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J Biol Chem, Vol. 274, Issue 40, 28542-28548, October 1, 1999
From the Department of Molecular Medicine, Institute for Molecular
and Cellular Regulation, Gunma University,
Maebashi 371-8512, Japan
A novel rabphilin-3-like gene, granuphilin, has
been identified in pancreatic beta cells by comparing genes expressed
in pancreatic alpha and beta cell lines using mRNA differential
display. The domain structure of the protein products of the
granuphilin gene contains an amino-terminal zinc-finger motif and
carboxyl-terminal C2-domains, similar to that of the
rabphilin-3 gene. There are two isoforms: the larger isoform,
granuphilin-a, has two C2-domains, whereas the smaller one,
granuphilin-b, contains only the first C2-domain.
Granuphilin is specifically expressed in pancreatic beta cells and the
pituitary gland, but not in pancreatic alpha cells, the adrenal gland,
or other major organs such as the brain. A portion of granuphilin
associates with insulin-containing dense-core granules, but not with
synaptic-like microvesicles in beta cells. Thus, its distribution
pattern presents a striking contrast with that of rabphilin-3, which
associates with small synaptic vesicles in neurons. The first
C2-domain of granuphilin binds phospholipids in a
Ca2+-independent manner, whereas the second one does not.
These distinctive characteristics of granuphilin suggest that it is not
a simple counterpart of rabphilin-3 in endocrine cells and that it has a unique role in the regulated exocytosis of dense-core granules in
endocrine tissues.
A combination of genetics in yeast and biochemistry in animal
cell-free systems has revealed basic molecular mechanisms of constitutive exocytosis (1, 2). For its cells to adapt to changes in
environments and to communicate with other cells, a multicellular
organism further differentiates a regulated exocytosis that occurs only
in the presence of secretagogues (3). This pathway is highly developed
in neuronal, endocrine, and exocrine cells, where neurotransmitters,
hormones, and digestive enzymes are released by exocytosis in response
to an intracellular Ca2+ signal generated by secretagogues.
These cells must be equipped with specific machinery that permits them,
for example, to sense the Ca2+ signal and then release
their cargo and replenish the stored material. Biochemical studies of
synaptic vesicles at the nerve terminal have identified a number of
molecules possibly involved in these processes (4). A group of proteins
with two C2-domains has drawn particular attention because
this motif has biochemical properties that sense a Ca2+
signal and then bind membranes through interaction with phospholipids (5). Furthermore, some C2-domains interact with other
proteins in a Ca2+-dependent manner (5). These
C2-domain-containing molecules include synaptotagmins (5),
rabphilin-3 (6), Doc2 (7), and RIM (8), although their precise
functions remain elusive.
Both neuron and endocrine cells contain at least two types of secretory
vesicles (9). One type of vesicle, the small synaptic vesicle
(SSV)1 in neurons and the
synaptic-like microvesicle (SLMV) in endocrine cells, releases
classical neurotransmitters. These are relatively small and
electron-lucent and cycle between the plasmalemma and the early
endosome compartment. The other type of vesicle, the large dense-core
vesicle (LDCV) or the secretory granule in neuroendocrine cells, mainly
secretes polypeptides, although some of these in specific
subpopulations secrete monoamines. These LDCVs directly form from the
trans-Golgi network and have an electron-dense core. The
physiologic and pharmacologic properties of the membranes of the two
types of vesicles differ. For example, LDCV exocytosis is triggered by
the lower Ca2+ concentrations reached away from the sites
of Ca2+ entry (3-30 µM calcium for
half-maximal stimulation for release), whereas SSV exocytosis is
triggered by high Ca2+ concentrations close to the entry
site (200 µM) (10). The synaptic events occur as brief as
60 µs after stimulation for SSVs, whereas the latency of LDCVs in
neuroendocrine cells is 100 times longer (10). Furthermore, in motor
nerve terminals, the exocytosis of acetylcholine-containing SSVs is
sensitive to To analyze the molecular machinery of regulated exocytosis of
LDCVs/granules, we studied pancreatic beta cells, which secrete insulin
in response to blood glucose levels. Because beta cells are one of the
most physiologically characterized endocrine cells and possess both
insulin-containing dense-core granules and Cell Culture and Tissue Preparation--
Pancreatic beta cell
lines ( RNA Preparation and Northern Blotting--
Total RNA from cell
lines (80% confluent culture at a density of 1-2 × 107 cells/10-cm plate) and tissues was prepared as
described previously (17). For Northern blotting, 20 µg of total RNA
was separated on a 1% agarose and 6.7% formaldehyde gel and
transferred onto Hybond-N membranes (Amersham Pharmacia Biotech,
Buckinghamshire, United Kingdom). Radioactive probes were generated
with [ Molecular Cloning of Granuphilin cDNAs--
Expression
levels of genes in Reverse Transcription-Polymerase Chain Reaction
(RT-PCR)--
Total RNA (1 µg) was reverse-transcribed using
oligo(dT)17 primer as described previously (15). The 5'-
and 3'-primers used for PCR were derived from sequences flanking the
granuphilin-a-specific region so that the sizes of PCR products from
the two isoforms are discriminated. The 5'-primer,
5'-ACTTTCCTTGGAGAGGCGGA-3', was combined with either of the 3'-primers,
5'-CATCATCTACCTTGATACCC-3' or 5'-CCCTGGGTACTGTCGCATCT-3'. The expected
sizes of PCR products with the former 3'-primer are 936 base pairs (bp)
for granuphilin-a and 518 bp for granuphilin-b, and those with the
latter 3'-primer are 588 bp for granuphilin-a and 170 bp for
granuphilin-b. PCR was carried out in 20 µl of 50 mM KCl,
10 mM Tris-HCl, pH 8.3, 1.5 mM
MgCl2, 200 µM each dNTP, 1.5 units of
Taq DNA polymerase (Roche Molecular Biochemicals, Mannheim,
Germany), 12.5 pmol of the 5'- and 3'-primers, and the RT mixture
described above. Following incubation for 5 min at 94 °C for
denaturation, the samples were incubated at 94 °C for 30 s,
56 °C for 30 s, and 72 °C for 1 min for 27 cycles. The
products were resolved on 2% agarose gels and visualized with ethidium bromide.
Antibodies, Immunostaining, and Immunoblotting--
Rabbit
antibodies against the carboxyl-terminal granuphilin-a peptide,
CTLQLRSSMVKQKLGV, were produced as described previously (15).
Rabbit antibodies against vesicle-associated membrane protein (VAMP)-2
(18) and synaptotagmin III (19) were gifts from Dr. M. Takahashi
(Mitsubishi Kasei Institute of Life Sciences). Mouse monoclonal
antibodies against synaptophysin (SY 38) and guinea pig anti-glucagon
serum were purchased from Progen Biotechnik (Heidelberg, Germany) and
Linco Research (St. Charles, MO), respectively. Guinea pig anti-porcine
insulin serum was a gift from Drs. K. Wakabayashi and H. Kobayashi
(Institute for Molecular and Cellular Regulation, Gunma University).
Immunostaining on the mouse pancreas specimen was performed as
described previously (15).
For immunoblotting, proteins were solubilized in lysis buffer
containing 50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 1%
sodium deoxycholate, 150 mM NaCl, 5 mM EDTA,
and the following protease inhibitors: 1 mM
phenylmethylsulfonyl fluoride and 10 µg/ml each aprotinin, pepstatin
A, and leupeptin. The supernatant of the whole-cell extract was
prepared by sedimenting the insoluble material by centrifugation at
15,000 rpm for 15 min. After SDS-polyacrylamide gel electrophoresis,
proteins were transferred onto an Immobilon-P membrane (Millipore
Corp., Bedford, MA). Immunoblotting was performed using enhanced
chemiluminescent Western blotting detection reagents (Amersham
Pharmacia Biotech).
Equilibrium Sucrose Density Gradient--
SLMVs and dense-core
granules were separated according to the method described previously
(12), with modifications. Briefly, MIN6 cells (1-2 × 107 cells/10-cm plate) were resuspended in 1.5 ml of
homogenization buffer (0.3 M sucrose, 20 mM
MOPS, pH 7.0, and the protease inhibitors described above) and
homogenized by 25 strokes with a Dounce glass homogenizer. The
homogenate was centrifuged at 2500 × g for 15 min at
4 °C. The resultant post-nuclear supernatant was loaded on top of a
continuous sucrose density gradient (0.45-2 M sucrose and
20 mM MOPS, pH 7.0) and then centrifuged at 30,000 rpm for 15 h at 4 °C in an RPS40T rotor (Hitachi Koki, Katsuta, Japan). Fractions (870 µl) were collected from the bottom of the gradient. The sucrose molarity of each fraction was determined by measuring its
refractive index. Fractions were analyzed by SDS-polyacrylamide gel
electrophoresis, followed by immunoblotting. Immunoreactive insulin was
extracted with acidic ethanol (sample/ethanol/concentrated HCl = 28.5:70:1.5) overnight at 4 °C. After sonication and centrifugation, the insulin was measured using anti-insulin antibodies as described previously (16).
Immunoprecipitation of Secretory Vesicles--
This was
performed according to the method described previously (19). Briefly,
the post-nuclear supernatant was prepared from five confluent 10-cm
plates of MIN6 cells as described above with the addition of
resuspension in 3 ml of homogenization buffer (0.3 M
sucrose, 10 mM HEPES, pH 7.2, 1.5 mM
MgCl2, 1% bovine serum albumin, and the protease
inhibitors described above). A portion of the supernatant (0.6 ml) was
incubated with the indicated antibodies at 4 °C for 2 h. The
antibodies were then bound to protein G-Sepharose 4FF (Amersham
Pharmacia Biotech) at 4 °C for 1 h with gentle rotation. The
immunoprecipitates were washed four times with the homogenization buffer. Tricine/SDS-polyacrylamide gel electrophoresis (20) was used to
detect small peptides such as insulin, and proteins were transferred
onto an Immobilon-PSQ membrane (Millipore Corp.).
Immunoblotting was then performed using anti-insulin antibodies.
Production of Glutathione S-Transferase (GST)-fused Recombinant
Proteins--
Two cDNA fragments of granuphilin-a encoding amino
acids 315-502 and 487-673 that contained the first and second
C2-domains, respectively, were amplified by PCR using a
full-length granuphilin-a cDNA as a template. Amplified cDNA
fragments were subcloned into pGEX4T-1 (Amersham Pharmacia Biotech). A
cDNA fragment encoding the first C2-domain of rat
synaptotagmin I (amino acids 139-267), synthesized by RT-PCR using rat
brain RNA as a template, was similarly fused to GST (21). GST fusion
proteins were expressed in Escherichia coli BL21 and
purified by affinity chromatography with glutathione-Sepharose 4B
(Amersham Pharmacia Biotech).
Phospholipid Binding Assay--
Preparation of
3H-labeled liposomes and measurement of phospholipid
binding were performed according to the methods described previously
(21). All phospholipids were purchased from Avanti Polar Lipids
(Alabaster, AL). Liposomes were made from 1.25 mg of
phosphatidylcholine, 0.42 mg of phosphatidylserine, and 20 µCi of
1,2-dipalmitoyl-L-3-phosphatidyl[N-methyl-3H]choline
(Amersham Pharmacia Biotech). When indicated, phosphatidylserine was
replaced by either phosphatidylinositol or phosphatidylethanolamine. Phospholipids dissolved in chloroform were mixed and dried under a
stream of nitrogen gas. After resuspension in 5 ml of buffer A (50 mM HEPES, pH 7.4, and 0.1 M NaCl) by vigorous
shaking for 1 min, phospholipid suspensions were sonicated for 15 s and centrifuged at 10,000 × g for 10 min to remove
aggregates. The supernatant was transferred to a new tube; 5 ml of
buffer A was added; and then it was stored at 4 °C for as long as 1 month.
The phospholipid binding assay contained 15 µg of recombinant protein
bound to 10 µl of glutathione beads. Beads were prewashed twice with
the respective test solutions and resuspended in 0.1 ml of buffer A
containing 3H-labeled liposomes (~0.1 µCi/16.7 µg of
phospholipid) and 2 mM EGTA with or without the addition of
2.1 mM CaCl2. The mixture was incubated at room
temperature for 10 min with vigorous shaking and then briefly
centrifuged at 2200 rpm in a tabletop centrifuge. The beads were washed
three times with 1 ml of the incubation buffer without liposomes. After
eluting the proteins with 250 µl of buffer containing 1% SDS and 10 mM EDTA, liposome binding was quantified by liquid
scintillation counting.
Molecular Cloning of a Novel Rabphilin-3-like Gene--
Although
the pancreatic alpha and beta cells are developmentally very close,
they have an opposite role in maintaining glucose homeostasis: the
alpha cells secrete glucagon in response to a low glucose level, and
the beta cells increase insulin secretion in response to a high glucose
level. Therefore, there must exist fundamental differences in their
glucose recognition and metabolism and in their regulation of peptide
hormone secretion. A comparison of the genes expressed in the two cells
may reveal the molecules involved in these processes. It is difficult
to purify considerable amounts of these cells from islets, and so we
instead used cultured alpha and beta cell lines (
Molecular cloning of the full-length cDNAs revealed one novel gene,
granuphilin, which encodes a protein whose domain structure is similar
to that of rabphilin-3 (6). Two isoforms were found among the multiple
independent clones in the
Rabphilin-3 is specifically localized on the synaptic vesicles at the
nerve terminals and is associated with Rab3A through the N-terminal
fragment containing the zinc-finger motif (14, 22). Although the amino
acid conservation in the N-terminal regions of granuphilin and
rabphilin-3 is only 31%, the distribution of cysteine residues in
granuphilin (Fig. 1, boxed),
CX2CX12-13CX2CX4CX2CX10-18CX2C, is similar to that in rabphilin-3 (6), RIM (8), and FGD1 protein (23),
all of which bind small G proteins. Crystal structural analysis of the
Rab3A·rabphilin-3 complex has revealed that a deep pocket in Rab3A,
called the Rab complementarity-determining region, directly interacts
with an SGAWFF motif in rabphilin-3 (24). Although the SGAWFF motif is
conserved among other members of the rabphilin-3 family, including RIM
(8) and Noc2 (25), the corresponding sequence of granuphilin is
distinct, TGDWFY (Fig. 1, boxed). Like rabphilin-3, there is
a single stretch of glutamate residues between the zinc-finger motif
and the first C2-domain, although its functional role
remains unknown (Fig. 1, dashed underlining). The primary
sequences within the C2-domains of granuphilin and
rabphilin-3 also vary considerably (31% conservation). Particularly,
the five aspartate residues participating in Ca2+ binding
in the C2A-domain of synaptotagmin I (5) are only partially
conserved in the C2-domains of granuphilin (Fig. 1, asterisks).
Preferential Expression of Granuphilin in Pancreatic Beta
Cells--
Tissue expression of granuphilin was examined. Northern
blot analysis revealed that it was not expressed in major tissues such
as the brain, kidney, liver, heart, muscle, and spleen (Fig. 2A). As expected from our
finding using mRNA differential display (data not shown), it was
specifically expressed in
A polyclonal antibody was produced against the C-terminal peptide of
granuphilin-a. In this study, the larger isoform, granuphilin-a, was
further characterized using this antibody. Immunoblot analysis showed
that the antibody recognized a single 78-kDa protein (the value of
which is consistent with the calculated molecular mass of
granuphilin-a) in the extracts of
Northern blot analysis in cultured cell lines suggests that granuphilin
is expressed in pancreatic beta cells, but not in alpha cells.
Transformed cell lines, however, may acquire properties different from
those of the original progenitors, as in the case of the insulin
receptor-related receptor, which is expressed in pancreatic alpha
cells, but not in Association of Granuphilin-a with Insulin-containing Dense-core
Granules--
Next, the subcellular localization of granuphilin-a was
biochemically addressed. Because of the difficulty in obtaining large amounts of materials from pancreatic islets and because of the heterogeneous cell components in the islets, biochemical studies were
performed using the beta cell lines
To obtain further evidence for the association of granuphilin-a with
dense-core granules, intact secretory vesicles were incubated with
anti-granuphilin-a antibodies. The immunoprecipitates were then
examined for the presence of insulin immunoreactivity. Granuphilin-a immunoprecipitates contained both insulin and proinsulin (Fig. 4B, lane 3). The association is specific because
neither control IgG nor anti-synaptophysin precipitates contained
detectable insulin immunoreactivity (Fig. 4B, lanes
1 and 5). On the other hand, anti-synaptotagmin III and
anti-VAMP-2 precipitates possessed insulin and proinsulin (Fig.
4B, lanes 2 and 4). Synaptotagmin III
is localized in the insulin granules (19), whereas VAMP-2 exists in
both granules and SLMVs in beta cells (31). These results indicate an
association of granuphilin-a with insulin-containing dense-core
granules in beta cells.
Ca2+ and Phospholipid Binding of Granuphilin--
As
described above, the primary sequences of the C2-domains of
granuphilin varied from those of the C2A-domain of
synaptotagmin I (Fig. 1), including the amino acids corresponding to
the five aspartate residues that participate in Ca2+
binding (5). In addition, the granuphilin-b isoform lacks the second
C2-domain. To determine the biochemical property of each
C2-domain of granuphilin, recombinant proteins fused to GST were produced in E. coli (Fig.
5A). A recombinant protein
consisting of the first C2-domain of rat synaptotagmin I
(GST-SytC2A) was similarly produced as a positive control.
GST-SytC2A showed Ca2+-dependent
binding of the 3H-labeled liposomes (Fig. 5B).
Binding was detected only in liposomes containing negatively charged
phospholipids such as phosphatidylserine and phosphatidylinositol, but
not in those containing phosphatidylethanolamine, as described
previously (21). A protein derived from the first C2-domain
of granuphilin (GST-GrpC2A) bound the
3H-labeled liposomes, but in a Ca2+-independent
manner (Fig. 5B). The binding is specific because it also
required negatively charged phospholipids. Surprisingly, a recombinant
protein from the second C2-domain of granuphilin (GST-GrpC2B) did not have significant affinity for the
phospholipid (Fig. 5B). Although it could be that the
protein does not have a proper conformation, a similar construction
used for each C2-domain suggests that the
C2B-domain of granuphilin does not bind phospholipids and
has a distinct role other than membrane binding.
In this study, we have identified and characterized a novel
rabphilin-3-like gene, granuphilin. Granuphilin and rabphilin-3 share
several sequence characteristics: an N-terminal zinc-finger domain,
C-terminal C2-domains, and a glutamate stretch between them. Granuphilin, however, has several properties distinct from those
of rabphilin-3; the tissue distribution pattern and subcellular localization are particularly different. Rabphilin-3 is specifically expressed in brain and adrenal gland tissues and is associated with
SSVs at nerve terminals. On the other hand, granuphilin is selectively
expressed in pancreatic beta cells and pituitary tissue. It is not
significantly expressed in the brain, pancreatic exocrine cells, or
other endocrine tissues such as pancreatic alpha cells, adrenal gland,
testis, and ovary. It is associated with dense-core granules, but not
SLMVs, in pancreatic beta cells. So far, no concomitant expression of
granuphilin and rabphilin-3 has been found in tissues, suggesting that
each protein is separately differentiated based on the properties of
its associated secretory vesicles. To our knowledge, granuphilin is the
first protein strictly associated with the membrane of dense-core
granules in endocrine cells. Synaptotagmin III is expressed in
pancreatic islets at low levels and associates with insulin-containing
granules in MIN6 cells, but is abundantly expressed in brain tissue
(19, 32). Noc2, which has a zinc-finger motif but no
C2-domain, is expressed only in endocrine tissues, but is
not associated with insulin-containing granules in MIN6 cells (25).
The distribution pattern of granuphilin-a suggests that its function is
restricted to those dense-core granules that contain a polypeptide
hormone in endocrine cells. Although it is well known that SSVs and
LDCVs differ in the membrane protein constituents of their neurons (9),
little is known about the difference of dense-core granules in
endocrine cells. Because it is relatively easy to obtain the materials,
most biochemical studies of endocrine cells have been performed on
adrenal chromaffin cells or on their derivative PC12 cell lines, whose
granules contain monoamines. These granules, however, may have
characteristics more closely resembling SSVs. For example, in
chromaffin and PC12 cells, synaptophysin exists in both SLMVs and
granules (33, 34), whereas in pancreatic beta cells, it is not present
in the insulin granules, but is restricted to the SLMVs (12, 19, 31).
Rabphilin-3 associates with SSVs in neurons (14) and with granules in
chromaffin cells (35), but is not expressed in beta cells (27).
Granuphilin may have a role for the regulated exocytosis of a specific
type of dense-core gran-ules in endocrine tissues. The reason for the differential expression of granuphilin between pancreatic alpha and
beta cells is currently unknown. It is possible that it reflects the
different regulatory mechanism for hormone secretion in response to
glucose. It will also be important to determine which cells in the
pituitary, another multicellular endocrine tissue, express granuphilin.
Despite the similarity of the domain structure, the primary amino acid
sequences of granuphilin and rabphilin-3 vary considerably. The five
aspartate residues participating in Ca2+ binding in the
C2A-domain of synaptotagmin I are mostly conserved in
synaptotagmins, rabphilin-3, and Doc2 (5), but not in either RIM (8) or
granuphilin. This sequence feature is consistent with the
Ca2+ independence of the phospholipid binding in the first
C2-domain of granuphilin-a. A protein consisting of the
second C2-domain had no affinity for phospholipids,
suggesting that this second C2-domain may have a functional
role distinct from membrane binding, possibly relevant to differential
roles between the two isoforms. Further characterization of
granuphilin-b, which lacks the second C2-domain, will
require antibodies specific to this isoform.
Rabphilin-3 is bound to Rab3A/C through the N-terminal region (14, 22).
All known members of Rab3 (A, B, C, and D) are expressed in rat
pancreatic islets and beta cell lines (27). Because rabphilin-3 is not
expressed in these cells (27), granuphilin may interact with Rab3 in
place of rabphilin-3. We could not detect, however, such an
interaction, at least with Rab3A, in MIN6
cells.3 Furthermore, the
SGAWFF motif in rabphilin-3, which has a crucial role in the
interaction with Rab3A (24), is not conserved in granuphilin. Thus, it
is likely that granuphilin has different Rab partners.
In summary, we have identified a novel rabphilin-3-like protein,
granuphilin, that is specifically associated with dense-core granules
in pancreatic beta cells. Although the biological function of the
protein remains unknown, it has several functional domains and
biochemical properties that are both common to and distinct from other
proteins thought to be involved in the regulatory secretion of synaptic
vesicles. The primary amino acid sequences within these domains of
granuphilin and rabphilin-3 vary considerably. These domains interact
with specific sets of other molecules and work as functional modules
(5), suggesting that granuphilin interacts with a different set of
molecules compared with rabphilin-3. By identifying these interacting
molecules, we may gain an understanding of the biological function of granuphilin.
We thank Dr. M. Hosaka (Institute for
Molecular and Cellular Regulation, Gunma University) for useful
suggestions and critical reading of the manuscript, Dr. M. Takahashi
for anti-VAMP-2 and anti-synaptotagmin III antibodies, and Drs. K. Wakabayashi and H. Kobayashi for anti-porcine insulin antibodies.
*
This work was supported by a grant-in-aid for scientific
research from the Ministry of Education, Science, Sports, and Culture of Japan and in part by the Ministry of Health and Welfare of Japan
(Research on Human Genome and Gene Therapy) and by grants from the
Japan Diabetes Foundation and the Tanabe Medical Frontier Conference
(to T. I.).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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB005756, AB025258, and AB025259.
2
J. Wang, T. Takeuchi, I. Hirayama, K. Hamaguchi,
and T. Izumi, unpublished observations.
3
J. Wang, Z. Yi, and T. Izumi, unpublished observations.
The abbreviations used are:
SSV, small synaptic
vesicle;
SLMV, synaptic-like microvesicle;
LDCV, large dense-core
vesicle;
RT-PCR, reverse transcription-polymerase chain reaction;
bp, base pair(s);
VAMP, vesicle-associated membrane protein;
MOPS, 3-(N-morpholino)propanesulfonic acid;
Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine;
GST, glutathione S-transferase.
Novel Rabphilin-3-like Protein Associates with Insulin-containing
Granules in Pancreatic Beta Cells*
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-latrotoxin, the toxin of black widow spider venom,
which does not affect the exocytosis of calcitonin gene-related
peptide-containing LDCVs (11). Thus, the secretory pathways of
SSVs/SLMVs and LDCVs/granules are different. It is also likely that
their protein compositions are at least partly distinct; however, due
to their low abundance, there has been less biochemical
characterization of LDCVs/granules.
-aminobutyric acid-containing SLMVs (12), they are a good model for dissecting both
types of secretory vesicles. The limited number and anatomical position
of beta cells, however, have hampered their biochemical analysis.
Therefore, we attempted molecular characterization by analyzing the
genes expressed in the cells. The expression levels of beta cell genes
were then compared with those in closely related pancreatic alpha cells
by mRNA differential display (13), which resulted in the
identification of several novel genes that are preferentially expressed
in beta cells.2 In this
study, one of these genes, granuphilin, was characterized. This gene
encodes a protein whose domain structure is similar to that of
rabphilin-3. Rabphilin-3 is specifically expressed in neurons, contains
two C2-domains, and is thought to be involved in the
regulated secretion of SSVs (6, 14). Granuphilin, however, has a number
of distinct characteristics compared with rabphilin-3: it is
specifically expressed in pancreatic beta cells and in the pituitary,
but not in other major organs such as the brain. Furthermore, it is
associated with insulin-containing dense-core granules, but not with
the SLMVs in beta cells. In addition to a difference in the
distribution, other distinctive properties of granuphilin suggest that
it is not a simple counterpart of rabphilin-3 in endocrine cells and
that it has a unique role in the regulated exocytosis of dense-core
granules in endocrine tissues.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
HC9 and MIN6) and an alpha cell line (TC1.6) were cultured
in Dulbecco's modified Eagle's medium supplemented with 10% fetal
calf serum under 5% CO2 atmosphere at 37 °C as
described previously (15). Tissues were immediately excised from
8-12-week-old C57BL/6J mice killed by cervical dislocation, frozen in
a liquid nitrogen bath, and stored at 
80 °C. Pancreatic islets
were isolated as described previously (16).
-32P]dCTP (1000-3000 Ci/mmol; Amersham
Pharmacia Biotech) using the Rediprime DNA labeling system (Amersham
Pharmacia Biotech), followed by centrifugation through Sephadex G-50
spin columns to remove unincorporated dNTPs.
HC9 cells were compared with those in TC1.6
cells by mRNA differential display (13). Using one of the cDNA
fragments preferentially expressed in HC9 cells as a probe
(DDBJ/GenBankTM/EBI accession number AB005756), granuphilin
cDNAs were cloned from a HC9/MIN6 cell cDNA library (15).
Sequencing was performed using an ABI PRISM cycle sequencing kit
(Perkin-Elmer).
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
TC1.6 and HC9)
derived from transgenic mice. Using mRNA differential display (13),
we identified several novel genes that are specifically or
preferentially expressed in HC9 cells.2
HC9/MIN6 cDNA library. One
(granuphilin-a) encodes a protein of 673 amino acids that has a
zinc-finger motif at the N terminus and two C2-domains at the C terminus (Fig. 1). The other
(granuphilin-b) lacks 418 nucleotides (from positions 1641 to 2058) of
granuphilin-a, which causes a frameshift at residue 486, located
between the two C2-domains of granuphilin-a, and generates
a stop codon at residue 503. Thus, granuphilin-b is a protein of 502 amino acids and has only the first C2-domain of
granuphilin-a and a different C-terminal sequence, GSVMAKWWTGWIRLVKK
(Fig. 1). The calculated molecular mass and isoelectric point for
granuphilin-a are 76,000 Da and 9.2, and those for granuphilin-b are
57,000 Da and 9.2, respectively. Despite the similarity of the domain
structure, the overall identity of the amino acid sequences of
granuphilin-a and rabphilin-3 only amounts to 22%.
View larger version (49K):
[in a new window]
Fig. 1.
Amino acid sequence of granuphilin. The
amino acid sequence of mouse (m) granuphilin is aligned with
that of mouse rabphilin-3 (DDBJ/GenBankTM/EBI accession
number D29965) (26). Dashes indicate individual amino acid
gaps to generate optimal alignment. Two isoforms, granuphilin-a and -b,
are found. Granuphilin-b lacks 418 bp of the internal sequence of
granuphilin-a. This causes a frameshift at residue 486, which generates
a 17-amino acid different sequence, followed by a stop codon. Thus,
granuphilin-b contains only the first C2-domain. Conserved
cysteine residues in the zinc-finger motifs are boxed. An
SGAWFF structural element of rabphilin-3 (24) and the corresponding
sequence of granuphilin are also boxed. The
C2-domains are underlined. The amino acids
corresponding to the five aspartate residues participating in the
Ca2+ binding of synaptotagmin I C2A-domains
(5) are indicated by asterisks. Consecutive stretches of
glutamate residues found between the zinc-finger motif and the
C2-domains are indicated by dashed underlining.
These sequence data are available from DDBJ/GenBankTM/EBI
under accession numbers AB025258 (granuphilin-a) and AB025259
(granuphilin-b).
HC9 cells, but not in TC1.6 cells. The
double bands of signals in HC9 cells suggest that both granuphilin-a
and -b are transcribed. This interpretation was further supported by
RT-PCR analysis. The PCR products consisted of two discrete bands,
whose sizes correspond to those expected for the two isoforms, in the
pancreatic islets, HC9 cells, and MIN6 cells (Fig. 2B,
lanes 2-4 and 7-9). In contrast, they were not
found in either TC1.6 cells or brain (Fig. 2B,
lanes 1, 5, 6, and 10).
These results indicate that both isoforms are expressed in pancreatic
beta cells.
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Fig. 2.
Tissue expression of granuphilin.
A, 20 µg of total RNA from each tissue and cell was
electrophoresed, transferred to a nylon membrane, and hybridized with a
granuphilin cDNA probe that was derived from a common sequence in
the two isoforms (nucleotides 677-1198). Arrowheads
indicate the positions of 28 S (4.7 kilobases) and 18 S (1.9 kilobases)
rRNAs. Note that the signal in the HC9 cells is a doublet
(arrows), reflecting expression of both isoforms.
B, RT-PCR analysis was performed on RNA from TC1.6 cells
(lanes 1 and 6), HC9 cells (lanes 2 and 7), MIN6 cells (lanes 3 and 8),
mouse pancreatic islets (lanes 4 and 9), and
brain (lanes 5 and 10) using two sets of primers
derived from sequences flanking the granuphilin-a-specific region. The
expected sizes of PCR products using one set of primers are 936 bp for
granuphilin-a and 518 bp for granuphilin-b (lanes 1-5), and
those using the other set are 588 bp for granuphilin-a and 170 bp for
granuphilin-b (lanes 6-10). Note that both beta cell lines
and islets specifically express two isoforms. The first and
last lanes show a 100-bp DNA ladder and
X174/HinfI-digested DNA markers, respectively.
C, an equal amount of protein (100 µg) from the whole-cell
extract of each tissue and cell was loaded onto a 10% polyacrylamide
gel. Immunoblotting was performed using anti-granuphilin-a
antibodies.
HC9 cells (Fig. 2C) and MIN6 cells (data not shown, but see Fig. 4A). Consistent
with the finding from the Northern blot analysis (Fig. 2A),
there were no immunoreactive proteins in either TC1.6 cells or other
major organs (Fig. 2C). Among the endocrine tissues
examined, granuphilin-a was most abundantly expressed in pancreatic
islets. It was also expressed in the pituitary, but was not
significantly expressed in the testis, ovary, and adrenal gland. This
expression pattern is in striking contrast to that of rabphilin-3,
which is specifically expressed in the brain, but is not expressed in
endocrine tissues, except for the adrenal gland (6, 26, 27).
TC1.6 cells (15). Therefore, we performed
immunohistochemical analysis directly on the mouse pancreas specimen.
Granuphilin-a was highly expressed in the insulin-positive beta cells
(Fig. 3, a, c, and
e). In contrast, granuphilin-a immunoreactivity was not
detected in either alpha or exocrine cells (Fig. 3, b, d, and f).
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[in a new window]
Fig. 3.
Immunofluorescent detection of granuphilin-a
in the pancreas. The pancreases of C57BL/6J mice (12-week-old
females) were immunostained with anti-insulin (a),
anti-glucagon (b), and anti-granuphilin-a (c and
d) antibodies. e and f are images of
double staining of a and c and b and
d, respectively. Note that granuphilin-a is specifically
expressed in beta cells, but not in alpha or acinar cells.
Bar, 50 µm.
HC9 and MIN6. Both cell lines
were established from transgenic mice expressing simian virus 40 large
T antigen under the control of the rat insulin promoter, and they
preserve some distinctive characteristics of the normal progenitors,
such as glucose-inducible insulin secretion (28, 29). MIN6 cells were
mainly used for biochemical analysis in this study because they contain
a larger number of insulin granules and have a higher insulin content
than do HC9 cells (30). MIN6 cells were homogenized and then
fractionated on continuous sucrose gradients. Similar procedures have
permitted the separation of the two types of secretory vesicles present
in beta cells: insulin-containing dense-core granules and
-aminobutyric acid-containing SLMVs (12). Insulin immunoreactivity
peaked at fraction 10 (Fig. 4A, upper panel),
whereas synaptophysin was enriched at fractions 6 and 7 (lower
panel). Synaptophysin, an integral membrane protein of SSVs in
neurons, is absent in the insulin granules, but present in the SLMVs in
beta cells (12, 19, 31). Thus, the two types of secretory vesicles were
clearly separated. Granuphilin-a immunoreactivity showed two peaks,
fractions 2-4 and fractions 9 and 10 (Fig. 4A, middle
panel). These results suggest that a portion of granuphilin-a associates with the insulin granules. The two separate fractions of
granuphilin-a may reflect its different functional states.
View larger version (31K):
[in a new window]
Fig. 4.
Subcellular localization of
granuphilin-a. A, a post-nuclear supernatant of MIN6
cells was separated on a continuous sucrose density gradient. Fourteen
fractions were collected, and the sucrose molarity and the
immunoreactive insulin in a portion of each fraction were measured
(upper panel). Equal volumes of the fractions were analyzed
by SDS-polyacrylamide gel electrophoresis (10% polyacrylamide gel),
followed by immunoblotting using antibodies against granuphilin-a
(78-kDa protein; middle panel) and synaptophysin (36-38-kDa
protein; lower panel). B, intact vesicles from
MIN6 cells were incubated with control IgG (lane 1),
anti-synaptotagmin III (lane 2), anti-granuphilin-a
(lane 3), anti-VAMP-2 (lane 4), or
anti-synaptophysin (lane 5) antibodies, followed by protein
G-Sepharose. After washing the beads, immunoprecipitates were analyzed
by immunoblotting using anti-insulin antibodies. Human proinsulin
(lane 6) and bovine insulin (lane 7) were loaded
as standards. Although it is not clear in this figure, longer exposure
revealed that the immunoprecipitates with anti-synaptotagmin III and
anti-granuphilin-a antibodies contained proinsulin as well as insulin.
Furthermore, immunoblotting using anti-C-peptide antibodies showed that
the immunoprecipitates with anti-synaptotagmin III, anti-granuphilin-a,
or anti-VAMP-2 antibodies contained proinsulin, whereas those with
anti-synaptophysin antibodies did not (data not shown).
View larger version (20K):
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Fig. 5.
Phospholipid binding of granuphilin.
A, recombinant proteins were produced in E. coli
and purified with glutathione-Sepharose 4B. GST-GrpC2A
(lane 1), GST-GrpC2B (lane 2), GST
(lane 3), and GST-SytC2A (lane 4)
were electrophoresed on a 10% polyacrylamide gel and stained with
Coomassie Blue. B, assays for
Ca2+-dependent phospholipid binding to
recombinant proteins were performed. GST, GST-SytC2A,
GST-GrpC2A, and GST-GrpC2B bound to
glutathione-Sepharose 4B were incubated with 3H-labeled
liposomes in the absence (open bars) or presence
(closed bars) of Ca2+. 3H-Labeled
liposomes were composed of phosphatidylcholine (PC) mixed
with phosphatidylserine (PS), phosphatidylinositol
(PI), or phosphatidylethanolamine (PE).
Phospholipid binding was measured by scintillation counting of the
beads after extensive washing. Error bars indicate
S.D.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
To whom correspondence should be addressed: Dept. of Molecular
Medicine, Inst. for Molecular and Cellular Regulation, Gunma University, 3-39-15 Showa-machi, Maebashi, Gunma 371-8512, Japan. Tel.:
81-27-220-8856; Fax: 81-27-220-8896; E-mail:
tizumi@akagi.sb.gunma- u.ac.jp.
ABBREVIATIONS
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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