1 Jul 26, 2006 Proteomic analysis of apical and basolateral plasma membranes of rat renal ...

55 Pages·2006·1.58 MB·English

MCP Papers in Press. Published on August 9, 2006 as Manuscript M600177-MCP200
1

Jul 26, 2006

Proteomic analysis of apical and basolateral plasma membranes of rat renal collecting duct
cells

Ming-Jiun Yu1, Trairak Pisitkun1, Guanghui Wang2, Rong-Fong Shen2, and Mark A. Knepper1

D
1 Laboratory of Kidney and Electrolyte Metabolism, National Heart, Lung and Blood Institute, ow
n
lo
a
d
National Institutes of Health, Bethesda, MD 20892, USA ed
fro
m
2 Proteomics Core Facility, National Heart, Lung and Blood Institute, National Institutes of http
://w
w
Health, Bethesda, MD 20892, USA w
.m
c
p
o
nlin
e
.o
rg
b/
y
Running title: IMCD plasma membrane proteome gu
e
s
t o
n
J
a
n
u
a
ry
1
3
, 2
Address for correspondence: 0
1
9
Mark A. Knepper, M.D. Ph.D.
National Institutes of Health
Bldg. 10, Room 6N260
10 CENTER DR, MSC-1603
BETHESDA, MD 20892-1603
Phone: [phone] FAX [phone] e-mail: [email protected]
2

Abbreviations
ACE angiotensin-converting enzyme
ACVRL1 activin A receptor type-II like I
ANPEP aminopeptidase N
APC adenomatous polyposis coli protein
AQP aquaporin
CA4 carbonic anhydrase 4
CD40LG CD40 ligand
CDDB collecting duct database
CLIC4 chloride intracellular channel protein 4 D
o
w
DAPI 4',6-diamidino-2-phenylindole n
lo
a
d
ENaC epithelial sodium channel e
d
fro
ERC2 ERC protein 2 m
h
FZD1 frizzled 1 precursor ttp
://w
GPI glycosylphosphatidylinositol w
w
.m
GPR64 G-protein coupled receptor 64 precursor cp
o
n
GRIN2D glutamate [NMDA] receptor subunit epsilon 4 precursor lin
e
.o
H/K ATPase potassium-transporting ATPase brg/
y
HCN1 potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 1 gu
e
s
IMCD inner medullary collecting duct t o
n
J
a
Kv4.3 potassium voltage-gated channel subfamily D member 3 n
u
a
ry
LPL lipoprotein lipase 1
3
, 2
LRP4 low-density lipoprotein receptor-related protein 4 0
1
9
MAPK12 mitogen-activated protein kinase 12
MCT2 monocarboxylate transporter 2
Na/K ATPase sodium/potassium-transporting ATPase
NHE2 sodium/hydrogen exchanger 2
NOS1 nitric oxide synthase 1
PamlT palmitoyltransferase ZDHHC7
phosphatidylinositol-4-phosphate 3-kinase C2 domain-containing gamma
PIK3-C2γ
polypeptide
PIK3-Cβ phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit beta isoform
PKA-βC cAMP-dependent protein kinase, beta-catalytic subunit
3

PODXL podocalyxin
ROMK ATP-sensitive inward rectifier potassium channel 1
S1P-R sphingosine-1-phosphate receptor Edg-8
SCN5A sodium channel protein type 5 alpha subunit
SNAP29 synaptosomal-associated protein 29
SPA-1 signal-induced proliferation-associated 1-like protein 1
TauT sodium- and chloride-dependent taurine transporter
TGF-β transforming growth factor-beta
THIK-1 potassium channel subfamily K member 13
TYRO3 tyrosine-protein kinase receptor TYRO3
D
UT-A urea transporter A o
w
n
VAPA vesicle-associated membrane protein-associated protein A loa
d
e
d
VKγC vitamin K-dependent gamma-carboxylase fro
m
WASPIP Wiskott-Aldrich syndrome protein interacting protein h
ttp
Wnt4 wingless-type MMTV integration site family member 4 ://w
w
w
.m
c
p
o
n
lin
e
.o
rg
b/
y
g
u
e
s
t o
n
J
a
n
u
a
ry
1
3
, 2
0
1
9
4

Summary
We employed biotinylation and streptavidin affinity chromatography to label and enrich
proteins from apical and basolateral membranes of rat kidney inner medullary collecting ducts
(IMCDs) prior to LC-MS/MS protein identification. To enrich apical membrane proteins and
bound peripheral membrane proteins, IMCDs were perfusion-labeled with primary amine-
reactive biotinylation reagents at 2 °C using a double-barreled pipette. The perfusion-
biotinylated proteins, and proteins bound to them were isolated with CaptAvidin-agarose beads,
D
o
w
n
separated with SDS-PAGE, and sliced into continuous gel pieces for LC-MS/MS protein loa
d
e
d
identification (LTQ, Thermo Electron Corp.). 17 integral and GPI-linked membrane proteins from
h
ttp
and 44 non-integral membrane proteins were identified. Immunofluorescence confocal ://w
w
w
.m
microscopy confirmed ACVRL1, H/K ATPase α1, NHE2, and TauT expression in the IMCDs. cp
o
n
lin
e
Basement membrane and basolateral membrane proteins were biotinylated via incubation of .org
b/
y
g
IMCD suspensions with biotinylation reagents on ice. 23 integral and GPI-linked membrane u
e
s
t o
n
J
proteins and 134 non-integral membrane proteins were identified. Analyses of non-integral an
u
a
ry
1
membrane proteins preferentially identified in the perfusion-biotinylated and not in the 3
, 2
0
1
9
incubation-biotinylated IMCDs reveals protein kinases, scaffold proteins, SNARE proteins,
motor proteins, small GTP-binding proteins and related proteins that may be involved in
vasopressin-stimulated AQP2, UT-A1, and ENaC regulation. A WWW-accessible database was
constructed of 222 membrane proteins (integral and GPI-linked) from this study and prior
studies.
5

Introduction
The renal collecting duct is the terminal part of the renal tubule. Its major function is to
transport water and solutes in a regulated manner. Although there are many regulatory factors
that affect collecting duct transport functions, one of the most important factors is vasopressin, a
peptide hormone secreted by the posterior pituitary gland. Vasopressin regulates several
transport proteins including aquaporin 2 (1,2), aquaporin 3 (3), the epithelial sodium channel
D
ENaC (4,5) , and the urea transporter UT-A (6,7). Abnormalities of regulatory processes in the o
w
n
lo
a
collecting ducts are responsible for a large number of clinically important disorders of salt and de
d
fro
m
water balance (8,9). h
ttp
://w
w
The terminal portion of the collecting duct is the IMCD. Investigation of the mechanisms w
.m
c
p
o
n
of vasopressin action in the IMCD is benefiting from analysis of the proteome of the IMCD lin
e
.o
rg
cells. The work so far has identified a large number of IMCD proteins that have been included b/
y
g
u
e
s
in a publicly accessible database, the IMCD Proteome Database t o
n
J
a
n
u
(http://dir.nhlbi.nih.gov/papers/lkem/imcd/index.htm). Because most of the proteomic methods ary
1
3
, 2
used so far (10-13) to construct this database are biased against integral membrane and 0
1
9
glycosylphosphatidylinositol (GPI)-linked membrane proteins, these classes of proteins appear
underrepresented. The difficulty in detecting membrane proteins has arisen largely because of
the difficulty of solubilizing them in detergents that are compatible with 2-dimensional
electrophoresis. However, shotgun proteomics using LC-MS/MS offers greater efficiency in
identification of integral membrane proteins since they can be solubilized using the strong ionic
detergent SDS and then separated on 1-dimensional gels prior to trypsinization. However,
6

biochemical approaches are needed to isolate specific membrane fractions prior to identification.
One objective of the present study was to devise an approach that increases identification of
integral membrane proteins and GPI-linked proteins in plasma membrane domains as well as
proteins that are bound to integral membrane proteins.
Plasma membrane segregation into apical and basolateral domains at the tight junctions
provides the key functionality of epithelial cells. Discrete proteomes are expected in the apical
and the basolateral membranes to account for structural and functional differences including D
o
w
n
lo
hormone responses and vectorial transport across the epithelia. In the kidney, Cutillas et al were a
d
e
d
fro
the first to profile the apical and basolateral proteomes of renal cortex tissue using samples m
h
ttp
prepared from differential centrifugation and free-flow electrophoresis (14). Because of the ://w
w
w
.m
c
relative abundance of proximal tubules in the renal cortex, the findings from this study are po
n
lin
e
.o
probably applicable to the proximal tubule but not the collecting duct. Here, we devised rg
b/
y
g
methods combining surface biotinylation and streptavidin affinity chromatography to label and ue
s
t o
n
J
enrich proteins from apical and basolateral membranes of IMCDs prior to LC-MS/MS protein an
u
a
ry
1
identification. 62 integral and GPI-linked membrane proteins were identified. Subtractive 3, 2
0
1
9
comparison of non-integral membrane proteins identified in the apical and not the basolateral
membrane reveals 25 potential signaling and trafficking proteins involved in vasopressin-
regulated AQP2, UT-A, and ENaC regulation.
7

Experimental Procedures
Animals. Pathogen-free male Sprague-Dawley rats (Taconic Farms Inc., Germantown,
NY) were maintained on ad libitum rat chow (NIH-07; Zeigler, Gardners, PA) and drinking
water in the Small Animal Facility, National Heart, Lung, and Blood Institute (NHLBI). Animal
experiments were conducted under the auspices of the animal protocol H-0110 approved by the
Animal Care and Use Committee, NHLBI. Adult animals weighing between 200 and 250 g were
injected intraperitoneally with furosemide (5 mg/rat) 20 min before decapitation and removal of D
o
w
n
lo
kidneys. Furosemide dissipates the medullary osmolality, thereby preventing osmotic shock to a
d
e
d
fro
the cells upon isolation of the inner medullas (15). Immediately after the inner medullas were m
h
ttp
excised from the kidneys, they were transferred in ice-cold isolation solution (in mM: 250 ://w
w
w
.m
c
sucrose, 10 Tris, pH 7.4) to a cold room (2 ºC) for apical surface biotinylation. Some excised po
n
lin
e
.o
inner medullas were used to prepare IMCD suspensions for basolateral surface biotinylation. rg
b/
y
g
u
e
Perfusion biotinylation of IMCDs. In the cold room, each inner medulla was placed on a s
t o
n
J
a
n
porous support that allows drainage of excess fluid and in between two stacks of filter paper that u
a
ry
1
3
moisturizes the tissue (Fig. 1A). To introduce biotinylation reagents to the lumens of IMCDs, a , 2
0
1
9
double-barreled pipette was made from a theta glass capillary (TST 150-6, World Precision
Instruments, Inc., Sarasota, FL). The tip of the pipette was bent close to 90 degrees and drawn to
a spindle shape with a diameter of 100 µm tapering to 30 µm at its opening to fit the openings of
the IMCDs (ducts of Bellini) at the inner medullary tip. The geometry of the pipette tip was
made such that the body of the spindle seals the duct of Bellini to prevent back flow of the
perfused fluid. Two surface biotinylation reagents (thiol-cleavable sulfo-NHS-SS-biotin or non-
8

cleavable sulfo-NHS-LC-biotin, Pierce Biotechnology, Rockford, IL) were used in different
experiments at a concentration of 1.5 mg/ml in phosphate buffered saline (PBS; in mM: 5.1
Na HPO , 1.2 KH PO , 154 NaCl). These reagents are covalently link biotin to surface proteins
2 4 2 4
through the N-hydroxysuccinimide (NHS) group that reacts with primary amines at the N’-
termini and on side chains of lysine residues. They are believed to be excluded from the cell
interior due to their negatively charged sulfonate groups. However, our preliminary
experiments showed that two of these nominally “membrane impermeant” reagents readily
D
o
w
n
entered the IMCD cells. Preliminary experiments also showed that a brief fixation of the cell lo
a
d
e
d
membrane lipids with 4% paraformaldyhyde (16) prior to biotinylation prevented intracellular fro
m
h
ttp
biotinylation at 2 ºC (Fig. 1C, D). The optimized labeling process took place in the following ://w
w
w
.m
sequence. One barrel delivered the fixative to the IMCD lumen for 5 min followed by the other c
p
o
n
lin
e
barrel delivering the biotinylation reagent (sulfo-NHS-LC-biotin) for another 5 min. After the .o
rg
b/
y
biotinylation step, the solution was switched back to the fixative that flushed out the gu
e
s
t o
n
biotinylation reagents and stayed in the lumen while other ducts of Bellini were being perfusion- Ja
n
u
a
ry
biotinylated. On average, between 6 and 8 ducts of Bellini of a single inner medulla were 13
, 2
0
1
9
perfusion-biotinylated. Two non-toxic food dyes (FD&C blue No. 1 and FD&C red No. 3) were
used in the perfusates to visualize solution change. Another pipette (single-barreled) was
situated at the top of the perfusion pipette to drip Tris-buffered isolation solution onto the tissue
in order to moisturize the tissue and to quench the reactive NHS group of the biotinylation
reagents if back flow occurred. After perfusion-biotinylation, the inner medullas were
immediately frozen on dry ice. A total of 12 inner medullas from 6 rats were collected. Some
non-fixed inner medullas were perfused with sulfo-NHS-SS-biotin. Both fixed and non-fixed
9

perfusion-biotinylated IMCDs were prepared for LC-MS/MS analysis in different experiments.
Basolateral biotinylation of IMCDs. To label the basement membrane and basolateral
membrane proteins with biotin, an IMCD suspension was prepared from the excised inner
medullas as described previously (17). Briefly, the inner medullas were minced and digested
with 2 mg/ml hyaluronidase and 3 mg/ml collagenase B. A 60 xg centrifugation was then
carried out to precipitate the heavier IMCD segments from the non-IMCD components of the
inner medulla (loops of Henle, interstitial cells, vasa recta, and capillaries). The isolated IMCD D
o
w
n
lo
suspension was fixed with 4% paraformaldehyde for 5 min on ice before incubation with 1.5 a
d
e
d
fro
mg/ml sulfo-NHS-LC-biotin for 5 min on ice to label selectively the basement membrane and m
h
ttp
basolateral membrane proteins as previously described (18). Another IMCD suspension ://w
w
w
.m
c
remained non-fixed was labeled with 1.5 mg/ml sulfo-NHS-SS-biotin on ice for 5 min. Both po
n
lin
e
.o
fixed and non-fixed incubation-biotinylated IMCD suspensions were used for LC-MS/MS rg
b/
y
g
protein identification in different experiments. ue
s
t o
n
J
a
n
Plasma membrane-enriched fraction. To enrich for plasma membrane components of the u
a
ry
1
3
IMCD cells, a high-density membrane fraction was prepared using differential centrifugation as , 2
0
1
9
described previously (3,19). Perfusion-biotinylated inner medullas were homogenized in liquid
nitrogen using a mortar and a pestle. The inner medulla homogenate was suspended in ice-cold
isolation buffer containing protease inhibitors (0.1 mg/ml PMSF and 1 µg/ml leupeptin) and
centrifuged at 1,000 xg for 10 min at 4 °C to remove incompletely homogenized fragments and
nuclei. The supernatant was collected and centrifuged again at 17,000 xg for 20 min. The
17,000 xg pellet is a high-density membrane fraction that was previously reported to be enriched
10

for plasma membrane (19).
Incubation-biotinylated IMCD suspensions were homogenized in the ice-cold isolation
buffer containing the protease inhibitors using a tissue homogenizer (TH, Omni International,
Marietta, GA). The high-density membrane fraction was prepared as described above.
Isolation of biotinylated proteins. When the non-cleavable biotinylation reagent was
used, high-density membrane fractions were prepared and the membranes were solubilized with
D
1 ml lysis solution (in mM: 150 NaCl, 5 EDTA, 50 Tris, pH 7.4) containing 1% NP-40 plus o
w
n
lo
a
protease (0.1 mg/ml PMSF and 1 µg/ml leupeptin). The solubilized membrane fraction was de
d
fro
m
centrifuged at 10,000 xg for 10 min at 4 °C to remove insoluble components. 100 µl of the h
ttp
://w
w
resulting supernatant was saved as pre-isolation control and 900 µl of it was mixed with 200 µl w
.m
c
p
o
sediment CaptAvidin-agarose beads (Invitrogen, Carlsbad, CA) that bind biotinylated proteins nlin
e
.o
rg
(20). After removal of unbound proteins, the CaptAvidin-agarose beads were washed with the b/
y
g
u
e
s
following solutions (900 µl for each wash) to remove non-specifically bound proteins: 1) lysis t o
n
J
a
n
u
solution containing 1% NP-40 3 times, 2) high salt solution (in mM: 500 NaCl, 5 EDTA, 50 Tris, a
ry
1
3
, 2
pH 7.4) 2 times, and 3) no salt solution (10 mM Tris, pH 7.4) 1 time. The biotinylated proteins 0
1
9
were eluted from the CaptAvidin-agarose beads twice, each time with 45 µl alkaline solution (50
mM Na CO , pH 10.1) plus 10 mM D-biotin (Invitrogen) that competes the biotin binding sites
2 3
on the CaptAvidin molecules thereby enhancing elution of biotinylated proteins (20).
When the thiol-cleavable biotinylation reagent was used, the isolation procedures for the
biotinylated proteins were similar to those for the non-cleavable biotinylated proteins except 1)
RIPA detergent solution (0.1% SDS, 0.5% sodium deoxycolate, and 1% NP-40) was used in the