(Received for publication, August 23, 1995)
From the
We have identified a major calmodulin (CaM)-binding protein in
rat liver endosomes using I-CaM overlays from
two-dimensional protein blots. Immunostaining of blots demonstrates
that this protein is the polymeric immunoglobulin receptor (pIgR). We
further investigated the interaction between pIgR and CaM using
Madin-Darby canine kidney cells stably expressing cloned wild-type and
mutant pIgR. We found that detergent-solubilized pIgR binds to
CaM-agarose in a Ca
-dependent fashion, and binding is
inhibited by the addition of excess free CaM or the CaM antagonist W-13 (N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide),
suggesting that pIgR binding to CaM is specific. Furthermore, pIgR is
the most prominent
S-labeled CaM-binding protein in the
detergent phase of Triton X-114-solubilized, metabolically labeled
pIgR-expressing Madin-Darby canine kidney cells. CaM can be chemically
cross-linked to both solubilized and membrane-associated pIgR,
suggesting that binding can occur while the pIgR is in intact
membranes. The CaM binding site is located in the membrane-proximal
17-amino acid segment of the pIgR cytoplasmic tail. This region of pIgR
constitutes an autonomous basolateral targeting signal. However,
binding of CaM to various pIgR mutants suggests that CaM binding is not
necessary for basolateral targeting. We suggest that CaM may be
involved in regulation of pIgR transcytosis and/or signaling by pIgR.
The plasma membrane of polarized epithelial cells is divided
into apical and basolateral surfaces, each having a distinct protein
and lipid composition. Cells use two pathways to deliver proteins to
the correct surface. First, newly made proteins are packaged in the
trans-Golgi network (TGN) ()into vesicles that deliver them
directly to either the apical or basolateral surface. Second, proteins
can be delivered first to one surface and then endocytosed and
transcytosed to the opposite surface. At least in the well polarized
Madin-Darby canine kidney (MDCK) cell line, targeting to the
basolateral surface generally requires a sorting signal located in the
cytoplasmic domain of the protein. For a number of basolateral proteins
it has been found that mutations in the cytoplasmic domain prevent TGN
to basolateral targeting(1) . The first basolateral signal to
be identified is the 17-amino acid membrane proximal segment (residues
653-670) of the cytoplasmic domain of the polymeric
immunoglobulin receptor (pIgR)(2) . This protein is normally
delivered from the TGN to the basolateral surface, and then endocytosed
and transcytosed to the apical surface. Deletion of most of this
17-residue segment prevents TGN to basolateral delivery. Moreover, this
segment can be transplanted to a heterologous reporter molecule, which
is then retargeted from the apical to the basolateral
surface(2) . In at least one other case, the low density
lipoprotein receptor, autonomous basolateral sorting signals have been
identified(3) .
The pIgR basolateral targeting signal has
been systematically analyzed by alanine scanning
mutagenesis(4) . Mutation of His-656, Arg-657, or Val-660 to
Ala substantially diminishes, but does not eliminate, TGN to
basolateral targeting. Mutation of other residues had little or no
effect. The structure of a synthetic 17-residue peptide corresponding
to this signal has been determined by two-dimensional nuclear magnetic
resonance spectroscopy(4) . This peptide tends to adopt a
putative type 1 -turn, encompassing residues 658-661,
followed by a nascent helical structure. In MDCK cells polarized
sorting takes place in both the TGN and after endocytosis. Mutations
that diminish TGN to basolateral sorting and increase TGN to apical
sorting have a similar effect on sorting in the endocytotic pathway,
decreasing recycling to the basolateral surface and increasing
transcytosis to the apical surface(5) . These data indicate
that the basolateral targeting signal also functions as a signal to
retrieve the pIgR from the endocytotic pathway to the basolateral
surface. Very similar results have been obtained with the two
basolateral sorting signals of the low density lipoprotein
receptor(6) . This suggests that the same sorting machinery
operates in both the TGN and the endocytotic pathway, or that sorting
in both pathways actually occurs in a common compartment.
Transcytosis of pIgR is regulated by several mechanisms. Phosphorylation of Ser664, which is part of the basolateral sorting signal, stimulates transcytosis. Mutation of Ser-664 to Ala (S664A) reduces transcytosis, while mutation to Asp (S664D), whose negative charge may mimic a phosphate, stimulates transcytosis(7) . Phosphorylation apparently works by weakening the basolateral signal, since the S664D mutant exhibits decreased TGN to basolateral sorting, increased TGN to apical sorting(5) , as well as decreased recycling and increased transcytosis after endocytosis.
Binding of
the ligand, dimeric IgA (dIgA) to the pIgR also stimulates
transcytosis. Ligand binding stimulates transcytosis of the wild-type
pIgR, as well as S664D and S664A(8, 9) .
Ligand-dependent stimulation suggests that the pIgR may transduce a
signal to the cytoplasm. Indeed, we have recently found that dIgA
binding causes tyrosine phosphorylation of a
phosphatidylinositol-specific phospholipase C1, activation of
protein kinase C, and production of inositol 1,4,5-trisphosphate. (
)Transcytosis of pIgR and other molecules is stimulated
either by activation of protein kinase C (10) or by increase in
intracellular free Ca
, (
)so both arms of
the phospholipase C signaling pathway may redundantly stimulate
transcytosis. Delivery to the apical surface, either by transcytosis or
directly from the TGN, is also stimulated by the heterotrimeric G
protein, G
(11, 12) , as well as cAMP and
protein kinase A(13, 14) . Both the G
and G
subunits are stimulatory, at least for transcytotic
apical delivery(11) . Transcytosis in MDCK cells is stimulated
by bradykinin,
while in animals transcytosis is stimulated
by several hormones and neurotransmitters (15, 16, 17, 18) . Presumably, these
extracellular signals work through the various second messengers
mentioned above.
Transcytosis of the pIgR can be divided by both morphological and biochemical assays into at least three steps. Step 1 is internalization via clathrin-coated pits into basolateral early endosomes. Step 2 is delivery to a compartment located immediately beneath the apical plasma membrane (``apical recycling compartment'')(19, 20) . Step 3 is delivery to the apical plasma membrane. It has previously been observed that phosphorylation of Ser-726 regulates step 1(21) , phosphorylation of Ser-664 regulates both steps 2 and 3(22) , whereas dIgA(22) , protein kinase C(10) , and cAMP (14) act primarily on step 3.
We are interested in molecules
that bind to the cytoplasmic domain of the pIgR, particularly its
basolateral sorting signal, as such molecules might be involved in
basolateral sorting and/or regulation of traffic. The best evidence for
a candidate molecule capable of recognizing and deciphering a
basolateral targeting signal is a protein of 220 kDa apparent
molecular mass that has been cross-linked to the cytoplasmic domain of
the vesicular stomatitis virus G protein (VSVG) (23) . A
peptide corresponding to the cytoplasmic domain of VSVG prevented
cross-linking and also inhibited TGN to basolateral transport of VSVG
in permeabilized MDCK cells, while a peptide corresponding to a mutant
VSVG that is not basolaterally targeted prevented neither cross-linking
nor basolateral transport. However, a peptide corresponding to the pIgR
17-amino acid basolateral targeting signal did not inhibit basolateral
transport of VSVG(23) , suggesting that this protein might not
recognize the pIgR basolateral targeting signal.
Studies on MDCK
cells using CaM antagonists suggest that CaM is important in endosome
function(24, 25) . We now report that in the presence
of Ca, calmodulin (CaM) binds to the basolateral
sorting signal of the pIgR. An analysis of mutant pIgRs reveals that
the sequence requirements for basolateral targeting of the pIgR and CaM
binding are not identical, suggesting that the role of CaM binding is
not to target the pIgR to the basolateral surface, but rather may be
involved in regulation of pIgR transcytosis.
Figure 1:
Identification of
pIgR as the major CaM-binding protein on blots of two-dimensional gels
from rat liver endosomes. An RRC endosome fraction from rat liver was
subjected to two-dimensional electrophoresis and stained with Coomassie
Blue (A), or transferred to nitrocellulose filters and probed
with I-CaM (B) or a monoclonal antibody (SC 166)
against pIgR (C). Numbers indicate major
polypeptides: 1, unidentified protein found in RRC, MVB, and
CURL; 2, 5`-nucleotidase; 3, group of polypeptides,
including annexin VI, present in all endosome and plasma membrane
fractions analyzed; 4, pIgR. The M
and pH
positions are indicated.
The sample (500 µl) was combined
with 450 µl of binding buffer and 50 µl of a 20% slurry of
CaM-agarose, which had been equilibrated with binding buffer. Samples
were incubated for 1 h at 4 °C on a rotator. After pelleting the
CaM-agarose beads briefly in a cold microcentrifuge and aspirating the
liquid, the beads were washed once with 1 ml of binding buffer
containing 0.5% Triton X-100 and once with binding buffer containing
0.05% Triton X-100. At this point, a 5-min incubation was carried out
at 4 °C with binding buffer containing 0.05% Triton X-100, with or
without 3 mM EGTA. The liquid was separated from the beads as
before, and the beads were washed once with binding buffer with or
without EGTA (no detergent). The beads (10 µl packed) were boiled
in 20 µl of sample buffer, and half of the sample (10 µl)
was analyzed by SDS-PAGE and fluorography or phosphorimaging. Total
pIgR in the starting material for CaM-agarose was determine by
immunoprecipitation of a duplicate sample. Starting material, typically
500 µl, was diluted with an equal volume of 2.5% Triton dilution
buffer (2.5% Triton X-100, 100 mM triethanolamine-HCl, pH 8.6,
100 mM NaCl, 5 mM EGTA, 1% Trasylol, 0.02%
NaN
) and analyzed by pIgR immunoprecipitation, followed by
SDS-PAGE and fluorography or phosphorimaging.
To correct for differences in pIgR concentrations in the starting material due to differing expression levels among clones or different partitioning behavior of constructs in Triton X-114, unlabeled preparations were analyzed by Western blotting to determine the relative concentration of each construct in the preparations. For clones that yield more pIgR in the starting material, the volume of Triton X-114 preparation added was reduced appropriately so that approximately equal amounts of each pIgR (wild-type or mutant) were added to CaM beads. The total amount of Triton-X114 starting material added was kept at 500 µl by adding the appropriate volume of an identical preparation from non-transfected MDCK cells.
In order to address the mechanism(s) by which CaM regulates
endosomal function, we were interested in identifying major CaM-binding
proteins in endosomes. Previous studies showed a major CaM-binding
protein of approximately 115 kDa could be demonstrated by I-CaM overlay of two-dimensional protein blots of an
endosome-rich fraction from rat liver(38) . We discovered that
this protein can also be labeled by staining with a monoclonal antibody
(SC166) against the polymeric immunoglobulin receptor (pIgR) (Fig. 1). Similar results were obtained with a rabbit antiserum
raised against rat SC (data not shown).
Figure 2:
Binding of pIgR to CaM-agarose.
CaM-agarose beads were incubated under various conditions with Triton
X-114 preparations of metabolically labeled proteins from
pIgR-transfected MDCK cells as described under ``Materials and
Methods.'' After washing, the beads were boiled in SDS sample
buffer and the eluate was analyzed by SDS-PAGE. A,
Ca dependence of CaM binding. The
S-labeled proteins in the Triton X-114 fraction added to
CaM beads are shown (starting material). The amount of pIgR in
the starting material was analyzed by immunoprecipitation and SDS-PAGE (IP). The total
S-labeled proteins bound to
CaM-agarose after washing in buffer containing Ca
or
EGTA are shown. Note that pIgR is the major protein detected on the
CaM-agarose beads. Molecular weight standards are shown at left. B, proteins bound to CaM-agarose in the absence
or presence of 1 µM free CaM, as
indicated.
Binding of pIgR to CaM-agarose is inhibited by addition of EGTA (Fig. 2A), excess CaM (Fig. 2B), or the
CaM antagonist W-13 (N-(4-aminobutyl)-5-chloro-2-naphthalenesulfonamide; data not
shown), further supporting the specificity of binding. If CaM binding
to pIgR is physiologic, then we would expect the CaM binding site to be
on the cytoplasmic domain of pIgR. To test this, we used a pIgR
construct encoding the extracellular and transmembrane domains of pIgR,
but lacking virtually all of the cytoplasmic tail (pIgR R655-STOP). As
shown in Fig. 3, this ``tail-minus'' construct shows
no detectable binding to CaM. We used other mutants to localize the
site of CaM binding on the pIgR cytoplasmic tail (Fig. 3).
Remarkably, a mutant lacking the membrane-proximal 14-amino acid
segment of the cytoplasmic tail required for basolateral targeting
(655-668) also fails to bind CaM. Conversely, a mutant in
which the cytoplasmic domain consists only of the membrane-proximal
17-amino acid basolateral targeting signal (pIgR T670-STOP) exhibits
CaM binding indistinguishable from that of full-length pIgR.
Figure 3:
CaM binds to the 17-amino acid basolateral
targeting signal of pIgR. CaM binding of pIgR constructs was analyzed
as in Fig. 2A. Constructs indicated are as follows: wild-type, wild-type pIgR; pIgR 655-668, a
construct in which 14 amino acids of the 17-amino acid basolateral
targeting signal have been deleted; pIgR R655-STOP, a
construct which contains only the first two amino acids of the
cytoplasmic tail; pIgR T670-STOP, a construct which lacks all
but the 17-amino acid basolateral targeting segment of the cytoplasmic
tail. No binding was detected for pIgR
655-668 or pIgR
R655-STOP. The cytoplasmic domain of the protein encoded by each
construct is indicated schematically at the right.
Figure 4:
CaM can be chemically cross-linked to
detergent-solubilized pIgR. Triton X-114 preparations of metabolically
labeled proteins from MDCK cells expressing pIgR constructs (see
``Materials and Methods'') were incubated in the presence or
absence of 1 µM exogenous CaM, with or without 3 mM EGTA as indicated, at 4 °C for 1 h, followed by incubation for
another hour in the presence or absence of BS as indicated.
Samples were quenched with 100 mM glycine and boiled in SDS,
and pIgR was immunoprecipitated and analyzed by SDS-PAGE. With
wild-type pIgR, an additional band of
125 kDa (arrow),
which represents pIgR cross-linked to CaM, appears only in the presence
of CaM, BS
, and free Ca
. This treatment
does not produce an additional band for pIgR
655-668 or pIgR
R655-STOP.
In order to determine if CaM can bind to pIgR that
is still embedded in the membrane, we performed cross-linking
experiments using crude MDCK membranes containing unlabeled pIgR.
Radioiodinated CaM is incorporated into a complex of 125 kDa in a
Ca-dependent manner with membranes containing
wild-type pIgR, but not with pIgR
655-668 (Fig. 5) or
cells not expressing pIgR (data not shown). These results attest to the
likelihood of physiologic binding of pIgR to CaM in intact cells.
Figure 5:
CaM can be chemically cross-linked to
membrane-associated pIgR. Crude membrane preparations from MDCK cells
expressing pIgR constructs were incubated with I-CaM in
the presence or absence of EGTA. After 1 h at 4 °C, BS
was added to 0.5 mM, incubation was continued for
another hour, glycine and SDS were added to 100 mM and 1%,
respectively, samples were boiled for 5 min, and pIgR was
immunoprecipitated and analyzed by SDS-PAGE and fluorography.
I-CaM was incorporated into a
125-kDa species in the
presence of membranes containing pIgR, but not membranes from MDCK
cells expressing pIgR
655-668.
Figure 6:
CaM binding to pIgR point mutants does not
correlate with their basolateral targeting phenotype. CaM-agarose
binding of pIgR mutants, in which alanine is substituted at the
indicated positions in the context of full-length pIgR, was analyzed as
for Fig. 2A. Lanes showing an immunoprecipitation of
pIgR from the starting material (IP), and S
proteins bound to CaM-agarose in the presence of Ca
or EGTA are indicated. The percentage of each construct bound to
CaM-agarose, relative to wild-type, and basolateral targeting phenotype
(*, determined previously; (4) ) of each mutant: (+)
normal basolateral targeting;(-), disrupted basolateral
targeting, are indicated at right. Values represent the mean
of two experiments (see ``Materials and Methods'' for
details).
CaM binding of several proteins is known to be modulated by phosphorylation(39) . The pIgR has a major phosphorylation site at Ser-664 in the basolateral targeting signal, and phosphorylation of this residue stimulates transcytosis of pIgR(7, 8) , presumably by inactivating the basolateral targeting signal(5) . We therefore assayed the ability of a non-phosphorylatable mutant, S664A, and a mutant that mimics the presence of a phosphate residue, S664D, to bind to CaM. Both of these mutants bind to CaM as well as wild-type pIgR (data not shown). Thus phosphorylation of Ser-664 probably does not alter CaM binding.
CaM binds to and regulates the function of a wide variety of
proteins. In most cases this binding occurs only when CaM is
``activated'' after binding Ca. Because the
affinity of CaM for Ca
(
10
M) is above the resting
[Ca
]
(
10
M), CaM is a sensor for transient increases in
[Ca
]
, binding to target
proteins only when [Ca
]
is
elevated(39) . CaM has been suggested to play several possible
roles in membrane traffic in mammalian cells. Endocytosis in neurons is
dependent on the phosphorylation state of dynamin(40) , which
is in turn a substrate for calcineurin (phosphatase 2B)(41) , a
phosphatase that is regulated by CaM binding. Cyclosporin A, an immune
suppressant that functions by binding calcineurin, inhibits
Ca
/CaM-dependent secretion from pancreatic acinar
cells, thereby implicating calmodulin/calcineurin in regulation of this
secretion(42) . More generally,
[Ca
]
is clearly important in
many intracellular membrane traffic events, including both regulated
exocytosis of granules (43) and synaptic vesicles(44) ,
as well as classically ``constitutive'' processes, such as
endoplasmic reticulum to Golgi transport (45) and nuclear
envelope fusion (46) (at least in in vitro systems).
It is not known if [Ca
]
acts on
CaM and/or some other target in these events.
We have found that CaM
binds to the basolateral targeting signal of the pIgR, making CaM the
first identified protein shown to bind specifically to a basolateral
sorting signal. Binding is strictly dependent on Ca.
The pIgR is the major CaM-binding protein detected in membranes of a
highly purified rat liver endosome fraction, and is also the major
CaM-binding protein detected in a Triton X-114 detergent phase extract
from total metabolically labeled MDCK cells that are transfected with
pIgR. These striking results suggest that the interaction of CaM and
pIgR is of high specificity. We also detected the CaM-pIgR interaction
by cross-linking, both in detergent extracts and, more significantly,
in non-solubilized crude membranes prepared from MDCK cells. That CaM
can bind to pIgR that is still in its native membrane makes it quite
likely that under the appropriate in vivo conditions of
elevated [Ca
]
, CaM should bind
to pIgR. Capturing the interaction of CaM with pIgR in intact cells may
be quite difficult, as elevations of
[Ca
]
are generally very
transient and spatially localized(47) .
Using a series of
mutant pIgRs expressed in MDCK cells, we mapped the CaM binding site to
the membrane-proximal 17 amino acids of the cytoplasmic domain of the
pIgR. This same segment has previously been shown to be necessary and
sufficient to direct the pIgR to the basolateral surface from either
the TGN or the endocytotic pathway(2) . However, an analysis of
Ala point mutants in this segment indicates that there is not a precise
correlation between the residues needed for basolateral sorting and
those needed for CaM binding. The sequence of this 17-residue segment
is generally comparable with other known CaM binding sites. CaM often
binds to a sequence with a preponderance of basic residues near the N
terminus, and hydrophobic residues more concentrated near the C
terminus(39) . This is largely true of this 17-residue segment
of the pIgR. It is remarkable that this short region of the cytoplasmic
domain of the pIgR therefore has at least two separable functions,
basolateral targeting and CaM binding. This segment also has a third
function, as a rather weak signal for endocytosis, centered around
tyrosine 668(48) . Based on our knowledge of how sorting occurs
in other systems, it seems likely that a specific protein complex
recognizes the basolateral sorting signal. This complex would be
functionally and perhaps structurally homologous to the adaptor
proteins found in clathrin-coated vesicles, and to the coatomer
involved in traffic in the early portion of the biosynthetic pathway. A
likely candidate for a component of the coat involved in basolateral
sorting is the p200 protein found in the TGN(49) . Such
complexes presumably recognize specific signals on the cytoplasmic
domains of integral membrane proteins. In the case of the AP2/HA2
adaptor found in the plasma membrane, the signal contains a type 1
-turn(50, 51, 52, 53) , perhaps
somewhat similar to the type 1
-turn that is an essential part of
the basolateral signal of the pIgR(4) .
In contrast, CaM
generally does not function as an adaptor, i.e. it does not
recognize a feature on one protein, and then bind to another protein.
Rather, when CaM binds to a site on a protein, a common consequence is
that it simply masks that site, preventing it from interacting with
anything else(39) . We speculate that one possible role of CaM
is to sequester the basolateral signal on the pIgR. In the absence of
elevated [Ca]
, CaM would not
bind to the pIgR. The hypothetical complex that recognizes the
basolateral signal would bind to the signal and direct the pIgR to the
basolateral surface. However, when
[Ca
]
is elevated, perhaps due
to the binding of dIgA to the pIgR or to another hormone signaling
event, CaM would bind to pIgR and thereby prevent the hypothetical
complex from binding. By masking the basolateral signal, the pIgR would
be allowed to be transcytosed to the apical surface.
In pancreatic
epithelial cells, the elevation of
[Ca]
in response to
extracellular signals is greatest in the most apical region of the
cell(54) . With strong stimulation a wave of elevated
[Ca
]
can then propagate through
the cell. It seems likely that in MDCK cells elevation of
[Ca
]
would similarly be
concentrated in the most apical region of the cytoplasm. As described
in the Introduction, the last known stage of pIgR transcytosis (i.e. step 3) is movement from the apical recycling
compartment (located immediately beneath the apical plasma membrane) to
the apical surface. It is possible that CaM binds primarily to the pIgR
in this compartment and is involved in stimulating this step of
transcytosis. Consistent with this hypothesis, we have observed that
this is the step of transcytosis that is stimulated by dIgA binding (22) , as well as by artificially raising
[Ca
]
.
This
localized increase in [Ca
]
could avoid the binding of CaM to pIgR in other locations, e.g. the TGN, which might lead to inappropriate delivery of
pIgR to the apical surface. Exocytosis of synaptic vesicles at nerve
terminals represents an extreme example of how a highly localized
increase in [Ca
]
leads to
membrane traffic at a precise location.
Signaling by dIgA binding to
the pIgR or other extracellular signals leads to activation of several
second messenger pathways, which may redundantly stimulate
transcytosis. In particular artificial elevation of
[Ca]
stimulates apical
transcytosis of several molecules (e.g. transferrin), although
these effects are in general smaller than those observed with
pIgR.
The molecular mechanisms of these effects are
unknown. We suggest that the specific binding of CaM to pIgR provides
an additional regulatory mechanism, which amplifies the stimulation of
transcytosis of pIgR.
CaM binding to the pIgR could have another,
non-mutually exclusive role in regulation of pIgR traffic. When dIgA
binds to pIgR, the pIgR initiates a signaling cascade involving
tyrosine phosphorylation. Preliminary data indicate that
mutations in the 17-residue basolateral targeting signal of the pIgR
prevent this signaling, suggesting that this segment has an additional
role in signal transduction. Perhaps CaM binding to the pIgR serves to
sequester this segment and thereby is part of a negative feedback loop
that shuts off signaling by the pIgR.
Taken together our data
indicate that the CaM binds to a portion of the pIgR that has multiple
functions in regulation of polarized traffic. It will be interesting to
learn if other receptors bind to CaM and if this is involved in
regulation of their traffic by
[Ca]
.