©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Evidence That the Rab3a-binding Protein, Rabphilin3a, Enhances Regulated Secretion
STUDIES IN ADRENAL CHROMAFFIN CELLS (*)

Sul-Hee Chung (1), Yoshimi Takai (2), Ronald W. Holz (1)(§)

From the (1)Department of Pharmacology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0632 and the (2)Department of Molecular Biology and Biochemistry, Osaka University Medical School, Suita 565, Osaka, Japan

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Rabphilin3a had been identified in brain as a Rab3a-binding protein and may serve as an effector for Rab3a function. We have cloned a splice variant of brain-Rabphilin3a from a bovine adrenal chromaffin cell cDNA library and investigated the function of the protein in regulated exocytosis in bovine chromaffin cells. The predicted amino acid sequence of chromaffin cell (c-) Rabphilin3a was identical with that of brain (b-) Rabphilin3a except for a 6-amino-acid insert VFSLSA in the amino-terminal half of the protein. An antibody directed against a carboxyl-terminal peptide recognized an 85-kDa protein in COS7 cells transfected with the cDNA in a mammalian expression vector. A band of similar mobility was enriched in a fraction of highly purified chromaffin granule membranes, consistent with the Rabphilin3a being associated with chromaffin granule membranes. Overexpression of either chromaffin cell or brain Rabphilin3a by transfection with the corresponding cDNAs in mammalian expression vectors enhanced DMPP-induced secretion of co-expressed human growth hormone (GH) approximately 30%. Chromaffin cells transfected with a plasmid with the entire coding sequence of c-Rabphilin3a inserted in the antisense orientation inhibited secretion of co-expressed GH by approximately 30%. Rabphilin3a mutants lacking one or both of the carboxyl-terminal C2 domains strongly inhibited DMPP-stimulated exocytosis. The single C2 domain deletion also strongly inhibited Ca-dependent secretion from digitonin-permeabilized cells. These data indicate that Rabphilin3a is a positive regulator of exocytosis. Because the C2 deletion mutants contain the amino-terminal Rab3a-GTP binding domain, they may inhibit secretion by competing with endogenous Rabphilin3a for interaction with Rab3a-GTP without being able to mimic the functional effects of full-length Rabphilin3a.


INTRODUCTION

The convergence of genetic studies in yeast and biochemical studies in mammalian cells has revealed two sets of proteins implicated in vesicular trafficking including regulated exocytosis (reviewed in Refs. 1-4). One set involves proteins of the SNARE()complex and includes the vesicle membrane proteins VAMP and synaptotagmin, the plasma membrane proteins SNAP25 and syntaxin, and the cytosolic proteins NSF and - and -SNAPs. The SNARE hypothesis (4, 5, 6) proposes that vesicle SNAREs interact specifically with cognate target SNAREs. Docking and fusion would proceed as a consequence of sequential interactions modulated by NSF.

The other set of proteins consists of members of the Rab class of low molecular weight GTPases. Distinct Rab proteins are necessary for vesicular trafficking in the constitutive secretory (7, 8, 9) and endocytosis pathways(10, 11, 12, 13, 14) . Although Rab family members are required for vesicular trafficking, they are not core components of the SNARE complex. In mammalian cells there is growing evidence that members of the Rab3 subclass of proteins modulate regulated exocytosis(15, 16, 17) . We and others have demonstrated that Rab3a acts as an inhibitory modulator of secretion in bovine adrenal chromaffin (16, 17) and PC12 (17) cells. We had found that overexpression of Rab3a inhibited the earliest rates of Ca-dependent secretion from permeabilized chromaffin cells(16) . Since the initial secretory rates reflect secretion that is already primed by the previous action of ATP (18, 19), overexpression of Rab3a may reduce the number of chromaffin granules in the readily releasable pool.

Rabphilin3a was identified in brain as a possible effector protein for Rab3a. It binds specifically to the GTP-bound form of Rab3a(20, 21) and also binds to Rab3c(22) . Cloning of bovine brain Rabphilin3a revealed a protein of 704 amino acids(23) . Rabphilin3a is a peripheral membrane protein (22, 24) which is composed of at least two functionally distinct domains: the amino-terminal region (1-280 amino acids) which binds to Rab3a and the carboxyl-terminal domain (281-704 amino acids) which contains two C2 domains that confer Ca-dependent lipid binding(25) . The interaction of Rabphilin3a with Rab3a inhibits GTPase-activating protein activity (26) and enhances guanine nucleotide exchange(27) . These effects should maintain Rab3a in the GTP-bound state within the cell. Rab3a and Rabphilin3a are both localized to synaptic vesicles (22, 24, 28, 29) where they may interact. Removal of the Rab3a gene in mice results in loss of Rabphilin3a in brain suggesting that Rab3a stabilizes Rabphilin3a(30) . These data support the notion that Rabphilin3a is an effector for Rab3a.

Bovine chromaffin cells are primary, nondividing secretory cells that store and secrete catecholamine and a variety of proteins including chromogranins, opiate peptides, and opiate precursors. Secretion is normally stimulated by an influx of Ca and occurs in permeabilized cells upon incubation with micromolar and higher Ca concentrations. In a previous study we demonstrated that Rab3a can act as an inhibitory modulator of secretion in bovine chromaffin cells by using a transient transfection technique. A plasmid of interest was co-transfected with a plasmid encoding human growth hormone (GH), a reporter for the regulated secretory pathway. Although only approximately 0.1% of the cells were transfected, a sensitive immunological assay for GH could readily detect its secretion(31) . Analysis of the effects of various mutants suggested that the GTP-bound form of Rab3a was responsible for the inhibition of secretion(16, 17) . These results prompted us to investigate the role of Rabphilin3a in secretion from the cells. In the present study, we isolated from a bovine adrenal medullary cDNA library an apparent splice variant of the bovine brain form of the protein and demonstrated that Rabphilin3a is expressed in chromaffin cells. Using the transient transfection approach, we provide the first evidence for a role of Rabphilin3a in the regulated secretory pathway.


MATERIALS AND METHODS

cDNA Cloning and Sequencing

cDNA encoding Rabphilin3a from bovine adrenal medulla was identified on the basis of its structural homology to bovine brain Rabphilin3a (23) using PCR screening in a bovine adrenal medullary library pCDSP6T7 (from Dr. Michael Brownstein, NIH). The nucleotide sequence of a cloned cDNA of about 4.5 kb, containing a full-length coding sequence for chromaffin cell Rabphilin3a (c-Rabphilin3a), was obtained.

Construction of Expression Vectors

The 4.5-kb BamHI fragment of c-Rabphilin3a was inserted into the BamHI site of the plasmid pRK7 to express Rabphilin3a cDNA under the control of the CMV promoter (pRK7-cRP). Other cDNA constructs were made by the polymerase chain reaction (PCR) using specific oligonucleotide primers to generate sense (amino acids 1-710; pCMV-HAcRP), antisense(1-710), and deletion mutant constructs (1-286, 1-536, and 287-710) of c-Rabphilin3a. The constructs were inserted into a plasmid under the control of the CMV promoter and containing the HA1 epitope (YPYDVPDYA) in-frame at the amino terminus. The construction of the mammalian expression vector for bovine brain Rabphilin3a, pCEV4-Rabphilin3a, has been described previously(23) .

Generation of Anti-Rabphilin3a Antibody

A rabbit antibody against the 16 carboxyl-terminal amino acids (IERWHQLQNENHVSSD) common to both the chromaffin cell and brain forms of Rabphilin3a was generated from a KLH-peptide immunogen emulsified by mixing with Freund's adjuvant. High titers of antibody were obtained at 10 and 14 weeks as determined by an enzyme-linked immunosorbent assay against the peptide.

Protein Expression and Immunocytochemistry

Protein expression was examined by transient transfection of COS7 cells in 35-mm diameter dishes by calcium phosphate precipitation. After 3 days, cells were harvested into sample buffer and separated on 5-15% gradient SDS-polyacrylamide gel electrophoresis and transferred to Immobilon. The blot was incubated sequentially with a rabbit anti-Rabphilin3a antiserum (1:500 dilution) and I-goat anti-rabbit antibody. Chromaffin granule membranes were purified from a P2 fraction from fresh bovine adrenal medulla by centrifugation through 1.7 M sucrose(32) . The chromaffin granule pellet was resuspended in hypotonic solution, and the chromaffin granule membranes were pelleted by centrifugation.

Immunocytochemistry to detect human GH and the HA1 epitope was performed as described previously(16) .

Cell Preparation, Transfection, and Secretion Experiments

Chromaffin cell preparation, transient transfection, and secretion experiments were performed as described previously(16, 31) . Secretion experiments were performed 5-6 days after the transfection at 27 °C.

Human growth hormone was measured with a high sensitivity, chemiluminescence assay kit from Nichols Institute (San Juan Capistrano, CA). Endogenous catecholamine secretion was measured with a fluorescence assay(33) . Secretion was expressed as the percentage of the total cellular human GH or catecholamine that was released into the medium. There was usually 0.2-1 ng of GH and 80-100 nmol of catecholamine/35-mm diameter dish.


RESULTS

Isolation of Rabphilin3a cDNA Clone from Bovine Chromaffin Cells

A cDNA encoding Rabphilin3a from bovine adrenal medulla was identified on the basis of its structural homology to bovine brain Rabphilin3a (b-Rabphilin3a) using PCR screening in a bovine adrenal medullary library pCDSP6T7. The nucleotide sequence of a cloned cDNA of about 4.5 kb was determined and contained a full-length coding sequence for chromaffin cell Rabphilin3a (c-Rabphilin3a).

The nucleotide sequence of c-Rabphilin3a is identical with that of b-Rabphilin3a except for an 18-bp insert in the coding region (Fig. 1) and an A for T substitution 20 nucleotides upstream from the ATG start codon. The insertion site starts with the consensus GT sequence for splicing suggesting that the chromaffin cell and brain forms of Rabphilin3a result from alternative splicing. The predicted amino acid sequence of c-Rabphilin3a is identical with that of b-Rabphilin3a except for the 6-amino-acid insert VFSLSA (encoded by the 18-bp insertion) following Arg-206 in the amino-terminal half of the protein (underlined in Fig. 1). This insert may be a phosphorylation site although a specific protein kinase is not suggested by the sequence or the neighboring amino acids.


Figure 1: Chromaffin cell Rabphilin3a is a likely splice variant of brain Rabphilin3a. The nucleotide sequence of a clone from a chromaffin cell c-DNA library was identical with that of brain Rabphilin3a (23) except for an 18-bp insert in the coding region and an A for T substitution 20 nucleotides upstream from the ATG start codon. Shown are the nucleotide and predicted amino acid sequences for brain and chromaffin cell Rabphilin3a in the coding region that encompasses the insert (underlined). The numbering corresponds to the amino acid number in the sequence.



Expression of Rabphilin3a in COS7 Cells and Chromaffin Granules

We constructed a plasmid encoding c-Rabphilin3a under the control of the CMV promoter (pCMV-HAcRP). COS7 cells were transfected with a plasmid encoding either the brain form of Rabphilin3a (pCEV4-Rabphilin3a) or the chromaffin cell form of Rabphilin3a (pCMV-HAcRP). The anti-Rabphilin3a antibody recognized a protein at 85 kDa in COS7 cells transfected with plasmids encoding b- and c-Rabphilin3a (Fig. 2A, lanes B and C, respectively) but not in the cells transfected with the control plasmid (Fig. 2A, lane D). These experiments indicate that the cloned cDNA from bovine chromaffin cells encodes a protein of appropriate molecular weight for Rabphilin3a. The antibody also recognized a protein of similar molecular weight in a highly purified chromaffin granule membrane preparation (Fig. 2A, lane A).


Figure 2: Expression of transiently transfected plasmid encoding Rabphilin3a in COS7 cells and in bovine adrenal chromaffin cells. A, antibody against the carboxyl-terminal domain of Rabphilin3a (see ``Materials and Methods'') identifies Rabphilin3a in chromaffin granules and in COS7 cells expressing cDNA encoding the brain and chromaffin cell isoforms in immunoblots. Lane A, highly purified chromaffin granule membranes (50 µg of protein); B, COS7 cells (7.5 µg of protein) 72 h after transfection with plasmid encoding brain Rabphilin3a; C, COS7 cells (7.5 µg of protein) 72 h after transfection with a plasmid encoding chromaffin cell Rabphilin3a; D, COS7 cells (7.5 µg of protein) 72 h after transfection with control plasmid. The plasmid encoding c-Rabphilin3a was under the control of the CMV promoter; the plasmid encoding b-Rabphilin3a was under the control of the SR promoter. The antibody recognizes an 85-kDa protein (indicated as Rabphilin3a) in COS7 cells transfected with the plasmid encoding either brain or chromaffin cell Rabphilin3a (lanes B and C) and in chromaffin granule membranes (lane A). Preimmune antibody did not detect the band. B, HA1-tagged Rabphilin3a derived from an adrenal medullary library can be co-expressed with human GH in chromaffin cells. Bovine chromaffin cells were co-transfected with pXGH5 encoding human GH and pCMV-HAcRP encoding HA1-tagged c-Rabphilin3a. Five days later, cells were fixed and permeabilized with methanol. Rabbit anti-human GH (with donkey anti-rabbit lissamine rhodamine) and mouse monoclonal anti-HA1 (12CA5, with goat anti-mouse fluorescein isothiocyanate) was used to detect GH (middle) and the HA1-tagged c-Rabphilin3a (right). The phase image of the field is on the left.



Overexpression of c- and b-Rabphilin3a Enhances Secretion from Chromaffin Cells

We investigated the effects on GH secretion of co-transfection with plasmids encoding Rabphilin3a and pXGH5 (encoding human GH). To determine whether the transfected Rabphilin3a is co-expressed with human growth hormone in chromaffin cells, immunocytochemistry was performed on chromaffin cells. Plasmid encoding c-Rabphilin3a (pCMV-HAcRP) was co-transfected with pXGH5. An epitope tag (HA1) was attached to the amino terminus of the Rabphilin3a protein to allow detection in the cells. Fig. 2B demonstrates that Rabphilin3a could be co-expressed with human GH when it was co-transfected with pXGH5 in chromaffin cells. Approximately 90% of the growth hormone-expressing cells expressed detectable amounts of epitope-tagged protein as determined by fluorescence microscopy.

The effects on secretion of transfection with plasmids encoding bovine adrenal chromaffin cell Rabphilin3a (pCMV-HAcRP) and bovine brain Rabphilin3a (pCEV4-Rabphilin3a) were examined. Expression of both b-Rabphilin3a and c-Rabphilin3a (Fig. 3) enhanced DMPP (20 µM)-induced secretion. Expression of pRK7-cRP (non-epitope-tagged c-Rabphilin3a) enhanced secretion similarly (data not shown). The effects were observed in 10 of 10 experiments with average enhancements of secretion of 34 ± 6% and 32 ± 10% between 0-2 min and 2-16 min, respectively (p < 0.001 during each time interval versus transfection with pCMV.neo). The enhancement of secretion by c- and b-Rabphilin3a is a specific effect of expression of the protein since expression of numerous other exogenous proteins including -galactosidase, certain mutants of Rab3a (Rab3aCAC and Rab3aV55E), and Rabin3a do not alter GH secretion in transfected chromaffin cells. The latter protein, Rabin3a, was recently cloned from a rat brain cDNA library as a Rab3a-binding protein using the yeast two-hybrid system(34) .


Figure 3: Expression of brain Rabphilin3a and chromaffin cell Rabphilin3a enhances secretion from chromaffin cells. Chromaffin cells were co-transfected with pXGH5 encoding human GH and either pCEV4-Rabphilin3a encoding bovine brain Rabphilin3a or pCMV-HAcRP encoding HA1-tagged bovine chromaffin cell-Rabphilin3a. Six days later, cells were incubated in physiological salt solution for 2 min ± 20 µM DMPP. There were 4 dishes/group. a, p < 0.05 versus GH secretion from cells transfected with control plasmid, pCMV.neo.



Transfection with the Antisense Construct of c-Rabphilin3a Inhibits Secretion from Chromaffin Cells

Since overexpression of Rabphilin3a enhances secretion, reduced expression of endogenous Rabphilin3a by transfection with an antisense Rabphilin3a construct should reduce stimulated secretion. Fig. 4suggests that this is, indeed, the case. Chromaffin cells were transfected with a plasmid with the entire coding sequence of c-Rabphilin3a inserted in the reverse orientation in the expression vector downstream from a CMV promoter. Six days later, DMPP-induced secretion was inhibited by approximately 30% (Fig. 4). In four experiments, the average inhibition of secretion between 0-2 min and 2-16 min was 27 ± 4% during each time interval (p < 0.001 during each time interval, compared to transfection with pCMV.neo).


Figure 4: Transient transfection with antisense Rabphilin3a inhibits secretion of co-transfected and expressed GH. GH secretion was determined from chromaffin cells that were co-transfected with the plasmid encoding human GH (pXGH5) and either a plasmid constructed with the entire coding region for chromaffin cell Rabphilin3a in the antisense orientation (Anti-Sense) or with the parent plasmid (CMV.neo). Six days after transfection, cells were incubated with and without 20 µM DMPP for 2 min in physiological salt solution, and DMPP-dependent secretion was determined. There were 7-8 dishes/groups. a, p < 0.01 versus pCMV.neo.



Effects of Deletion Mutants of Rabphilin3a on Secretion

Chromaffin cells were co-transfected with pXGH5 and several deletion mutants of Rabphilin3a (Fig. 5A). The constructs lacking one (RpC2B) or both (RpC2AB) of the carboxyl-terminal C2 domains but containing the Rab3a binding domain of the amino-terminal portion of the Rabphilin3a strongly inhibited secretion in the first 2 min. The construct without the Rab3a binding domain, Rp(287-710), was without effect although immunocytochemistry revealed that the transfected mutant was co-expressed with human growth hormone in chromaffin cells (data not shown). Truncated Rabphilin3a lacking one or both C2 domains retains the amino-terminal sequence necessary for binding Rab3a-GTP and is likely to bind Rab3a-GTP similarly to the full-length protein. Thus, the results with the deletion mutants raise the possibility that truncated Rabphilin3a competitively inhibits the function of endogenous Rabphilin3a.


Figure 5: Effects of Rabphilin3a mutants on secretion from intact (A) and permeabilized (B) cells. Chromaffin cells were co-transfected with pXGH5 encoding human GH and either pCMV.RpC2B encoding Rabphilin3a(1-536) without the second C2 domain, pCMV.RpC2AB encoding Rabphilin3a(1-286) without both C2 domains, pCMV.Rp(287-710) encoding just the carboxyl-terminal half of the protein, or pCMV.neo, the parent plasmid. Six days later, cells were incubated in physiological salt solution ± 20 µM DMPP. DMPP-induced GH secretion was determined between 0-2 min and 2-16 min (A) or were permeabilized for 4 min with digitonin-containing potassium glutamate solution with and without 30 µM Ca (B). There were four 35-mm diameter dishes/group. a, p < 0.01 versus pCMV.neo.



One of the mutants (RpC2B) was further investigated in permeabilized cells (Fig. 5B). Its expression strongly inhibited Ca-dependent secretion. Thus, it is likely that the inhibition was a direct effect on the Ca-dependent secretory pathway and not on the Ca signal.


DISCUSSION

Previous studies demonstrating an inhibitory role of Rab3a in Ca-dependent exocytosis in bovine chromaffin cells prompted us to investigate the function of the Rab3a-binding protein, Rabphilin3a, as a possible effector for Rab3a function. The present study identified Rabphilin3a in bovine adrenal chromaffin cells, probably associated with chromaffin granules. Overexpression of Rabphilin3a by transient transfection of the cDNA enhanced secretion, and transfection with antisense Rabphilin3a cDNA inhibited secretion. The data suggest that Rabphilin3a is likely to be a positive regulator of secretion.

Bovine Chromaffin Cells Express a Splice Variant of the Bovine Brain Rabphilin3a

The predicted amino acid sequence of the Rabphilin3a cDNA clone identified in a bovine adrenal medullary cDNA library was identical with that of bovine brain Rabphilin3a except for a 6-amino-acid insert VFSLSA (encoded by the 18-bp insertion) following Arg-206 in the amino-terminal half of the protein (underlined in Fig. 1). The insert probably resulted from alternative splicing of the mRNA. Both forms of Rabphilin3a enhanced secretion from bovine chromaffin cells (Fig. 3). Alternative splicing probably also occurs in rat brain Rabphilin3a (22) in which there is a 3-amino-acid insert in the amino-terminal region. The expression of Rabphilin3a was confirmed by protein immunoblots. These results, together with the identification of Rabphilin3a in PC12 cells(35, 36) and mRNA analysis in an insulin-secreting cell line (HIT-T15 cells), suggest a function for Rabphilin3a in endocrine cells as well as in brain.

A comparison of the predicted amino acid sequences of bovine brain (23), rat brain(22) , and mouse (36) Rabphilin3a indicates that the protein is highly conserved across species. The corresponding cysteine-rich region in the amino-terminal region and the C2 domains in the carboxyl-terminal region are at least 98% identical. Thus, the biochemical characteristics of the proteins from the different species are likely to be similar, and the highly conserved regions may be important for the physiological function of the protein. Indeed, as discussed below, both C2 domains are necessary for Rabphilin3a function in secretion.

Opposite Effects on Secretion of Rab3a and Rabphilin3a Suggest a Model with Sequential Events

The high affinity and specific binding of Rab3a-GTP with Rabphilin3a in vitro(20, 23) , the association of both Rab3a and Rabphilin3a with synaptic vesicles and probably chromaffin granules, and the evidence for Rab3a-Rabphilin3a interaction in vivo(30) suggest that the function of these proteins in secretion is likely to be governed by their interaction in the cell. If the inhibitory effect of overexpression of Rab3a on secretion were directly mediated by its interaction with Rabphilin3a, then overexpression of Rabphilin3a should have also inhibited secretion. The opposite effects of Rab3a to inhibit and of Rabphilin3a to enhance secretion indicate that the functional relationship between the proteins is probably more complex. One explanation that is consistent with the data is that there is a sequence of events in which the formation of Rab3a-GTPRabphilin3a complex is necessary for secretion but the complex must dissociate (perhaps because of GTP hydrolysis) in order for secretion to occur. Free, activated Rabphilin3a would be a positive effector for secretion. Overexpression of Rab3a would inhibit secretion by rapidly rebinding uncomplexed Rabphilin3a. The model is compatible with the inhibition of secretion by expression of the Rabphilin3a deletion mutants without one or both of the C2 domains. Because these mutants bind Rab3a-GTP, they may inhibit secretion by competing with endogenous Rabphilin3a for interaction with Rab3a-GTP without being able to mimic the functional effects of full-length Rabphilin3a. Reduced binding of endogenous Rabphilin3a with Rab3a may also result in increased degradation of either or both endogenous Rabphilin3a and Rab3a(22, 30) .

A sequential model for Rab3a-Rabphilin3a interaction is also consistent with the inability of expression of either Rab3aT36N (16, 17) or Rab3aCAC()to inhibit secretion. Rab3aT36N does not alter secretion because it is predominantly in the GDP-bound state that does not bind Rabphilin3a(21) ; unprenylated Rab3aCAC does not alter secretion because it does not bind to membranes.

The Presence of Both C2 Domains Is Necessary for Wild Type Rabphilin3a Function

The strong inhibition of secretion by expression of Rabphilin3a mutants without one or both of the C2 domains contrasts with the enhancement of secretion by overexpression of full-length Rabphilin3a. The results provide direct evidence for the importance of both of the C2 domains for the physiological function of Rabphilin3a. Both C2 domains are also necessary for synaptotagmin function(37) . A single C2 domain is sufficient to convey Ca-dependent effects to protein kinase C (38) and phospholipase A2(39) . Model studies with single C2 domains of synaptotagmin also demonstrate Ca-dependent lipid binding(40, 41) . The tandem C2 domains of Rabphilin3a (and of synaptotagmin) may confer specificity of binding to target proteins or permit the simultaneous binding to different membranes or membrane proteins. Such effects could be important in positioning the granule at the plasma membrane.

C2 domains may regulate Ca/phospholipid-dependent binding to specific proteins(42) . Indeed, it has been reported that Rabphilin3a interacts with -adducin in a Ca- and phospholipid-dependent manner(43) . -Adducin is associated with the subplasmalemmal cytoskeleton and probably catalyzes cytoskeleton assembly(44) . It is tempting to speculate that Rabphilin3a bound to the chromaffin granule membrane may reduce the cytoskeletal barrier for granule interaction with the plasma membrane by inhibiting -adducin function.

Rab3a, Rabphilin3a, and SNAREs

Although at this time direct physical interaction of Rab proteins with SNARE proteins has not been demonstrated, genetic and biochemical evidence in yeast suggest that the Rab pathway interacts in some manner with the v-SNARE/t-SNARE pathway(45, 46, 47) . To date, a protein homologous to Rabphilin3a has not been identified in yeast. However, it is possible that just as the Rab protein YPT1 enhances the interaction of two v-SNAREs (Bos1p and Sec22p)(46) , Rab3a enhances the functional interaction of Rabphilin3a with a SNARE as well as with other proteins.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant RO1 DK27959 (to R. W. H.) and a postdoctoral fellowship from the American Heart Association of Michigan (to S.-H. C.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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

The abbreviations used are: SNARE, receptor for soluble NSF attachment protein; SNAP25, synaptosome-associated protein of 25 kDa; b-Rabphilin3a, brain Rabphilin3a; c-Rabphilin3a, chromaffin cell Rabphilin3a; CMV, human cytomegalovirus; GH, growth hormone; GAP, GTPase activating protein; Rp, Rabphilin3a; kb, kilobase(s); bp, base pair(s); PCR, polymerase chain reaction; DMPP, 1,1-dimethyl-4-phenylpiperazinium.

D. L. Scheuner and R. W. Holz, unpublished observations.


ACKNOWLEDGEMENTS

We are grateful for the ongoing discussions about this work with Dr. Ian G. Macara (Dept. of Pathology, University of Vermont Medical School, Burlington, VT), Dr. Mary A. Bittner (Dept. of Pharmacology, University of Michigan Medical School), and Dr. Michael D. Uhler (Dept. of Biological Chemistry and the MHRI, University of Michigan).


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