From the Mass Spectrometry Resource, Division of Endocrinology,
Diabetes, and Metabolism, Department of Medicine, Washington University
School of Medicine, St. Louis, Missouri 63110
Received for publication, November 16, 2000, and in revised form, January 2, 2001
A cytosolic 84-kDa group VIA phospholipase
A2 (iPLA2
) that does not require
Ca2+ for catalysis has been cloned from several sources,
including rat and human pancreatic islet
-cells and murine P388D1
cells. Many potential iPLA2
functions have been
proposed, including a signaling role in
-cell insulin secretion and
a role in generating lysophosphatidylcholine acceptors for arachidonic
acid incorporation into P388D1 cell phosphatidylcholine (PC). Proposals
for iPLA2
function rest in part on effects of inhibiting
iPLA2
activity with a bromoenol lactone (BEL) suicide
substrate, but BEL also inhibits phosphatidate phosphohydrolase-1 and a
group VIB phospholipase A2. Manipulation of
iPLA2
expression by molecular biologic means is an
alternative approach to study iPLA2
functions, and we
have used a retroviral construct containing iPLA2
cDNA to prepare two INS-1 insulinoma cell clonal lines that stably
overexpress iPLA2
. Compared with parental INS-1 cells or
cells transfected with empty vector, both
iPLA2
-overexpressing lines exhibit amplified insulin
secretory responses to glucose and cAMP-elevating agents, and BEL
substantially attenuates stimulated secretion. Electrospray ionization
mass spectrometric analyses of arachidonic acid incorporation into
INS-1 cell PC indicate that neither overexpression nor inhibition of
iPLA2
affects the rate or extent of this process in
INS-1 cells. Immunocytofluorescence studies with antibodies directed against iPLA2
indicate that cAMP-elevating agents
increase perinuclear fluorescence in INS-1 cells, suggesting that
iPLA2
associates with nuclei. These studies are more
consistent with a signaling than with a housekeeping role for
iPLA2
in insulin-secreting
-cells.
 |
INTRODUCTION |
Phospholipases A2
(PLA2)1 catalyze
hydrolysis of the sn-2 fatty acid substituent from
glycerophospholipid substrates to yield a free fatty acid and a
2-lysophospholipid (1-7). PLA2 are a diverse group of
enzymes, and the first members to be well characterized have low
molecular masses (~14 kDa), require millimolar [Ca2+]
for catalytic activity, and function as extracellular secreted enzymes
designated sPLA2 (3, 6). The first PLA2 to be
cloned that is active at [Ca2+] that can be achieved in
the cytosol of living cells is an 85-kDa protein classified as a group
IV PLA2 and designated cPLA2 (3, 5). This
enzyme is induced to associate with its substrates in membranes by
rises in cytosolic [Ca2+] within the range achieved in
cells stimulated by extracellular signals that induce Ca2+
release from intracellular sequestration sites or Ca2+
entry from the extracellular space, is also regulated by
phosphorylation, and prefers substrates with sn-2
arachidonoyl residues (5).
A second cytosolic PLA2 has been cloned (8-10) that does
not require Ca2+ for catalysis, and it is classified as a
group VIA PLA2 and has been designated iPLA2
(3, 4). The iPLA2 enzymes cloned from hamster (8), mouse
(9), and rat (10) cells represent species homologs, and all are 84-kDa
proteins containing 752 amino acid residues with highly homologous
sequences. Each contains a GXSXG lipase consensus
motif and eight stretches of a repeating motif homologous to a
repetitive motif in the integral membrane protein-binding domain of
ankyrin (8-10). Each of these iPLA2 enzymes is susceptible to inhibition (8-10) by a bromoenol lactone (BEL) suicide substrate (11, 12) that is not an effective inhibitor of sPLA2 or
cPLA2 enzymes at comparable concentrations (4, 11-14). It
has been proposed that this enzyme now be designated
iPLA2
to distinguish it from a membrane-associated,
Ca2+-independent PLA2 that contains a
peroxisomal targeting sequence and is designated iPLA2
(15, 16).
Proposed functions for iPLA2
include a housekeeping role
in phospholipid remodeling that involves generation of lysophospholipid acceptors for incorporation of arachidonic acid into phospholipids of
murine P388D1 macrophage-like cells (4, 17, 18). This proposal (4)
derives from experiments involving inhibition of iPLA2
activity in P388D1 cells with BEL (17) or with an antisense oligonucleotide (18). Inhibition of P388D1 cell iPLA2
activity suppresses incorporation of [3H]arachidonic acid
into phospholipids and reduces
[3H]lysophosphatidylcholine (LPC) levels in
[3H]choline-labeled cells (17, 18). Arachidonate
incorporation (17, 18) reflects a deacylation/reacylation cycle (19,
20) of phospholipid remodeling rather than de novo synthesis
(21), and the level of LPC acceptors is thought to limit the rate of [3H]arachidonic acid incorporation into P388D1 cell
phosphatidylcholine (PC) (17, 18).
Many other potential iPLA2
functions have been proposed
(22-54), and the facts that multiple splice variants are
differentially expressed among cells and form hetero-oligomers with
distinct properties suggest that iPLA2 gene products might
have multiple functions (23, 31, 44, 45). Proposed iPLA2
functions include signaling in secretion (10, 22, 25, 29, 48-50), and
we and others (47-54) have found that, in pancreatic islets, BEL
attenuates glucose-induced insulin secretion, arachidonate release, and
rises in islet
-cell cytosolic [Ca2+]. Both pancreatic
islets and brain contain electrically active secretory cells that
express high levels of iPLA2
(10), and iPLA2
is the vastly predominant brain cytosolic
PLA2 (24, 34, 35). BEL also inhibits iPLA2
(15) and phosphatidate phosphohydrolase-1 (PAPH-1) (55), and the
ambiguity of pharmacologic studies makes manipulating
iPLA2
expression by molecular biologic means an attractive alternative to study iPLA2
functions. We
report here the preparation of two stably transfected insulinoma cell
lines that overexpress iPLA2
. We have studied insulin
secretory responses, arachidonate incorporation into
phosphatidylcholine, and iPLA2
subcellular location in
these lines.
 |
EXPERIMENTAL PROCEDURES |
Materials--
ECL detection reagents and
1-palmitoyl-2-[14C]linoleoyl-sn-glycero-3-phosphocholine
(55 mCi/mmol) were purchased from Amersham Pharmacia Biotech.
Phosphatidylcholine standards were obtained from Avanti Polar Lipids
(Birmingham, AL) and arachidonic acid from Nu-Chek Prep (Elysian, MN).
BEL iPLA2 suicide substrate
(E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one was purchased from Cayman Chemical (Ann Arbor, MI). Tissue culture media (CMRL-1066, RPMI, and minimal essential medium), penicillin, streptomycin, Hanks' balanced salt solution, and
L-glutamine were purchased from Life Technologies, Inc.
Fetal bovine serum was obtained from HyClone (Logan, UT) and Pentex
bovine serum albumin (BSA, fatty acid-free, fraction V) from ICN
Biomedical (Aurora, OH). ATP, ampicillin, IBMX, propranolol, and
kanamycin were obtained from Sigma and forskolin from Calbiochem (La
Jolla, CA). Krebs-Ringer bicarbonate buffer (KRB) contained 25 mM HEPES, pH 7.4, 115 mM NaCl, 24 mM NaHCO3, 5 mM KCl, 1 mM MgCl2.
Cell Culture--
INS-1 insulinoma cells provided by Dr.
Christopher Newgard (University of Texas, Dallas, TX) were cultured as
described (56-58) in RPMI 1640 medium containing 11 mM
glucose, 10% fetal calf serum, 10 mM Hepes buffer, 2 mM glutamine, 1 mM sodium pyruvate, 50 mM
-mercaptoethanol, 100 units/ml penicillin, and 100 µg/ml streptomycin. RetroPack PT 67 cells
(CLONTECH, Palo Alto, CA) were maintained in
Dulbecco's modified Eagle's medium (4.5 mg/ml glucose) containing 10% fetal bovine serum, 4 mM L-glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin.
Preparation of Recombinant Retrovirus Containing the cDNA
Encoding the Rat Pancreatic Islet iPLA2
--
A
retroviral system (59, 60) was used to stably transfect INS-1 cells
with iPLA2
cDNA and achieve overexpression. To construct the retroviral vector, iPLA2
cDNA (10) was
subcloned into EcoRI-BglII multiple cloning sites
of pMSCVneo vector using the CLONTECH murine stem
cell retrovirus (MSCV) expression system. Full-length rat pancreatic
islet iPLA2
cDNA was excised from pBK-CMV-iPLA2
vector and subcloned into the retroviral
vector pMSCVneo at the recognition sites for restriction endonucleases EcoRI and XhoI. The construct containing the
iPLA2
cDNA (pMSCVneo-iPLA2
) was
transfected into CLONTECH RetroPack PT 67 packaging
cells with a GenePORTER transfection system according to the
manufacturer's instructions (Gene Therapy Systems, San Diego, CA).
Upon transfection of packaging cells, pMSCVneo integrated into the
genome and expressed a transcript containing viral packaging signal, a
neomycin resistance gene that confers resistance to the selection agent
G418, and iPLA2
cDNA. This transcript is recognized
by viral proteins in packaging cells. Introduction of
pMSCVneo-iPLA2
into PT 67 cells results in production of
high titer, replication-incompetent infectious virus particles that
were released into the culture medium, collected, and used to infect
INS-1 cells.
Infection of INS-1 Cells with Recombinant Retroviral Particles
and Selection of Stably Transfected Cells That Overexpress
iPLA2
--
INS-1 cells were plated on 100-mm Petri
dishes at a density of 3-5 × 105 cells/plate 12-18
h before infection. Freshly collected, retrovirus-containing medium was
passed through a 0.45-µm filter and added to INS-1 cell monolayers.
Polybrene (final concentration 4 µg/ml) was added to culture medium,
and medium was replaced after 24 h of incubation. To select stably
transfected cells that expressed high levels of iPLA2
,
retrovirally infected cells were cultured with G418 (0.4 mg/ml) for
1-2 weeks. After G418-resistant colonies became apparent, cell culture
was continued for several days. Individual colonies were transferred to
a 48-well plate for expansion of clonal cells. Two
iPLA2
-overexpressing (iPLA2-X) lines were
obtained that exhibit similar properties not shared by parental cells
or clonal lines selected after transfection with empty vector.
Assay of INS-1 Cell iPLA2 Activity--
Seeded INS-1
were washed with phosphate-buffered saline (PBS) and detached by
trituration. Cells were collected by centrifugation and disrupted by
sonication (Vibra Cell High Intensity Processor, five 1-s pulses,
amplitude 12%) in homogenization buffer (250 mM sucrose,
40 mM Tris-HCI, pH 7.1, 4 °C). Homogenates were
centrifuged (15,000 × g, 45 min, 4 °C) to yield a
cytosolic supernatant. Protein content was measured with Coomassie
regent (Pierce) against bovine serum albumin standard.
Ca2+-independent PLA2 activity in aliquots of
cytosol (25 µg of protein) was assayed by ethanolic injection (5 µl) of
1-palmitoyl-2-[14C]linoleoyl-sn-glycero-3-phosphocholine
(final concentration 5 µM) in assay buffer (40 mM Tris, pH 7.5, 5 mM EGTA; total volume 200 µl). Assay mixtures were incubated (3 min, 37 °C, with shaking) and reactions terminated by adding butanol (0.1 ml) and vortexing. After centrifugation (2,000 × g, 5 min), products in
the butanol layer were analyzed by silica gel G TLC in petroleum
ether/ethyl ether/acetic acid (80/20/1). The TLC plate region
containing free fatty acid was identified with iodine vapor and scraped
into a scintillation vial. Released [14C]fatty acid was
measured by liquid scintillation spectrometry, and PLA2
specific activity was calculated from dpm of released fatty acid and
protein content as described (61).
Immunoblotting Analyses of INS-1 Cell iPLA2
Protein--
INS-1 cell cytosolic proteins were analyzed by SDS-PAGE
and transferred to a nylon membrane that was subsequently blocked (3 h,
room temperature) with Tris-buffered saline plus Tween (TBS-T, 20 mM Tris-HCl, 137 mM NaCl, pH 7.6, 0.05% Tween
20) containing 5% milk protein. The blot was then washed (TBS-T, 5 min, five times) and incubated (1 h, room temperature) with a
polyclonal antibody (1:2000 dilution in TBS-T) to iPLA2
generated by multiple antigen core technology against peptides in the
iPLA2
deduced amino acid sequence, as described below.
The nylon membrane was then washed in TBS-T (5 min, five times) and
incubated (1 h, room temperature) with a secondary antibody coupled to
horseradish peroxidase (Roche Molecular Biochemicals) at 1:40,000
dilution in TBS-T containing 3% BSA. The antibody complex was
visualized by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech).
Determination of Insulin Secretion by INS-1 Insulinoma
Cells--
Culture medium from INS-1 cells seeded in 24-well plates
was removed, and the cells were washed twice in KRB medium and
incubated (1 h, 37 °C, under an atmosphere of 95% air, 5%
CO2) in KRB medium (1 ml). Medium was then removed and
replaced with KRB medium containing glucose (0-18 mM) with
or without forskolin (2.5 µM), IBMX (100 µM), or dibutyryl cAMP (1 mM). In experiments
with BEL (10 µM) or propranolol (250 µM),
these agents were added both to preincubation medium and to medium for
the experimental incubation. After addition of final incubation medium,
cells were incubated (1 h, 37 °C) under the atmosphere described
above, and medium was then removed for measurement of insulin by
radioimmunoassay (62). Cells were then detached from the plate and
their acid-ethanol extractable insulin determined by radioimmunoassay
(63). Secreted insulin was expressed as a fraction of total cellular
insulin content (64).
Determination of INS-1 Cell cAMP Content--
After experimental
incubations, medium was removed from each well, and ice-cold ethanol
(0.4 ml) containing IBMX (100 µM) was added. After a
5-min room temperature incubation, cells were detached with a rubber
policeman, and ethanol and cells were placed in glass test tubes
(12 × 75 mm). Cells were sedimented by centrifugation (1500 × g, 10 min), and the supernatant was placed in a clean test tube, concentrated to dryness under nitrogen, and reconstituted in
50 mM phosphate buffer (0.4 ml). The cAMP content was
measured by enzyme immunoassay and normalized to cell protein
content (65-67).
Incubation of INS-1 Cells with Arachidonic Acid to Induce
Phospholipid Remodeling--
INS-1 cells were cultured in RPMI medium
containing penicillin, streptomycin, fungizone, and gentamicin (0.1%
w/v each). Cells (1.2 × 106/condition) were treated
(30 min, 37 °C) with vehicle only or with BEL (10 µM).
Medium was then removed and replaced with fresh medium containing no
supplements other than those described above or containing arachidonic
acid (final concentration 70 µM), and cells were cultured
at 37 °C. After 0, 2, 6, 8, or 24 h, cells were washed twice
with PBS, suspended in homogenization buffer, and disrupted by
sonication. Lipids were extracted (68) and the extract concentrated and
analyzed by NP-HPLC.
Chromatographic Analyses of Phospholipids--
Phospholipid
head-group classes were separated by NP-HPLC (47) analyses on a silicic
acid column (LiChrosphere Si-100 (10 µm, 250 × 4.5 mm);
Alltech, Deerfield, IL) with the solvent system hexane, 2-propanol, 25 mM potassium phosphate, pH 7.0, ethanol, acetic acid
(367/490/62/100/0.6) at a flow of 0.5 ml/min for 60 min and then 1.0 ml/min. The retention time of standard PC was 102 min.
Electrospray Ionization Mass Spectrometric Analyses of
Choline-containing Lipids--
PC species were analyzed as
Li+ adducts by ESI/MS on a Finnigan (San Jose, CA) TSQ-7000
triple stage quadrupole mass spectrometer with an ESI source controlled
by Finnigan ICIS software. Phospholipids were dissolved in
methanol/chloroform (9/1, v/v) containing LiOH (2 nmol/µl), infused
(1 µl/min) with a Harvard syringe pump, and analyzed under described
conditions (69, 70). For tandem MS, precursor ions selected in the
first quadrupole were accelerated (32-36 eV collision energy) into a
chamber containing argon (2.3-2.5 millitorr) to induce
collisional-activated dissociation, and product ions were analyzed in
the final quadrupole to identify PC species in the total ion current
profile (70).
Measurement of Nonesterified Arachidonic Acid in INS-1 Cells by
Isotope Dilution Gas Chromatography Negative Ion Electron Capture Mass
Spectrometry--
Parental and iPLA2
-overexpressing
INS-1 cells that had been incubated with supplemental arachidonic acid
for 24 h as described above were washed with 0.1% BSA in KRB four
times to remove unincorporated arachidonic acid and were then
preincubated for 30 min in KRB medium containing 10 µM
BEL or BEL-free vehicle. The precincubation medium was then removed,
and the cells were incubated for 1 h at 37 °C in KRB medium
containing 0.1% BSA, 2.5 mM CaCl2, and 2 or 11 mM glucose without or with IBMX (100 µM).
Incubations were terminated by extraction with 2 ml of
chloroform/methanol (1/1) containing 330 pmol of
[2H8]arachidonic acid internal standard.
Extracts were analyzed by RP-HPLC to isolate arachidonic acid, which
was converted to a pentafluorobenzyl ester derivative and analyzed by
GC/MS in negative ion electron capture mode (47). Selected monitoring of carboxylate anions of arachidonate (m/z 303) and
[2H8]arachidonate (m/z 311) was
performed to quantitate arachidonate by reference to a standard curve
(47). The amount of arachidonic acid was expressed as a ratio to the
lipid phosphorus content of the extract.
Immunocytofluorescence Localization of iPLA2
within INS-1 Cells--
We prepared an iPLA2
antibody
by multiple antigen core methods (Research Genetics) that link eight
peptide copies to an octameric lysine core (71). The
iPLA2
peptides coupled to this core for immunizing
rabbits were 25KEVSLADYASSERVRE41 and
489RMKDEVFRGSRPY501. In INS-1 cells that
overexpress iPLA2
, this antibody recognizes an 84-kDa
protein corresponding to full-length iPLA2
(Fig. 1), and
recognition of the protein is blocked by including the immunizing peptides in incubations with the antibody. To determine the subcellular location of iPLA2
, cells were allowed to attach to
chambered glass slides overnight and then treated with experimental
solutions. At the end of incubations, cells were rinsed in PBS, fixed
in 4% paraformaldehyde, washed with PBS, fixed in ice-cold methanol, washed, and blocked in a PBS solution containing globulin-free BSA
(1%), Triton X-100 (0.3%), and goat serum (3%). Primary antibodies (either preimmune immunoglobulin or anti-iPLA2
, 0.003 µg/ml) were then added, and cells were incubated (18 h, 4 °C) in a
humidified chamber. Cells were then washed and incubated (1 h, in the
dark) with a secondary antibody (affinity-purified goat anti-rabbit IgG, 1:200 dilution) coupled to the fluorophore Cy3. Cells were then
washed and covered with Antifade solution (Molecular Probes, Eugene,
OR). Slides were mounted with coverslips and examined by confocal
microscopy at excitation and emission wavelengths of 550 and 570 nm, respectively.
Statistical Analyses--
Student's t test was used
to compare two groups, and multiple groups were compared by one-way
analysis of variance with post hoc Newman-Keul's analyses.
 |
RESULTS |
Transfection of INS-1 insulinoma cells with a retroviral construct
containing the rat iPLA2
cDNA followed by selection
of G418-resistant cells resulted in the isolation of two stably
transfected clones that expressed severalfold more iPLA2
activity than parental INS-1 cells (Fig.
1A). Like the
iPLA2
activity in pancreatic islets and in other
insulinoma cell lines (10), the iPLA2
activity in the
stably transfected cells was stimulated by 1 mM ATP and virtually completely inhibited by 10 µM BEL (Fig.
1A). The iPLA2-X cell lines also exhibited an
increased content of an 84-kDa protein that was recognized by an
iPLA2
antibody after SDS-PAGE and immunoblotting analyses (Fig. 1B). Increased iPLA2
expression was a stable property of the transfected cells and persisted
on serial passage in culture.

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|
Fig. 1.
Overexpression of
iPLA2 in INS-1 insulinoma cells
stably transfected with iPLA2
cDNA in a retroviral vector. Two clonal INS-1 cell
lines were prepared by stable transfection with a retroviral vector
containing iPLA2 cDNA as described under
"Experimental Procedures," and levels of their iPLA2
activity (panel A) and immunoreactive protein
(panel B) were compared with those of parental
INS-1 cells. PLA2 activity was measured in the absence of
Ca2+ and presence of EGTA without (open
bars) or with 1 mM ATP alone
(cross-hatched bars) or ATP and 10 µM BEL (solid bars). The
leftmost set of bars reflects activity
from parental cells (control) and the center and
rightmost sets of bars reflect
activity from the two clonal INS-1 cell lines transfected with
iPLA2 cDNA in the retroviral construct
(#1-iPLA2-X and
#2-iPLA2-X, respectively). Immunoblotting
analyses (panel B) were performed after SDS-PAGE
analyses of INS-1 cell cytosolic protein. After transfer to nylon
membranes, proteins were probed with an iPLA2 antibody
and visualized with ECL as described under "Experimental
Procedures." Migration positions of molecular size markers are
illustrated at the left margin of
panel B, and the three
experimental lanes represent immunoreactive
proteins from parental INS-1 cells (control) and the two
iPLA2 -overexpressing lines
(#1-iPLA2-X and
#2-iPLA2-X), respectively. The arrow
at the right indicates the expected migration position of an
84-kDa protein corresponding to full-length rat
iPLA2 .
|
|
Both forskolin and IBMX alone and in combination induced an increase in
INS-1 cell cAMP content (Fig. 5). Despite
the greater insulin secretory responses to cAMP-elevating agents by
iPLA2-X cells compared with control INS-1 cells,
iPLA2-X cells did not exhibit greater rises in cAMP than
control cells when stimulated with forskolin or IBMX. This indicates
that augmented cAMP accumulation does not explain enhanced insulin
secretion by iPLA2-X cells and suggests that responsiveness
of the secretory apparatus to cAMP is increased by iPLA2
overexpression.
One effect of cAMP-elevating agents in INS-1 cells appears to be to
increase nuclear association of iPLA2
. Association of the group IVA PLA2 (cPLA2) with nuclei and
endoplasmic reticulum (ER) also occurs upon cellular stimulation with
agents that induce cPLA2 activation (79-81), and a similar
subcellular distribution is observed for other enzymes involved in
arachidonate metabolism in stimulated cells (81, 97). The
iPLA2
deduced amino acid sequence contains a bipartite
nuclear localization sequence (45) (511KREFGEHTKMTDVKKPK527)
similar to that in nucleoplasmin and some other nuclear proteins in
which two adjacent basic amino acids are followed by a flexible spacer
region that precedes a second cluster that contains three basic
residues (98, 99). Although this sequence might be expected to promote
entry into the nucleus and images in Fig. 12 suggest a perinuclear
location of iPLA2
, ultrastructural studies are required
to determine whether iPLA2
resides on the cytoplasmic or
nucleoplasmic face of the nuclear membrane. The ankyrin repeat domain
of iPLA2
could promote association with either face of the membrane.
Our findings are thus more consistent with a signaling rather than a
housekeeping role for iPLA2
in insulin-secreting
-cells. The observation that overexpressing iPLA2
in
-cells amplifies their insulin secretory responses could prove to be
useful in
-cell engineering. Recently, use of modified
immunosuppressive regimens has permitted successful transplantation of
human islets in seven consecutive patients with
insulin-dependent diabetes mellitus, and each patient
remained normoglycemic a year after transplantation without exogenous
insulin (105, 106). Widespread application of this therapy is precluded
by limited availability of donor organs, and
-cell lines that are
engineered to exhibit regulated insulin secretion could represent an
alternate source of cells for transplantation (73, 74, 106).
-Cells
with improved secretory responses and other properties have been
engineered by introducing genes in viral vectors and by clonal
selection strategies (104, 107-110). Identifying additional genes
whose products affect
-cell secretion might permit further progress,
and our findings suggest that iPLA2
gene products might
be useful in constructing engineered
-cell lines.
We thank Karen Green and Dr. Jeffrey Saffitz
of the Washington University Department of Pathology for assistance
with immunocytofluorescence studies, Dr. Matthew Baker of Research
Genetics, Inc. for assistance in peptide antigen selection and
generation of the iPLA2
antibody, Dr. Christopher
Newgard (University of Texas Southwestern Medical Center, Dallas, TX)
for providing the parental INS-1 cell line, and Denise Kampwerth for
assistance in preparing the manuscript.
Published, JBC Papers in Press, January 22, 2001, DOI 10.1074/jbc.M010423200
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