems with several rab3 isoforms (rab3A-D), a single family member homologue of rab3 is present in the rapidly dividing cleavage stage sea urchin embryo that localizes to numerous vesicles enriched at the cell cortex. We hypothesized that whereas the contents of these rab3-positive vesicles may contribute to the embryonic extracellular matrix, the membrane and its constituent proteins may be important for other aspects of cell division. We tested the function of rab3 in cell division by the microinjection of either antibodies or competing effector domain peptides to interfere with its function. We found that perturbing rab3 function results in cessation of cell division, whereas cells injected with either heat-inactivated antibodies or control scrambled peptides develop as normal. Moreover, neither endocytosis nor general membrane topology are affected by rab3 perturbation. Thus, we conclude that rab3-associated vesicles and/or their contents are critical for cell division.

Genomic Organization, Chromosomal Localization, and Expression of the Murine RAB3D Gene

Rab proteins, members of the Ras superfamily of small GTPases, play regulatory roles in intercompartmental vesicular transport. Each step of traffic seems to require the participation of at least one distinct Rab, with the Rab3 subfamily involved in stimulated exocytosis. We report our studies on the murine rab3D gene, one of the four mammalian Rab3 isoforms. We located this gene on chromosome 13, region A2–3. The rab3D gene consists of 5 exons spanning 10.6 kb, and the structural gene is contained in exons 2 through 5 with one canonical GTP-binding motif in each exon. Organization of the rab3D gene is identical to that of rab3A but different from other rab genes. Alternative poly-A+ signals in the 3′ untranslated region account for the identities of multiple transcripts detected by Northern blot analysis. Rab3D is expressed in all tissues studied, predominantly in heart, lung, and liver, and binding sites for multiple transcription factors are found in the TATA-less promoter region.

Rab3A triggers the acrosome reaction in permeabilized human spermatozoa.

The acrosome reaction is a regulated exocytotic process leading to a massive fusion between the outer acrosomal membrane and the cell membrane. In spite of the great amount of information available related to the acrosome reaction in several species, there is a remarkable paucity about the role of monomeric guanosine triphosphatases (GTPases) of the Rab family-well-established participants in exocytosis in other cell types-in the acrosome reaction. Western blot and immunofluorescence analysis indicate that Rab3A is present in human spermatozoa and localizes to the acrosomal region in the sperm head. One difficulty in studying the role of proteins in intact cells is the fact that they are unable to cross the cell membrane. Therefore, we established a working model of streptolysin O-permeabilized human spermatozoa. Permeabilized spermatozoa were able to respond in a regulated way to different stimuli, such as G protein activators and calcium. An acrosomal reaction was also triggered by a Rab3A peptide corresponding to the effector region. More important, recombinant Rab3A protein in the GTP-bound form caused acrosome exocytosis. The same protein loaded with GDP or Rab11 in the GTP-bound form was inactive. Also, recombinant GDI (GDP dissociation inhibitor)-a protein that releases Rab proteins from membrane-inhibited a GTPgammaS-stimulated acrosome reaction. Our results indicate that 1) permeabilized spermatozoa can be used to study the role of macromolecules in the acrosome reaction, 2) Rab3A is present in human spermatozoa, and 3) Rab3A or another Rab3 isoform is involved in the exocytosis of the acrosomal granule in human spermatozoa.

Disruption of Rab3-calmodulin interaction, but not other effector interactions, prevents Rab3 inhibition of exocytosis.

Rab GTPases regulate membrane traffic between the cellular compartments of eukaryotic cells. Rab3 is associated with secretory vesicles of neuronal and endocrine cells and controls the Ca(2+)-triggered release of neurotransmitters and hormones. To clarify the mode of action of Rab3 we generated mutants of the GTPase that do not interact efficiently with its putative effectors Rabphilin and RIM. Surprisingly, these mutants transfected in PC12 cells were still capable of inhibiting Ca(2+)-evoked secretion. Rab3 was shown previously to bind to calmodulin in a Ca(2+)-dependent manner. By replacing two arginines conserved between Rab3 isoforms, we generated a mutant with a reduced affinity for calmodulin. This mutant retained the capacity to interact with the Rab3 regulatory proteins, Rabphilin, RIM, Mss4 and RabGDI, and was correctly targeted to dense-core secretory granules. However, replacement of the two arginines abolished the ability of the GTP-bound form of Rab3 to inhibit exocytosis of catecholamine- and insulin-secreting cells. We propose that a Rab3-calmodulin complex generated by elevated Ca(2+) concentrations mediated at least some of the effects of the GTPase and limited the number of exocytotic events that occurred in response to secretory stimuli.

Expression and regulation of RAB3 proteins in osteoclasts and their precursors.

The ruffled membrane, the resorptive organelle of the osteoclast, is generated by fusion of intracytoplasmic acidifying vesicles with the plasma membrane, an event analogous to regulated exocytosis. While the ruffled membrane is essential to the bone resorptive process, the mechanisms governing its generation are unknown. However, regulated exocytosis is mediated, in part, by isoforms of the Rab3 subset of Rab GTPases. Because of similarities between exocytosis and ruffled membrane formation, we asked if Rab3 proteins are expressed by osteoclasts or their precursors, and if so, are these molecules regulated by agents known to prompt the osteoclast phenotype? We find murine osteoclast precursors, in the form of bone marrow macrophages (BMMs), express at least two Rab3 isoforms, namely A and B/C, which are individually enhanced by a variety of hematopoietic cytokines. Consistent with the osteoclastogenic properties of a number of these cytokines, differentiation of BMMs into osteoclasts, in vitro, is associated with increased expression of both isoforms, particularly Rab3B/C. Finally, Rab3B/C localizes with the avian osteoclast H+ATPase (vacuolar proton pump) and pp60c-src, both intracellularly and within acidifying vesicles derived largely from the ruffled membrane. Thus, expression of specific rab3 proteins, an event which may control formation of the osteoclast ruffled membrane, is modulated by cytokines during osteoclastogenesis.

Expression and localization of Rab3D in rat parotid gland

Rab3 proteins (isoforms A, B, C and D) are low molecular weight GTP-binding proteins proposed to be involved in regulated exocytosis. In the present study, Rab3 protein expression and localization was examined in rat parotid gland by reverse transcription (rt) PCR, Western blotting and immunocytochemistry. An ∼200 bp PCR product was obtained from parotid RNA by rtPCR and this fragment was cloned and sequenced. Nucleotide and deduced amino acid sequences obtained from five clones were identical to rab3D. Membrane and cytosolic fractions prepared from parotid acini were immunoblotted with antisera specific for each of the four Rab3 isoforms. A 28 kDa protein was detected with Rab3D-specific antisera in both fractions with staining being more intense in the membrane fraction. No other Rab3 isoforms were detected by immunoblotting, a result consistent with those obtained by rtPCR. Rab3D was enriched in zymogen granule membranes and Triton X-114 extraction revealed that this isoform is predominantly lipid-modified in parotid. Localization of Rab3D was done on frozen sections of parotid gland by immunofluorescence microscopy. Staining was observed primarily in the acinar cells and was adjacent to the acinar lumen. Incubation of dispersed acini with isoproterenol and substance P stimulated amylase secretion 4- and 2-fold above basal, respectively. Isoproterenol, but not substance P, induced redistribution of Rab3D from the cytosol to the membrane fraction in dispersed parotid acini. Consistent with these findings, isoproterenol injections into fasted rats also resulted in increased membrane-associated Rab3D in the parotid acini. These results indicate that Rab3D is: (1) the major Rab3 isoform expressed in rat parotid gland; (2) localized to zymogen granule membranes; and (3) involved with regulated enzyme secretion in acinar cells.

Cloning and Localization of Rab3 Isoforms in Bovine, Rat, and Human Parathyroid Glands

Rab3 proteins are small GTP-binding proteins known to play a role in regulated exocytosis processes. This study examines the expression of Rab3 mRNA and protein in bovine, rat and human parathyroid glands. mRNAs of several Rab3 isoforms were detected in bovine (Rab3A, Rab3B and Rab3C) and rat (Rab3A, Rab3B and Rab3D) parathyroid glands by RT-PCR and sequencing. Rab3A protein was detected in the cytosolic extract from bovine parathyroid gland by Western blotting using a monoclonal antibody for Rab3A. Rab3A protein was localized to parathyroid hormone-containing chief cells by immunohistochemical staining. Subcellular localization of Rab3A protein by immunogold electron microscopy revealed that the majority of Rab3A protein was not associated with dense-core vesicles, but localized in the cytosol of the chief cells. Altogether, our results demonstrate that Rab3 isoforms are expressed in parathyroid chief cells, suggesting that they may play a role in regulated exocytosis in these cells.

Subcellular distribution and function of Rab3A, B, C, and D isoforms in insulin-secreting cells.

Insulin-secreting cells express four GTPases of the Rab3 family. After separation of extracts of INS-1 cells on a sucrose density gradient, the bulk of the A, B, and C isoforms was recovered in the fractions enriched in insulin-containing secretory granules. Rab3D was also mainly associated with secretory granules, but a fraction of this isoform was localized on lighter organelles. Analyses by confocal microscopy of immunostained HIT-T15 cells transfected with epitope-tagged constructs confirmed the distribution of the Rab3 isoforms. Transfection of HIT-T15 cells with GTPase-deficient mutants of the Rab3 isoforms decreased nutrient-induced insulin release to different degrees (D>B>A>>C), while overexpression of Rab3 wild types had minor or no effects. Expression of the same Rab3 mutants in PC12 cells provoked an inhibition of K+-stimulated secretion of dense core vesicles, indicating that, in beta-cells and neuroendocrine cells, the four Rab3 isoforms play a similar role in exocytosis. A Rab3A/C chimera in which the carboxyterminal domain of A was replaced with the corresponding region of C inhibited insulin secretion as Rab3A. In contrast, a Rab3C/A chimera containing the amino-terminal domain of C was less potent and reduced exocytosis as Rab3C. This suggests that the degree of inhibition obtained after transfection of the Rab3 isoforms is determined by differences in the variable amino-terminal region.

Rab3D, a small GTPase, is localized on mast cell secretory granules and translocates to the plasma membrane upon exocytosis.

Although mast cell secretion has been intensively studied because of its pivotal role in allergic reactions and its advantages as a physiologic model, the molecular composition of the secretory machine is virtually unknown. In view of the guanine-nucleotide dependency of mast cell exocytosis and the participation of Rab3 proteins in synaptic vesicle release, we hypothesized that a Rab3 isoform regulates mast cell secretion. Fragments of Rab3A, 3B, and 3D were cloned from RBL-2H3 mast cells by reverse transcription- polymerase chain reaction (RT-PCR). Northern blot analysis revealed Rab3D transcripts to be relatively abundant, Rab3B substantially less so, and Rab3A and 3C undetectable. By ribonuclease (RNase) protection assay, Rab3D transcripts were at least 10-fold more abundant than those of other isoforms, and by immunoblot analysis, Rab3D protein was at least 60-fold more abundant than that of Rab3B. Rab3D was more abundant in RBL cells than in brain, but the total mass of Rab3 proteins in RBL cells was 10-fold less than in brain. Rab3D only partly colocalized with secretory granules in RBL cells, but fully colocalized in mature peritoneal mast cells. There was a descending concentration gradient of Rab3D from peripheral to central granules, and no cytoplasmic pool was detectable in resting mast cells. Following exocytotic degranulation, Rab3D translocated to the plasma membrane and remained there for at least 15 min. These studies suggest that Rab3D is a component of the regulated exocytotic machine of mast cells, and identify differences between mast cells and neurons in Rab3 expression and trafficking.

Differential Expression of Rab3 Isoforms during Differentiation of Pancreatic Acinar Cell Line AR42J

Evidence is accumulating that low molecular weight GTPases of the Rab3 subfamily regulate exocytosis in secretory cells. The different isoforms of Rab3 (Rab3A-D) probably have opposite roles in secretory processes. In the present study we have investigated differentiation-dependent expression of Rab3 isoforms in the pancreatic acinar carcinoma cell line AR42J. The data show that AR42J cells express all four different isoforms of Rab3. Dexamethasone treatment, which increases the number of secretory granules and secretagogue-induced exocytosis several-fold, was accompanied by down-regulation of Rab3A, Rab3C, and Rab3D mRNA, whereas Rab3B mRNA and Rab3 protein were found up-regulated at the mRNA and protein level, suggesting that Rab3B might be involved in the secretory function of AR42J cells.

Expression, localization and functional role of small GTPases of the Rab3 family in insulin-secreting cells.

We examined the presence of small molecular mass GTP-binding proteins of the Rab3 family in different insulin-secreting cells. Rab3B and Rab3C were identified by western blotting in rat and in human pancreatic islets, in two rat insulin-secreting cell lines, RINm5F and INS-1, as well as in the hamster cell line HIT-T15. In contrast, Rab3A was detected in rat pancreatic islets as well as in the two insulin-secreting rat cell lines but not in human pancreatic islets and was only barely discernible in HIT-T15 cells. These findings were confirmed by two-dimensional gel electrophoresis followed by GTP-overlay of homogenates of pancreatic islets and of the purified protein. Northern blotting analysis revealed that Rab3D is expressed in the same insulin-secreting cells as Rab3A. Separation of the cells of the rat islets by fluorescence-activated cell sorting demonstrated that Rab3A was exclusively expressed in beta-cells. Rab3A was found to be associated with insulin-containing secretory granules both by immunofluorescence, immunoelectron microscopy and after sucrose density gradient. Overexpression in HIT-T15 cells of a Rab3A mutant deficient in GTP hydrolysis inhibited insulin secretion stimulated by a mixture of nutrients and bombesin. Insulin release triggered by these secretagogues was also slightly decreased by the overexpression of wild-type Rab3A but not by the overexpression of wild-type Rab5A and of a Rab5A mutant deficient in GTP hydrolysis. Finally, we studied the expression in insulin-secreting cells of rabphilin-3A, a putative effector protein that associates with the GTP-bound form of Rab3A. This Rab3A effector was not detectable in any of the cells investigated in the present study. Taken together these results indicate an involvement of Rab3A in the control of insulin release in rat and hamster. In human beta-cells, a different Rab3 isoform but with homologous function may replace Rab3A.

Differential expression of rab3 isoforms in oligodendrocytes and astrocytes.

The small GTP-binding protein Rab3a is involved in regulated secretory pathways and is enriched in synaptic and neuroendocrine secretory vesicles. We have reported previously the developmental regulation of Rab3a in oligodendrocytes in culture and purified central nervous system myelin (Huber et al.: FEBS Lett 347: 273-278, 1994). Since multiple rab3 isoforms exist in the brain and may be associated with different secretory pathways, we have investigated the differential expression of the rab3 isoforms in oligodendrocytes, astrocytes, and Schwann cell line RT4-D6P2T. The expression of specific rab3 isoforms (rab3a-c) was detected by polymerase chain reaction (PCR) amplification and confirmed by sequence analyses. These data show that in addition to the previously reported expression in neurons, the two macroglial populations, astrocytes and oligodendrocytes, also express rab3 isoforms. Rab3b was preferentially amplified from purified, cultured astrocytes, while rab3a and rab3c were preferentially amplified from highly enriched populations of both cultured oligodendrocytes and those isolated directly from the brain by immunopanning. No novel rab3 isoform was detected in glia. These results indicate that glial cells in the brain express specific isoforms of the vesicular trafficking Rab3 protein family.

Differential expression of rab3 isoforms in oligodendrocytes and astrocytes

Differential expression of rab3 isoforms in oligodendrocytes and astrocytes

Expression of Rab3D in dispersed chief cells from guinea pig stomach.

Rab3 proteins are low molecular weight GTP-binding proteins that are expressed in neurons and other secretory cells. These proteins are localized to secretory vesicles and may play a role in regulated exocytosis. Presently, four highly homologous Rab3 isoforms (A, B, C, D) have been identified. We examined the expression of Rab3 isoforms in dispersed chief cells from guinea pig stomach. Immunoblotting with a specific monoclonal Rab3 antibody detected a 27-kDa protein in chief cell cytosolic and membrane fractions, but staining was more intense in membrane fractions. Using the Rab3 antibody, immunohistochemical staining was detected in chief cells but not in parietal or mucous cells. To determine which Rab3 isoform(s) is (are) expressed, chief cell cDNA was obtained by reverse transcription and subjected to PCR using degenerate primers that are specific for rab3 isoforms. The resulting PCR products were cloned and sequenced. The nucleotide sequences obtained were 89% homologous to the nucleotide sequence of mouse rab3D. The deduced amino acid sequence was identical to that of mouse Rab3D (amino acids 16-83). Moreover, Rab3D was the only isoform detected in chief cells by these methods. To identify rab3 transcripts, the guinea pig rab3D fragment obtained by reverse transcription PCR cloning was used as a probe for Northern blotting. The 4.0- and 2.3-kb transcripts identified in chief cells with the rab3 probe were the same size as those detected by others in mouse adipocytes using a rab3D-specific probe. These results indicate that Rab3D is expressed in gastric chief cells.

Distinct Functional Properties of Rab3A and Rab3B in PC12 Neuroendocrine Cells

Rab3A and Rab3B are highly homologous monomeric GTPases that are putative regulators of exocytosis in those tissues in which they are expressed. We have characterized and directly compared the targeting and functional properties of these isoforms in PC12 neuroendocrine cells. Rab3A and Rab3B both targeted to norepinephrine (NE)-containing large dense core vesicles (LDCVs) when stably expressed in PC12 cells, as determined by immunofluorescence and membrane fractionation. Both Rab3 isoforms also bound to recombinant rabphilin-3A in a GTP-dependent manner. The membrane association of rabphilin-3A was modestly enhanced in Rab3B-expressing PC12 cells relative to Rab3A-overexpressing cells. In addition, overexpression of Rab3A modestly inhibited Ca2+-evoked NE release, whereas Rab3B and a GTP binding mutant (Rab3B N135I) markedly stimulated the efficiency of [3H]NE secretion by PC12 cells (i.e. secretion normalized to total cell radioactivity). Expression of Rab3B and Rab3B N135I increased not only the efficiency of NE secretion but also the accumulation of [3H]NE into LDCVs (i.e. the secretory cargo available for secretion). Neither of these effects was attributable to changes in the numbers of LDCVs nor the docking of LDCVs at the plasma membrane. Our results indicate that Rab3A and Rab3B have similar membrane targeting properties and are capable of interacting with the same putative downstream effector; i.e. rabphilin-3A. However, these isoforms are functionally distinct monomeric GTPases with Rab3B stimulating a late step in Ca2+-evoked secretion when expressed in PC12 cells.

Rab3 proteins and SNAP-25, essential components of the exocytosis machinery in conventional synapses, are absent from ribbon synapses of the mouse retina.

GTP-binding rab proteins, present in synaptic vesicles and endocrine secretory granules, have been shown to be involved in the control of regulated exocytosis. We found rab3 proteins in immunoblots of diverse areas of the mouse central nervous system (spinal cord, olfactory bulb, hippocampus, cerebellum and neocortex). Immunohistochemical observations at light- and electron-microscopical levels in the hippocampus and other areas revealed rab3 proteins in virtually all synaptic fields and terminals of the areas investigated. In the retina, rab3A immunoreactivity was confined to the inner and outer plexiform layers. Ultrastructural examination revealed that rab3A was present in conventional terminals in the inner plexiform layer and in horizontal cell processes of the outer plexiform layer. In contrast ribbon synapses, which play a key role in transferring information from the photoreceptor cells to the central nervous system, were immunonegative. We also tested whether other proteins of the rab3 family are present in ribbon synapses. However, using an antibody recognizing rab3B and rab3C in addition to rab3A, we found no immunoreactivity in these synapses. Interestingly, we observed also no immunoreactivity for synaptosomal-associated protein 25 (SNAP-25) in ribbon synapses, but conventional synapses and horizontal cell processes were heavily stained. Our data show that the known rab3 and SNAP-25 isoforms, which are components of the secretory apparatus of conventional synapses, are absent from ribbon synapses of the retina. Our observations suggest different mechanisms of transmitter exocytosis in conventional and ribbon terminals.

Stimulation of inositol 1,4,5-trisphosphate production by peptides corresponding to the effector domain of different Rab3 isoforms and cross-linking of an effector domain peptide target.

Rab3 proteins are localized on secretory vesicles and appear to be involved in regulated exocytosis. We have previously shown that a modified peptide corresponding to the effector domain of the small molecular mass GTP-binding protein Rab3A, Rab3AAL, stimulates inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] production and amylase release in digitonin-permeabilized pancreatic acini. Experiments using monoclonal antibodies reveal that the Rab3-like protein present in pancreatic acini is not the Rab3A isoform. However, since the putative effector domains of the four as yet known Rab3 proteins (A, B, C and D) differ only in the C-terminal four amino acid residues, Rab3A effector domain peptide could mimic the action of the pancreas-specific Rab3 isoform. In the present study we report that peptides corresponding to the different Rab3 isoforms stimulate both Ins(1,4,5)P3 production and amylase secretion with an order of potency Rab3B/D > Rab3AAL > Rab3A = Rab3C. For Rab3A, B/D and C effector domain peptides the concentrations causing half-maximal response (EC50) were 3, 0.2 and 3 nM for Ins(1,4,5)P3 accumulation and 0.3, 0.02 and 0.3 nM for amylase release, respectively. A Rab1A effector domain peptide, Rab1AAL, and a scrambled peptide of Rab3AAL were less potent by several orders of magnitude in eliciting these responses compared with native Rab3 effector domain peptides. None of the peptides influenced Ins(1,4,5)P3 production and amylase release in intact acini. Cross-linking of 125I-Rab3B/D peptide to pancreatic acinar membranes showed a band at 70 to 75 kDa with maximum intensity at 75 kDa. Radiolabelling of the substrates could be displaced by unlabelled Rab3B/D peptide, and to a lesser extend by Rab3A peptide, whereas the scrambled peptide of Rab3AAL had no effect. These data suggest that phospholipase C and exocytosis might be regulated by Rab3B-or Rab3D-like proteins in pancreatic acinar cells. A 75 kDa protein that preferentially cross-linked to 125I-Rab3B/D effector domain peptide is a potential candidate as an effector protein of Rab3 effector domain peptides.

Double in situ hybridization reveals overlapping neuronal populations expressing the low molecular weight GTPases Rab3a and Rab3b in Rat brain.

The ras-related Rab3 gene subfamily codes for small GTP-binding proteins which control a late step of exocytosis during which vesicles become docked to the plasma membrane. Rab3a and Rab3b are the most abundant Rab3 isoforms expressed in the CNS of mammals. We have shown previously that the Rab3a protein was selectively distributed and expressed in various regions of the rat brain. Here we have determined the pattern of expression of Rab3b mRNA in the brain and compared it with that of Rab3a mRNA. In addition, we examined the co-expression of these two Rab within individual neurons. In general the Rab3b transcript was detected in many regions which also express Rab3a mRNA but at a lower level than Rab3a, except in the olfactory bulb and in the pituitary where the Rab3b hybridization signal was similar and higher respectively. Double in situ hybridization revealed that Rab3a and Rab3b mRNAs were co-localized in most neurons, in all brain areas examined. However, in each of these areas, subsets of neurons appeared to preferentially express either Rab3b or Rab3a, or some neurons did not express either Rab3 homologue at detectable levels. These data support the view of a functional specialization of Rab3a and Rab3b in the control of exocytosis in neuronal and neuroendocrine cells.