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J. Biol. Chem., Vol. 278, Issue 34, 32405-32412, August 22, 2003
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Spinophilin Stabilizes Cell Surface Expression of 
2B-Adrenergic Receptors*
From the
Departments of Pharmacology and
¶Molecular Physiology and Biophysics, Center for
Molecular Neuroscience, Vanderbilt University Medical Center, Nashville,
Tennessee 37232-6600, the ||Department of
Psychiatry, Yale University School of Medicine, New Haven, Connecticut 06508,
and the **Laboratory of Molecular and Cellular
Neuroscience, Rockefeller University, New York, New York 10021
Received for publication, April 22, 2003
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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The third intracellular (3i) loops of the
2A- and
2B-adrenergic receptor (AR) subtypes are critical for
retention of these receptors at the basolateral surface of polarized
Madin-Darby canine kidney (MDCKII) cells at steady state. The third
intracellular loops of the 2A, 2B, and
2C-AR subtypes interact with spinophilin, a multidomain
protein that, like the three 2-AR subtypes, is enriched at
the basolateral surface of MDCKII cells. The present studies provide evidence
that 2-AR interaction with spinophilin contributes to cell
surface stabilization of the receptor. We exploited the unique targeting
profile of the 2B-AR subtype in MDCKII cells: random
delivery to apical and basolateral surfaces with rapid
(t
60 min) apical versus slower
(t = 10–12 h) basolateral turnover. Apical
delivery of a spinophilin subdomain containing the
2-AR-interacting region (Sp151–483) by fusion with
apically targeted p75NTR extended the half-life of
2B-AR at the apical surface to 
3.6 h and eliminated the
rapid phase (0–60 min) of 2B-AR turnover on that
surface. Furthermore, we examined 2B-AR turnover at the
surface of mouse embryo fibroblasts derived from wild type
(Sp+/+) or spinophilin knock-out
(Sp–/–) mice. Two independent
experimental approaches demonstrated that agonist-evoked internalization of
HA-2B-AR was accelerated in mouse embryo fibroblasts derived
from Sp–/– mice. These findings
are consistent with the interpretation that endogenous spinophilin contributes
to the stabilization of 2B-AR and presumably all three
2-AR subtypes at the surface of target cells and may act as
a scaffold that could link 2-ARs to proteins interacting
with spinophilin via other domains. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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2-Adrenergic receptors
(ARs)1 are members of
the large superfamily of G protein-coupled receptors that contain seven
putative transmembrane spanning regions. There are three
2-AR subtypes (2A, 2B,
and 2C), each of which is activated by the endogenous
catecholamines, epinephrine and norepinephrine, and performs multiple
physiological functions via pertussis toxin-sensitive
Gi/Go proteins
(1). Cellular signaling
pathways regulated by 2A-AR in native cells include
inhibition of adenylyl cyclase, activation of receptor-operated K+
channels, inhibition of voltage-gated Ca2+ channels, and
activation of the mitogen-activated protein kinase cascade
(1–3).
Many cells that express 2-ARs are polarized, including renal
and intestinal epithelia, where the 2-AR serves to regulate
sodium and water resorption (4,
5), as well as neurons, where
these receptors act to suppress neurotransmitter release
(6). The physiological
functions mediated by 2-ARs in polarized cells are dependent
upon precise localization of the receptor at the basolateral surface to gain
access to neurally delivered and blood-delivered catecholamines.
The 2-AR subtypes demonstrate unique targeting and
retention profiles in polarized renal epithelial Madin-Darby canine kidney
(MDCKII) cells in culture (7).
Previous work in our laboratory has shown that the 2A-AR
subtype is directly targeted to the basolateral surface, where it exhibits a
half-life of 10–12 h (8).
Direct and exclusive basolateral targeting of 2A-AR was
found to be dependent upon several noncontiguous regions within or near the
bilayer, whereas retention of the receptor at the basolateral surface appears
dependent upon the third intracellular (3i) loop
(8). Deletion of the 3i loop
results in accelerated surface turnover (t =
4.5 h) of the 2A-AR at the basolateral surface
(9). Unlike the
2A-AR, the 2B-AR subtype is
randomly targeted to both the apical and basolateral subdomains and
then selectively retained at the basolateral surface of polarized MDCKII
cells, where the receptor has a half-life comparable with that of the
2A-AR subtype (t =
10–12 h) (7). Like
for the 2A-AR, the 3i loop of the 2B-AR
also is critical for basolateral surface stabilization of this subtype
(10). In contrast to stable
retention of the 2B-AR on the basolateral surface, the
half-life on the apical surface is estimated to be dramatically shorter, on
the order of minutes (7). Taken
together, these data suggest that the stabilization/retention of
2A- and 2B-AR at specific membrane domains
is most likely mediated through interactions of the 3i loop with other
proteins either within or underlying the basolateral membrane surface and not
present (or expressed at much lower density) at the apical surface.
The 2-AR 3i loop has been used as a ligand to identify
potential interacting proteins and has led to the identification of two
2-AR-interacting molecules: 14-3-3
(11) and spinophilin
(12). Spinophilin is an
817-amino acid, ubiquitously expressed, multidomain-containing protein with an
apparent molecular mass on SDS-PAGE of 130 kDa. It was originally
identified both as a protein phosphatase 1 (PP1)-binding protein localized to
dendritic spines, hence the name spinophilin
(13), as well as an
F-actin-binding protein (14).
Spinophilin (also known as neurabin II) is highly homologous to the
brain-specific protein, neurabin I
(14). In addition to the
domains described above, spinophilin contains a single PDZ (PSD-95,
Discs large, ZO-1) domain and three coiled-coil domains
at the C terminus, the latter of which mediate homo-multimerization in
vitro (14) and may allow
for the formation of multiprotein complexes in intact cells. Spinophilin
previously was identified as a D2 dopamine receptor-interacting protein using
the 3i loop of the D2 dopamine receptor as bait in a yeast two-hybrid screen
(15). The D2 dopamine
receptor-binding domain in spinophilin (residues 151–444), located
between the F-actin-binding domain and the PP1 regulatory domain, also
interacts with all three of the 2-AR subtypes
(12) and will be referred to
as the receptor-interacting domain. Because reports in the literature, as well
as our own observations, indicate that spinophilin is specifically enriched at
the basolateral surface of polarized epithelial cells
(12,
15,
16), we postulate that
spinophilin may be involved in tethering and/or stabilizing the receptor at
the cell surface via interactions with the 2-AR 3i
loops.
The present studies utilized two different biological systems to explore
the role of spinophilin in 2-AR stabilization at the cell
surface. First, the unique targeting profile of the 2B-AR
subtype in polarized MDCKII cells (random delivery with rapid turnover at the
apical surface) was exploited to determine whether redirection of the
receptor-interacting domain of spinophilin to the apical surface of polarized
MDCKII cells would result in enhanced apical retention of randomly delivered
2B-AR. Second, the role of spinophilin in
2-AR surface turnover was addressed by studying the
internalization profile of the 2B-AR in mouse embryonic
fibroblasts (MEFs) derived from wild type
(Sp+/+) or spinophilin knock-out
(Sp–/–) mice
(17). The findings from both
lines of investigation implicate spinophilin in the stabilization of the
2B-AR at the cell surface.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
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Materials
Transwell culture chambers (0.4-µm pore size) were purchased from Costar (Cambridge, MA). Doxycycline hydrochloride was purchased from Sigma. NHS-SS-Biotin and Immunopure Immobilized Streptavidin were purchased from Pierce. Both [35S]EasyTagTM Express Protein Labeling mix (1200 Ci/mmol) and [3H]methoxy-inulin (126.5 mCi/g) were from PerkinElmer Life Sciences. Cysteine- and methionine-free DMEM was from Cellgro Mediatech. Dulbecco's modified Eagle's medium was prepared by the Cell Culture Core, a facility sponsored by the Diabetes Research and Training Center at Vanderbilt University Medical Center. Fetal calf serum was purchased from Sigma. Mouse monoclonal 12CA5 antibody against the HA epitope was obtained from BABCo. Affinity matrix-coupled high affinity rat monoclonal anti-HA antibody (clone 3F10), rat monoclonal anti-HA antibody (clone 3F10), and mouse monoclonal HA.11 antibody (clone 16B12) were purchased from Roche Applied Science. Mouse monoclonal anti-c-Myc (clone 9E10) ascites was purchased from Covance Research Products Inc. (Denver, PA). Both the mouse anti-gp135 and mouse anti-EGFR were gifts from Peter J. Dempsey (Department of Pathology, University of Washington, Harborview Medical Center, Seattle, WA). Rabbit anti-spinophilin antibody raised against spinophilin amino acids 286–390 (18) was purified in our lab (for details see Ref. 12). Alexa Fluor 488-conjugated fluorescent goat anti-mouse, goat anti-rat, and goat anti-rabbit IgG were purchased from Molecular Probes (Eugene, OR). Cy3-conjugated donkey anti-mouse IgG was purchased from Jackson Immunochemicals. Sheep anti-mouse, donkey anti-rabbit, and goat anti-rat horseradish peroxidase-conjugated IgG were purchased from Amersham Biosciences. The rat p75NTR cDNA was a generous gift from Dr. Bruce Carter (Department of Biochemistry, Vanderbilt University). The retroviral vector pBabe-HA-
2B-AR was kindly provided by Drs. Dan Gil
and John Donello (Allergan, Irvine, CA).
MDCKII Cell Culture and Polarization
MDCKII cells were plated at a density of 1.2 x 106
cells/100-mm polycarbonate membrane filter (Transwell culture chambers,
0.4-µm pore size) and cultured in DMEM supplemented with 10% fetal calf
serum (Sigma) and 100 units/ml penicillin and 10 µg/ml streptomycin at 37
°C and 5% CO2 with medium changes every other day for 5–7
days. Cells grown under these conditions achieve a morphologically and
functionally polarized phenotype, as described previously
(19). Leak assays of
[3H]methoxy-inulin were performed as described previously
(20) prior to each half-life
experiment to verify that the MDCKII cells had developed tight junctions and
that the apical and basolateral compartments were functionally separated.
Generation of cDNAs Encoding Myc-p75-Spinophilin Fusion Proteins
The cDNA encoding the pTRE-Myc-p75-Sp151–483 fusion protein was
generated via overlapping PCR extension using Pfu Turbo DNA
polymerase (Stratagene). The Myc tag was inserted 5' to the coding start
site of full-length rat p75NTR and 3' of the N-terminal
cleavable signal sequence. Four glycine residues were engineered via PCR onto
the C terminus of p75NTR with the intention of permitting
independent folding of the spinophilin subdomain and decreased steric
hindrance for interacting with other potential binding partners. The pTRE cDNA
backbone (Clontech) has a tetracycline-inducible promoter that is intended to
confer regulated expression of the fusion construct by treatment with the
synthetic tetracycline analog, doxycycline. Two fusion proteins were
generated. Myc-p75-Sp151–483 includes the receptor-binding domain and
the PP1 regulatory domain of the full-length spinophilin, whereas
Myc-p75-Sp151–586 also contains the PDZ-binding domain (cf.
schematic of spinophilin domain structure in
Fig. 2B). The cDNAs
were sequenced in their entirety via 33P-Thermo Sequenase
Radiolabeled Terminator Cycle Sequencing Kit (U. S. Biochemical Corp.) to
confirm that the sequences were correct.
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Creation of Clonal MDCKII Cell Lines Expressing the Myc-p75-Spinophilin Fusion Protein
Permanent clonal cell lines were developed in MDCKII cells using FuGENE-6 (Roche Applied Science) transfection reagent, according to the manufacturer's protocol; 6 µg of linearized pTET-On plasmid (Clontech) was co-transfected with 6 µg of pTRE-Myc-p75-Spinophilin construct and 1 µg of a vector encoding a hygromycin resistance gene into MDCKII cells already stably expressing the HA-
2B-AR, generated as described previously
(7). Stably transfected cells
were selected through growth in 400 µg/ml hygromycin and assayed for
HA-2B-AR expression via radioligand binding analysis using
the antagonist [3H]rauwolscine
(21) and for
Myc-p75-spinophilin expression and inducibility via Western analysis and
immunofluorescence using anti-c-Myc antibody. Despite the use of an inducible
expression system, Myc-p75-spinophilin expression occurred even in the absence
of doxycycline. Nonetheless, the cells were treated overnight with 1 µg/ml
doxycycline before the day of the experiment to assure maximal
Myc-p75-spinophilin expression.
Immunofluorescence in Polarized MDCKII Cells
Polarized MDCKII cells stably expressing HA-2B-AR were
grown on 12-mm Transwells for 5–7 days (as described above) and
processed as described previously for immunolocalization of
HA-2B-AR and endogenous spinophilin
(12) and for detection of
endogenous apical (gp135) and basolateral (EGFR) marker proteins
(22) (see
Fig. 1). MDCKII cells
expressing Myc-p75-Sp151–483 fusion protein in the
HA-2B-AR background were treated with 1 µg/ml doxycycline
overnight to maximize the expression of Myc-p75-Sp151–483 prior to
staining. All of the steps were performed essentially as described previously
(22) except that a rat anti-HA
(clone 3F10) antibody (Roche Applied Science) diluted 1:1000 in blocking
buffer was used for the detection of HA-2B-AR (see
Fig. 2D).
Immunoprecipitation of Apically Targeted Myc-p75-Spinophilin Fusion
Proteins from Stable MDCKII Cell Lines
MDCKII cells lines stably expressing the HA-2B-AR alone
or Myc-p75-Sp151–483 or Myc-p75-Sp151–586 in the
HA-2B-AR background were grown to confluence in 100-mm
tissue culture dishes. All of the dishes were treated with 1 µg/ml
doxycycline for 16 h before harvesting the cells to maximize fusion protein
expression. On the day of the assay, the dishes were washed twice with
Dulbecco's phosphate-buffered saline supplemented with 1 mM
MgCl2 and 0.5 mM CaCl2 (DPBS/CM) (4 °C)
and scraped into 12 ml of lysis buffer (15 mM Tris-HCl, 5
mM EGTA, 5 mM EDTA, pH 7.6, with
N-methyl-D-glucosamine) with protease inhibitors (1
µg/ml soybean trypsin inhibitor, 0.5 µg/ml leupeptin, 100
µM phenylmethylsulfonyl fluoride). The cells were passaged
through a 20-gauge needle 10 times and then centrifuged for 20 min at 39,000
x g. The supernatant was aspirated, and the pellet was
resuspended in 500 µl of radioimmune precipitation buffer (RIPA) (150
mM NaCl, 50 mM Tris-HCl, pH 8.0, 5 mM EDTA,
1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS) plus protease inhibitors (same
as above) with five passages through a 20-gauge needle followed by 10 passages
through a 25-gauge needle. The detergent-extracted membranes were cleared of
RIPA-insoluble debris by centrifugation for 1 h at 100,000 x g.
The supernatants were precleared for 30 min with rotation at 4 °C with
protein G-agarose beads pre-equilibrated in RIPA buffer. Anti-c-Myc antibody
(1:100 dilution) was added to each of the precleared samples and incubated
overnight at 4 °C with rotation. The next day, 30 µl of a 1:1 slurry of
protein G-agarose (pre-equilibrated in RIPA buffer containing 2.5 mg/ml bovine
serum albumin (BSA)) was added to each tube and incubated for 2 h at 4 °C
with rotation. The protein G-agarose was pelleted and washed four times with 1
ml of ice-cold RIPA buffer plus protease inhibitors. Immunoisolated protein
from the protein G-agarose was eluted by two sequential incubations for 10 min
each with 25 µl of 1x SDS sample buffer (50 mM Tris-HCl,
pH 8.0, 2% SDS, 10% glycerol, 100 mM dithiothreitol, 0.1%
bromphenol blue) at 70 °C. The eluates were pooled, and the entire sample
was resolved by 10% SDS-PAGE and transferred to nitrocellulose for Western
blot analysis as described previously
(23). Myc-p75-Sp151–483
or Myc-p75-Sp151–586 was detected by incubation with rabbit
anti-Sp286–390 antibody (1: 1000) followed by donkey anti-rabbit
horseradish peroxidase-conjugated secondary antibody (1:2000) and visualized
by ECL (Amersham Biosciences).
Determination of Protein Half-life on the Apical Surface of Polarized
MDCKII Cells
HA-2B-Adrenergic
Receptors—A metabolic radiolabeling strategy was used to determine
receptor half-life of 2B-AR at the apical surface of
polarized MDCKII cells because of the low steady state density of the
2B-AR on this surface
(7). Cells grown 5–7 days
in 100-mm Transwell culture were treated overnight with 1 µg/ml
doxycycline. The day of the assay the cells were washed once in DPBS/CM and
then, simultaneous with performing a transepithelial
[3H]methoxy-inulin leak assay (see above), incubated for 2 h at 37
°C in serum-free, cysteine/methionine-free DMEM. The cells were then
pulsed for 45 min with 900 µl of 2 µCi/µl
[35S]cysteine/methionine in cysteine/methionine-free DMEM at 37
°C and 5% CO2. "Chasing" of the metabolically
labeled cells was achieved by adding serum-free DMEM containing 1
mM cysteine and 1 mM methionine (chase medium) (9 ml
apical/9 ml basolateral) and returning the dishes to 37 °C for 40 min to
allow delivery of all of the receptor labeled during the pulse phase to the
cell surface.
To begin the determination of the surface half-life of metabolically labeled receptors, the apical surface of polarized MDCKII cells was biotinylated as follows. Transwells were washed once with 4 °C DPBS/CM and transferred to the cold room on ice. After washing, the Transwells were equilibrated for 10 min in 4 °C TEA buffer (250 mM sucrose, 2 mM CaCl2, 2 mM MgCl2, 10 mM triethanolamine, pH 9.0) and then biotinylated for 20 min at 4 °C on the apical surface by incubating freshly made 1 mg/ml sulfo-NHS-SS-Biotin in TEA buffer in the apical chamber (an equal volume of TEA buffer without sulfo-NHS-SS-Biotin was also added to the basolateral chamber). The biotinylation step was repeated to assure quantitative labeling of the receptor. Washing the cells with 100 mM glycine in DPBS/CM for 10 min quenched the biotinylation reaction. After washing twice more with 4 °C DPBS/CM and once with 4 °C serum-free DMEM chase medium, the cells were transferred to 37 °C chase medium and returned to the 37 °C incubator for the indicated times.
At varying time points, the amount of biotinylated
HA-2B-AR on the apical surface was quantified by sequential
immunoisolation and streptavidin chromatography. Selected dishes were washed
twice for 10 min at 4 °C with DPBS/CM, scraped into 12 ml of lysis buffer
(15 mM Tris-HCl, 5 mM EGTA, 5 mM EDTA, pH
7.6, with N-methyl-D-glucosamine) containing protease
inhibitors (1 µg/ml soybean trypsin inhibitor, 0.5 µg/ml leupeptin, 100
µM phenylmethylsulfonyl fluoride), triturated 10 times through a
20-gauge needle, and then centrifuged at 39,000 x g for 20 min.
The pellet was resuspended in 1 ml of RIPA buffer plus protease inhibitors and
incubated on ice 30 min before centrifugation for 1 h at 100,000 x
g. Supernatants from this centrifugation (the solubilized
preparation) were incubated with 25 µl of a 1:1 slurry of pre-equilibrated
rat anti-HA affinity matrix overnight at 4 °C with rotation. The affinity
matrix was pelleted and washed four times with 1 ml of ice-cold RIPA buffer
plus protease inhibitors before elution of the immunoisolated
HA-2B-AR from the affinity matrix by incubation twice for 10
min with 100 µl of SDS sample buffer (1.6% SDS, 8.3% glycerol, 167
mM Tris, pH 8.0) at 70 °C. The eluates were pooled and brought
to 1.5 ml with RIPA (containing no SDS) plus protease inhibitors. The sample
was allowed to sit for 10 min at room temperature to equilibrate the component
detergents.
To isolate the apically biotinylated HA-2B-AR from the
entire immunoisolate, streptavidin chromatography was performed as follows.
The samples were incubated with a 1:1 slurry of streptavidin-agarose (50
µl) pre-equilibrated in RIPA buffer for 2 h at 4 °C with rotation.
Pelleted streptavidin-agarose was washed three times with 1 ml of ice-cold
RIPA buffer containing protease inhibitors, and biotinylated
HA-2B-AR was eluted by incubation twice for 20 min with 100
µl of SDS sample buffer containing 50 mM dithiothreitol at 90
°C. The eluted samples were then incubated 40 min at 50 °C.
N-Ethylmaleimide was added to a final concentration of 15
mM, and the samples were incubated for an additional 40 min at 50
°C. The rationale for the high dithiothreitol/N-ethylmaleimide
treatment is to alkylate all sulfhydryl residues, thus better resolving the
2-AR preparation on SDS-PAGE
(24).
The samples were resolved overnight for a total of 160mAmp-hr on a 7.5–20% gradient SDS-polyacrylamide gel. The gels were treated with En[3H]ance Intensifying Solution (PerkinElmer Life Sciences) according to the manufacturer's protocols, dried, and exposed to BioMAX MR film. The bands on the film were quantitated using SCION image software, and/or bands were cut from the gel and counted directly in scintillation mixture. Equivalent findings were obtained from either quantitation procedure.
Endogenous gp135—Because endogenous gp135 is expressed at a relatively high concentration on the apical surface of polarized MDCKII cells, the surface half-life of this protein was determined by surface biotinylation, extraction into RIPA at various time points, resolution by SDS-PAGE, and identification of biotinylated gp135 via Western blot analysis for gp135, using methods described previously (23).
Culturing of MEFs
A 13.5-day pregnant female mouse (Sp+/+ or
Sp–/–)
(17) was sacrificed, and the
embryos were collected. The soft, dark colored tissues (i.e. heart,
liver, and spleen) were dissected away from the embryo, and the head was
removed. The remaining tissue was transferred to the barrel of a 5-ml syringe
(five embryos/syringe) and passed through an 18-gauge needle into 3 ml of
DPBS. The tissue was further dissociated by trituration five times, and the
cell suspension was transferred to a 150-mm culture dish containing 25 ml of
complete medium (DMEM with 10% fetal calf serum, 100 units/ml penicillin, and
10 µg/ml streptomycin supplemented with 2 mM glutamine). The
cells were grown at 37 °C and 5% CO2 until the plates reached
confluency, at which point the cells were split 1:5, expanded to confluency,
and frozen at 2 x 106 cells/ml in freezing medium (50% fetal
calf serum, 12% Me2SO in DMEM).
Transduction of MEFs with a Retroviral Vector Encoding
HA-2B-AR
Primary cultures of MEFs (Sp+/+ or
Sp–/–)
(17) were seeded at 1.4
x 106 cells/100-mm dish the day before transduction with 4 ml
of one part retroviral supernatant containing
HA-2B-AR-encoding virions harvested from BOSC cells
(25) and one part complete
DMEM containing a final concentration of 12 µg/ml polybrene. The viral
application was repeated four times over the course of 8 h at 37 °C and 5%
CO2, empirically determined to yield optimal transduction.
Afterward, the cells were returned to 9 ml of complete medium. Three days
post-transduction, the cells were assayed for HA-2B-AR
expression via radioligand binding analysis, essentially as described
previously (9). For
specifically indicated experiments, the transduced cells were selected for
retroviral vector expression by treatment overnight with 4 µg/ml puromycin
(the pBabe retroviral vector carries the resistance gene for puromycin).
Measuring Turnover of the HA-2B-AR in MEFs
Intact Cell ELISA Assay—The day before the assay,
Sp+/+ or
Sp–/– MEFs (selected for
HA-2B-AR expression in 4 µg/ml puromycin) were plated on
poly-D-lysine-coated 96-well culture plates at a density of 4
x 104 cells/well in complete medium containing the
2-AR antagonist phentolamine (1 µM) to
eliminate effects of catecholamines that might be present in the
serum-containing DMEM. The day of assay, the cells were washed three times for
15 min at 37 °C in serum-free DMEM containing 0.1% BSA (200 µl/well) to
wash away phentolamine and twice for 10 min in serum-free DMEM containing no
BSA (200 µl/well). The cells were returned to 37 °C with 90 µl of
serum-free DMEM/well. The cells were stimulated at 37 °C for the indicated
times by the addition of the agonist epinephrine (100 µM) and
the 
-AR antagonist propranolol (1 µM) to exclude the
activation of endogenous -adrenergic receptors. The cells were then
fixed with 4% paraformaldehyde in 0.12 M sucrose in DPBS/CM (100
µl/well) for 20 min at room temperature and washed twice with DPBS/CM (200
µl/well). The cells were blocked for 30 min at 37 °C with 3% BSA in
DPBS/CM (blocking buffer). Primary antibody (rat anti-HA) was diluted 1:500 in
blocking buffer and incubated with the cells (50 µl/well) for 1 h at 37
°C. Following labeling with primary antibody, the cells were washed three
times for 5 min with DPBS/CM (200 µl/well). Incubation with secondary
antibody (anti-rat horseradish peroxidase) diluted 1:1000 in blocking buffer
was for 1 h at 37 °C (50 µl/well). Unbound secondary antibody was
removed by three 5-min washes with DPBS/CM (200 µl/well). The colorimetric
substrate o-phenylene-diamine dihydrochloride (1 mg/ml) was prepared
according to the manufacturer's instructions (Pierce) and was incubated with
cells for 10–20 min at room temperature (100 µl/well). Color
development was stopped by the addition of 2.5 M sulfuric acid (100
µl/well). Absorbance at 490 nm was read on a microtitre plate reader, and
the values were analyzed using Graph Pad Prism software.
Reversible Biotinylation—Surface HA-2B-AR
was labeled on ice with a disulfide cleavable biotin (sulfo-NHS-SS-Biotin),
stimulated by agonist at 37 °C, and then treated with the cell-impermeant
reducing agent, 2-mercaptoethanesulfonic acid (MESNA). Receptors that have
trafficked to the inside of the cell at the time of MESNA treatment are
protected from reduction and can be subsequently isolated via streptavidin
agarose. Total receptor available at the cell surface at time 0
(t0) was defined by the difference in the amount of
biotinylated receptor detected in the absence of MESNA treatment and that
detected following immediate reversal by MESNA (thereby revealing the quantity
of surface receptor biotinylation that MESNA can reverse efficiently).
Internalized receptor detected at various time points (represented by the
MESNA-insensitive fraction) was quantitated following Western analysis and
expressed as a fraction of the total surface receptor available at
t0.
For each experiment, Sp+/+ or
Sp–/– MEFs expressing
HA-2B-AR were plated at 1 x 106 cells/60-mm
dish the day before the assay (1 dish for each time point assayed) and treated
overnight with phentolamine (1 µM) as described above. The day
of the assay, the cells were washed three times for 10 min at 37 °C with
serum-free DMEM containing 0.1% BSA, placed on ice for 20 min, and then washed
once with 4 °C DPBS/CM. The cells were incubated at 4 °C for 30 min
with 100 µg/ml sulfo-NHS-SS-Biotin to label surface proteins. Following
biotinylation, the cells were washed twice with DPBS/CM at 4 °C and once
with 4 °C serum-free DMEM. Next, the cells were stimulated for the
indicated times by incubation at 37 °C in serum-free DMEM containing
epinephrine (100 µM) and propranolol (1 µM). The
incubation was terminated by replacement of the 37 °C medium with 4 °C
DPBS/CM, followed by two 20-min incubations at 4 °C with MESNA (250
mM) in DPBS/CM. Following incubation with MESNA, the cells were
washed once with 4 °C DPBS/CM, and residual MESNA was quenched by
incubation with 5 mg/ml iodoacetamide for 20 min at 4 °C. The cells were
then washed once more in DPBS/CM, scraped into 100 µl of DM-CHS
extraction buffer (4 mg/ml dodecyl -maltoside, 0.8 mg/ml cholesteryl
hemisuccinate, 20% glycerol, 25 mM glycylglycine, 20 mM
HEPES, 100 mM NaCl, 5 mM EGTA, 0.1 mM
phenylmethylsulfonyl fluoride, 10 units/ml aprotinin) and triturated five
times with a 25-gauge needle. Supernatant of a 1-h microcentrifugation at
12,000 rpm at 4 °C was incubated with a 1:1 slurry (40 µl) of
streptavidin-agarose (pre-equilibrated in DM-CHS extraction buffer
containing 2.5 mg/ml BSA) for 1 h with rotation at room temperature. The
streptavidin-agarose pass-through was saved, and the strepavidin-agarose resin
was washed three times with 500 µl of ice-cold DM-CHS wash buffer
(0.5 mg/ml dodecyl -maltoside, 0.1 mg/ml cholesteryl hemisuccinate, 25
mM glycylglycine, 20 mM HEPES, 100 mM NaCl, 5
mM EDTA). The biotinylated HA-2B-AR was eluted
twice at 90 °C into 30 µl of SDS sample buffer (50 mM
Tris-HCl, pH 6.8, 2% SDS, 10% glycerol, 0.1% bromphenol blue) containing 50
mM dithiothreitol, resolved by 10% SDS-PAGE, and transferred to
nitrocellulose for Western blot analysis as described previously
(23).
|
RESULTS AND DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTS AND DISCUSSIONREFERENCES |
|---|
Co-localization of Endogenous Spinophilin with HA-
2B-AR in Polarized MDCKII
Cells—Immunofluorescence staining and confocal laser scanning
microscopy were used to confirm the localization of both the
HA-2B-AR and endogenous spinophilin in the MDCKII cell line
used for these studies (Fig.
1). As has been shown previously, the HA-2B-AR
is localized to the basolateral surface in these polarized epithelial cells,
albeit with a small pool of presumably endocytosed 2B-AR
(7). Endogenous spinophilin
also is enriched at the basolateral surface, as has been suggested in the
literature (12,
14,
15). The co-localization of
these two proteins at the basolateral surface is indicated by the yellow
signal present in the overlay of the two images, particularly evident in the z
scan. We postulated that the basolateral localization of spinophilin in MDCKII
cells may contribute to retention of 2-AR subtypes at that
surface and that the lack of apical spinophilin may explain the rapid apical
turnover of randomly delivered 2B-AR.
Redirection of a Spinophilin Subdomain to the Apical Surface of MDCKII
Cells—To test the hypothesis that spinophilin contributes to cell
surface 2-AR retention, we explored whether redirection of
the receptor-interacting domain of spinophilin to the apical surface of
polarized MDCKII cells would lead to enhanced apical retention of randomly
delivered 2B-AR. Fig.
2B provides a schematic diagram of the domain structure
of the spinophilin protein
(14). A region of spinophilin
sequence (amino acids 151–483) including the receptor-interacting domain
was fused-in frame to Myc epitope-tagged p75NTR, a single
transmembrane spanning protein known to be expressed predominantly (80%) at
the apical surface of polarized MDCKII cells
(26). The spinophilin
sequences were separated from p75NTR via a tetraglycine linker to
permit independent folding and accessibility of the spinophilin domains. A
cDNA encoding this fusion protein was then stably expressed in MDCKII cells
already stably expressing HA-2B-AR, as described under
"Experimental Procedures."
Expression of the Myc-p75-Sp151–483 and Myc-p75-Sp151–586
fusion proteins in these HA-2B-AR-expressing MDCKII cell
lines was confirmed via immunoprecipitation and Western blotting. As can be
seen in Fig. 2C, the
Myc-tagged fusion proteins can be specifically enriched by immunoprecipitation
of detergent-solubilized membrane fractions with anti-c-Myc antibody and then
identified on Western blots with an antibody against an epitope within
spinophilin (amino acids 286–390) (lanes 2 and 3). As
expected, no Myc-spinophilin fusion protein is detected in the parental cell
line expressing only heterologous HA-a2B-AR (lane 1).
As shown in Fig.
2D, immunolocalization studies using an antibody against
the Myc epitope reveal that Myc-p75-Sp151–483 is expressed at the apical
surface of polarized MDCKII cells (Fig.
2D, first panel) in a manner similar to the
expression pattern of the known endogenous apical marker protein, gp135
(Fig. 2D, third
panel). A similar apical expression pattern also was detected for the
clonal cell line expressing Myc-p75-Sp151–586 (data not shown).
Fig. 2D (second
panel) shows that the apical expression pattern of
Myc-p75-Sp151–483 is in marked contrast to the basolateral expression
pattern of endogenous spinophilin, heterologous HA-2B-AR at
steady state (Fig. 1), or the
EGFR (Fig 2D,
fourth panel), a marker protein for the basolateral surface. Thus,
our fusion protein strategy successfully delivered the receptor-interacting
domain of spinophilin to the apical surface and did so without altering
expression of endogenous markers for the polarized phenotype, gp135, and the
EGFR.
An Apically Targeted Spinophilin Subdomain Extends the Apical Half-life
of Randomly Delivered 2B-AR—To
determine whether apical expression of the receptor-interacting domain of
spinophilin would extend the apical surface half-life of randomly delivered
2B-AR, a cell surface biotinylation strategy in
metabolically labeled, polarized MDCKII cells was used to quantify the loss of
apical HA-2B-AR over time (see Refs.
7 and
19 and "Experimental
Procedures"). The autoradiogram in
Fig. 3A shows that the
rapid loss of apical HA-2B-AR over the initial 60 min is
attenuated in cells expressing Myc-p75-Sp151–483.
Fig. 3B summarizes
data from multiple experiments and shows a time course extended over 6 h.
These data reveal a biphasic loss of HA-2B-AR from the
apical surface for cells not expressing exogenous apical spinophilin, such
that 50% of the apical HA-2B-AR was lost with a
t = 50 min. By contrast, in cells expressing
the apically targeted spinophilin fusion protein, the half-life of the
HA-2B-AR at the apical surface is extended to 3.6 h.
Coincidentally, in cells expressing Myc-p75-Sp151–483,
HA-2B-AR loss from the surface occurred at a constant rate,
similar to that of the slower phase observed in cells not expressing exogenous
apical spinophilin. The initial phase on the apical surface was comparably
lengthened in a cell line expressing a p75 fusion protein that also contains
the PDZ domain of spinophilin, Myc-p75-Sp151–586 (data not shown),
indicating that protein interactions fostered by the PDZ domain do not
contribute to 2B-AR retention, at least on the apical
surface. Moreover, because these spinophilin fusion constructs did not contain
coiled-coil or F-actin-binding domains, neither multimerization nor
F-actin-binding of spinophilin appears to be necessary to stabilize
2B-AR expression at the cell surface. In addition, the
extended apical half-life of the HA-2B-AR does not appear to
be a consequence of generalized changes in apical membrane turnover, because
the turnover of the endogenous apical marker protein, gp135, was not
significantly altered in cells expressing Myc-p75-Sp151–483
(Fig. 3C).
|
Previous studies of apical retention of the randomly delivered
HA-2B-AR had only revealed a single population of rapidly
(t = 15–45 min) turning over
HA-2B-AR (7).
The second, slower phase of the HA-2B-AR loss from the
apical surface detected in the present studies is not due to leak of the
biotinylating reagent to the basolateral compartment, because the slower phase
of HA-2B-AR loss from the apical surface
(t = 3.6 h) is entirely different from the
half-life of the HA-2B-AR on the basolateral surface
(t = 10–12 h). Our lack of detection of
the second, extended apical expression (t =
3.6 h) of a subfraction of the apically delivered 2B-AR
in these earlier studies may have been due to the lesser amount of biological
material evaluated (the present studies examined cells grown in 100-mm rather
than 24-mm Transwell cultures). It is not entirely surprising that the apical
half-life of the HA-2B-AR in cells engineered to express an
apically targeted receptor-interacting domain of spinophilin is extended
beyond 60 min to 3.6 h but is not restored to that of the half-life of
the HA-2B-AR at the basolateral surface
(t = 10–12 h); it is likely that
interactions of spinophilin and/or the receptor with additional proteins
underlying the basolateral surface also contribute to 2B-AR
residence time. For example, although the F-actin-binding and coiled-coil
domains are not necessary for stabilization of
2B-AR expression on the apical surface (see above),
multimerization of endogenous spinophilin and/or its interaction with F-actin
may contribute to the more extended half-life on the basolateral surface.
Despite the enhanced apical retention of HA-2B-AR in cells
harboring the apically targeted spinophilin fusion protein observed in
biochemical studies, we were unable to detect an apical pool of
HA-2B-AR via immunofluorescence studies, perhaps because
metabolic labeling and biotinylation are intrinsically more sensitive for
examining receptor turnover at the cell surface than is
immunofluorescence.
The most straightforward interpretation of our findings is that the
receptor-interacting domain of spinophilin itself is stabilizing the
2B-AR at the apical surface. However, we cannot rule out a
role for the PP1 interacting domain, which is inherent in each of the fusion
proteins examined.
Agonist-induced Internalization Is Enhanced in Spinophilin-deficient
(Sp–/–)
MEFs—As a complementary strategy to explore whether spinophilin
plays a role in stabilizing the 2B-AR at the cell surface,
we took advantage of primary MEFs derived from wild type
(Sp+/+) or spinophilin knock-out
(Sp–/–) mice
(17). Western blot analysis of
whole cell lysate using an antibody against endogenous spinophilin reveals
that spinophilin is absent from the
Sp–/– MEFs, whereas it is readily
detected in the wild type (Sp+/+) cells
(Fig. 4A).
|
Wild type (Sp+/+) and spinophilin
knock-out (Sp–/–) MEFs were
transduced with HA-2B-AR using a retroviral expression
system, and receptor expression was verified via radioligand binding analysis.
In contrast to the lack of effect of agonists on short term
2A-AR turnover in MDCKII
(8) or other
(27,
28) target cells, the
2B-AR subtype has previously been documented to rapidly
internalize in response to agonist treatment
(28–31).
We postulated that if spinophilin is important for stabilizing the receptor at
the cell surface, then internalization of the 2B-AR may be
accelerated in a cell background lacking spinophilin. We evaluated
HA-2B-AR internalization using two independent strategies in
Sp+/+ versus
Sp–/– MEFs: 1) cell surface
ELISA, which measured the loss of receptor from the cell surface, and 2)
reversible biotinylation, which examined HA-2B-AR that is
internalized and protected from MESNA-evoked removal of the biotin moiety from
the cell surface.
For the intact cell ELISA,
Sp+/+versus
Sp–/– MEFs were treated with an
2-AR agonist for the indicated times and then labeled with
primary antibody directed against the HA epitope. Consistent with previous
findings in cultured cell lines (all of which express endogenous spinophilin)
(28,
31), 30% of the
HA-2B-AR was lost from the cell surface of
Sp+/+ MEFs in response to agonist treatment
for 60 min (Fig. 4B).
In the absence of agonist, the HA-2B-AR remained at the cell
surface for the entire time course of the incubation (data not shown).
Importantly, however, 55% of the HA-2B-AR was lost from
the cell surface of Sp–/– MEFs
over the same 60-min incubation duration
(Fig. 4B). These data
provide additional evidence that the presence of spinophilin stabilizes the
2B-AR at the cell surface.
A reversible biotinylation strategy, which measures the amount of
internalized HA-2B-AR that occurred over time following
agonist exposure, was also exploited. For these studies,
HA-2B-AR expressing MEFs
(Sp+/+ and
Sp–/–) were labeled at 4 °C
with the membrane-impermeant, cleavable biotinylating reagent,
sulfo-NHS-SS-biotin, before incubation with agonist for varying amounts of
time. At the end of each incubation period, the cells were placed at 4 °C
and treated with a membrane-impermeant reducing agent, MESNA, to cleave
disulfide-linked biotin remaining on the surface. Receptors isolated via
streptavidin-agarose from detergent-solubilized cells represent the receptors
that remain biotinylated at each incubation point after MESNA treatment, which
reveals the fraction of the receptor pool that was protected from reversal of
biotinylation (i.e. internalized) during the course of the
experiment. As shown in Fig. 5,
this experimental strategy also reveals increased internalization of the
HA-2B-AR following agonist treatment in
Sp–/– MEFs as compared with
Sp+/+ MEFs.
|
Conclusion—The present studies demonstrate a role for
spinophilin in the stabilization/retention of the 2B-AR at
the cell surface both in a cultured cell system and in cells derived from
spinophilin knock-out mice. An apically targeted spinophilin subdomain
containing the receptor-interacting domain extends the half-life of randomly
delivered HA-2B-AR at the apical surface of polarized MDCKII
cells, where the receptor exhibits a rapid surface turnover under wild type
conditions in contrast to its long-lived retention
(t = 10–12 h) at the basolateral
surface. Presumably, the transient stabilization of the
HA-2B-AR at the apical surface caused by redistribution of
the receptor-interacting domain of spinophilin to that surface reflects the
role that endogenous spinophilin plays in basolateral stabilization of all
three 2-AR subtypes, because all three interact with
spinophilin via their 3i loops
(12). This interpretation is
further supported by studies utilizing mouse embryonic fibroblasts derived
from spinophilin knock-out mice in which two distinct lines of evidence were
each consistent with enhanced agonist-induced internalization of
HA-2B-AR in
Sp–/– cells as compared with wild
type Sp+/+ cells expressing endogenous
spinophilin. Taken together, these data suggest that spinophilin plays a role
in stabilizing the receptor at the cell surface. The precise mechanism for
this stabilization is not known but could involve physically anchoring the
receptor to the actin cytoskeleton, as is seen with the D2 dopamine receptor
and actin-binding protein-280 (ABP-280, also known as Filamin-A)
(32,
33). Alternatively,
spinophilin may impede interaction with other molecules, such as
-arrestin (34), that
would foster receptor internalization. Our interpretation that spinophilin
stabilizes the 2-AR at the surface likely extends to other
Gi/Go-coupled G protein-coupled receptors, because
spinophilin also interacts with the 3i loop of the D2 dopamine receptor
(15). Future studies can
establish whether the multidomain nature of spinophilin may serve to bring
other proteins into the receptor microcompartment that participate not only in
receptor localization but also in coordination of receptor-elicited signal
transduction.
|
FOOTNOTES |
|---|
* This work was supported by several National Institutes of Health Grants DK43879 (to L. E. L.) and NS37508 (to R. J. C.) and also by National Institutes of Health Grants MH40899 and DA10044 (to P. G. for the development of the spinophilin knock-out mice). Confocal microscopy was performed on Zeiss LSM 410 and 510 Laser Scanning Confocal Microscopes at the Vanderbilt University Medical Center Cell Imaging Core Resource, which was supported by National Institutes of Health Grants CA68485, DK20593, and DK58404 and the shared Instrumentation Grant 1S1ORR15682-1. Cell culture media were prepared by the Diabetes Research and Training Center cell culture core facility, which was supported by National Institutes of Health Grant DK20593. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Supported in part by Pharmacological Sciences Training Grants GM07628-19,
GM07628-20, and GM07628-21 as well as from an Advanced Predoctoral Fellowship
in Pharmacology and Toxicology from the Pharmaceutical Research and
Manufacturers of America Foundation.
To whom correspondence should be addressed: Dept. of Pharmacology, Vanderbilt
University Medical Center, 464 Robinson Research Bldg., Nashville, TN
37232-6600. Tel.: 615-343-3538; Fax: 615-343-7286; E-mail:
lee.limbird{at}vanderbilt.edu.
1 The abbreviations used are: AR, adrenergic receptor; 3i loop, third
intracellular loop; BSA, bovine serum albumin; CHS, cholesteryl hemisuccinate;
DM, dodecyl--D-maltoside; DPBS/CM, Dulbecco's
phosphate-buffered saline supplemented with 1 mM MgCl2
and 0.5 mM CaCl2; DMEM, Dulbecco's modified Eagle's
medium; EGFR, epidermal growth factor receptor; HA, hemagglutinin; MEF, mouse
embryo fibroblast; MDCKII, Madin Darby canine kidney II; p75NTR,
p75 neurotrophin receptor; PP1, protein phosphatase 1; RIPA, radioimmune
precipitation buffer; Sp, spinophilin; ELISA, enzyme-linked immunosorbent
assay; MESNA, 2-mercaptoethanesulfonic acid.
|
ACKNOWLEDGMENTS |
|---|
We thank Carol Ann Bonner for excellent and dedicated technical support, Dr. Bruce Carter (Department of Biochemistry, Vanderbilt University) for providing the cDNA encoding p75NTR, and Drs. Dan Gil and John Donello (Allergan) for providing us with the pBABE
2B-AR
retroviral vector, as well as for offering invaluable technical advice
throughout the course of these studies. We are also grateful to all of the
other members of the Limbird laboratory, both past and present, for their
enthusiasm and support. |
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