(Received for publication, August 14, 1995; and in revised form, October 31, 1995)
From the
Endocytic properties of the M-type 180-kDa receptor for
secretory phospholipases A (sPLA
) were first
investigated in rabbit myocytes that express it at high levels.
Internalization of the receptor was shown to be clathrin-coated
pit-mediated, rapid (k
= 0.1
min
), and ligand-independent. The signal sequence
for internalization was then identified upon transient and stable
expression of various receptor constructs with mutated cytoplasmic
sequences. Analysis of the internalization efficiency of the mutants
suggested that the NSYY motif encodes the major endocytic signal, with
the distal tyrosine residue playing the key role. Amino acid
substitutions at the putative casein kinase II phosphorylation site of
the receptor did not affect internalization. A chimeric protein
composed of the extracellular and transmembrane domains of the rabbit
sPLA
receptor and of the cytoplasmic domain of the
structurally homologous human macrophage mannose receptor retained the
high affinity for sPLA
and was internalization competent,
exhibiting 50% endocytic activity of the M-type sPLA
receptor. The results indicate the compatibility of the
structural domains of the two parent proteins and provide evidence for
the interchangeable character of their internalization signals.
Secretory phospholipases A (sPLA
s) (
)are implicated in a number of crucial physiological and
pathological processes in mammals (for recent reviews, see (1, 2, 3) ). The pancreatic-type sPLA
(group I), besides its well known digestive function, has been
implicated in fertilization(4) , cell
proliferation(5) , and contraction of vascular and airway
smooth muscles(6, 7) . Elevated levels of the
inflammatory-type sPLA
(group II) in serum, bronchial
lavage, and synovial fluids are associated with propagation of
inflammation and hypersensibilization of the organism (for a recent
review, see (8) ).
Different types of membrane receptors for
sPLAs have been recently identified using various types of
venom
sPLA
s(9, 10, 11, 12) .
The M-type receptor is a 180-kDa protein that was first identified in
rabbit muscle cells in culture (10) . It binds some venom
sPLA
s like OS
, one of the sPLA
s
from the Taipan snake venom with a K
value of 38 pM, but it does not bind bee venom
sPLA
(10) . The N-type receptor, which is abundant
in the brain (9) and is present in most tissues(12) ,
does not bind OS
, but it associates very tightly with bee
venom sPLA
(K
= 100
pM). It is made of polypeptide units of about 40-50 and
85 kDa(9) . The M-type receptor binds the pancreatic-type
sPLA
as well as the inflammatory-type sPLA
with K
values of about 1-10
nM(13, 14) . These sPLA
s are
probably the normal endogenous ligands of the M-type 180 kDa receptor.
Experiments with pancreatic-type sPLA
suggested some of its
effects, i.e. induction of DNA synthesis and cell
proliferation (5) contraction(6) , cell
migration(15) , and eicosanoid production (16) to be
mediated by the M-type receptor. However, the intracellular signal
transduction mechanisms linking these multiple effects to the event of
ligand-receptor interaction still remain obscure.
The high molecular
weight M-type sPLA receptors were cloned from
rabbit(13) , bovine(17) , mouse(18) , and human (19) tissues. It is a single subunit, type I plasma membrane
glycoprotein of 180-200 kDa. The molecule is made of a large
highly glycosylated extracellular N-terminal portion, comprising a
N-terminal cystein-rich region, a fibronectin-like type II domain, and
a repeat of eight carbohydrate-recognition domains in tandem followed
by a single transmembrane domain and a short intracellular C-terminal
tail. The overall molecular organization of the M-type receptor is
similar to that of the macrophage mannose receptor that mediates the
uptake of mannose-glycosylated ligands and the phagocytosis of
parasitic microorganisms (20, 21) and to the recently
discovered DEC-205 membrane protein, which is implicated in antigen
processing(22) . The structural similarity is especially
pronounced in the extracellular and membrane-spanning protein portions,
while the C-terminal cytoplasmic domains seem to be more specific for
each protein.
The mannose receptor and DEC-205 protein are known to
be internalized with high
efficiency(22, 23, 24) . Endocytosis is
intimately linked to their function as integral parts of the defense
and clearance systems. Internalization of the M-type sPLA receptor has also been observed(19, 25) , and it
could play an important role in receptor function. The purpose of this
work is to analyze in details the endocytic properties of the M-type
sPLA
receptor by determining its rate of internalization,
the utilized endocytic mode (ligand-induced or constitutive), as well
as the structural signals promoting the endocytic process.
For stable expression, HEK 293 cells (ATCC) were transfected with
mutant and wild-type sPLA receptor cDNA plasmid constructs
using the calcium phosphate precipitation method (28) . Cell
clones were selected based on acquired resistance to G418 (1 mg/ml
final concentration). Levels of M-type sPLA
receptor
expression were estimated by Scatchard plot analysis of sPLA
binding to cell membrane preparations. When used in ligand
binding and internalization experiments, HEK 293 cells were cultured on
polylysine-precoated plates.
Cell lysates were prepared by sonicating cell suspensions in 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride (lysis buffer) as described previously(10) . Membrane fractions were prepared by several cycles of freezing, thawing, and centrifugation of cells in lysis buffer(32) . Protein concentration was determined by the Bio-Rad adaptation of Bradford's dye-binding assay.
Fig. 1shows the results of a Western blotting of the rabbit
skeletal muscle cells membrane proteins and the indirect
immunofluorescence microscopy of cells using anti-sPLA receptor antibodies. In the blots, the antibodies recognized a
single protein band corresponding to a molecular mass of about 180 kDa.
This value is in a good agreement with earlier estimations of the
molecular mass of the mature highly glycosylated M-type sPLA
receptor(10, 11) . In immunofluorescence
experiments, the antibodies revealed an intensive cell-surface staining
typical for protein expressed at the plasma membrane.
Figure 1:
Expression of the
M-type sPLA receptor in rabbit skeletal muscle cells. A, Western blotting analysis of the endogenously expressed
M-type sPLA
receptor. The membrane protein fractions were
prepared from confluent primary cultures of rabbit skeletal muscle
cells. Aliquots (50 µg of total protein) were subjected to SDS-PAGE
(6.5% polyacrylamide) followed by Western blotting using guinea pig
anti-PLA
receptor antibodies (lane 1) or preimmune
serum (lane 2). The position of the molecular weight standards
is shown at left. B, indirect immunofluorescence staining for
the M-type sPLA
receptor in cultured rabbit skeletal muscle
cells. Confluent cells on coverslips were fixed with 4%
paraformaldehyde and then treated with anti-sPLA
receptor
antibodies and then fluorescein isothiocyanate-conjugated goat
anti-guinea pig IgG. Immunocomplexes were visualized by fluorescence
microscopy at a 400-fold magnification.
Fig. 2A shows the kinetics ofI-OS
internalization during the first 15
min after ligand addition. The internalization rate constant (k
) was 0.09 ± 0.005 min
(n = 5). Exposure of cells to a hypertonic medium
causes disassembly of membrane-associated clathrin lattices and, thus,
abolishes internalization mediated by clathrin-coated
pits(33) . Pretreatment of rabbit skeletal muscle cells with a
hypertonic sucrose-containing medium resulted in a complete blockade of
the receptor-mediated
I-OS
internalization (Fig. 2A). The observed blockade was obviously not due
to the modified ligand binding properties of the receptor since the
levels of total cell-associated radioactivity were the same in control
and sucrose-treated samples (not shown). These results suggest that
endocytosis of the sPLA
receptor occurs via clathrin-coated
pits.
Figure 2:
Internalization properties of the M-type
sPLA receptor in rabbit skeletal muscle cells. A,
kinetics of
I-OS
internalization. Cells kept
in the normal (open circles) or hypertonic sucrose-containing (closed circles) medium were exposed to
I-OS
(60 pM) for 3-15 min at
37 °C. The quantities of surface-bound and internalized ligand were
determined as described under ``Experimental Procedures.''
The results are mean values of three independent experiments, each
performed in quadruplicate. B, saturation plots for specific
cell-surface
I-OS
binding at 4 °C in
control (closed triangles) and monensin-treated (open
triangles) rabbit skeletal muscle cells. Cells were pretreated
with 10 µM monensin and processed as described under
``Experimental Procedures.'' All points were done in
duplicate. Nonspecific binding was subtracted from all experimental
values. Protein concentrations were determined on total cellular
lysates.
The effect of the long term exposure to the ligand was
investigated on cells pretreated for 30 min, 1, 2, and 24 h with 100
nM porcine pancreatic sPLA (K
= 10 nM)(13) . Following preincubation,
the cells were chilled on ice, washed with ligand-free medium, and
subjected to binding analysis using
I-OS
(K
= 38 pM(10) to
evaluate the number of remaining sPLA
binding sites. The
large difference in affinity of these respective ligands for the M-type
receptor ensured the quantitative substitution of the pancreatic enzyme
from the cell surface exposed ligand-receptor complex by
OS
. At all time points tested, the number of cell-surface
ligand binding sites in the sPLA
-pretreated samples was
0.1-0.13 pmol/mg of total protein, essentially the same as in
controls (data not shown). The results, thus, argue against receptor
sequestration and/or down-regulation upon prolonged exposure to the
pancreatic enzyme.
The possibility of a sequential uptake and
recycling of the unoccupied M-type sPLA receptor was
analyzed by comparing the quantity of the cell-surface PLA
binding sites in controls and in cells pretreated with a
carboxylic ionophore monensin, an inhibitor of trans-Golgi endosomal
trafficking(34) . Fig. 2B shows that
monensin-pretreatment caused a substantial, up to 57%, decrease in the
number of M-type sPLA
receptor molecules exposed at the
cell surface. The affinity for the ligand and the B
values estimated on total cell lysates were unaffected by the
inhibitor (not shown), ruling out any nonspecific inhibitory or
modifying effect of monensin on the sPLA
binding.
Figure 3:
Cytoplasmic tail of the M-type sPLA receptor and design of mutants. A, amino acid sequence
of the cytoplasmic domain of the M-type sPLA
receptor with
a scheme of the primary set of mutants. The arabic numbers above the sequence indicate the position of residues in the cytoplasmic
tail. The stretches of residues predicted to form tight
-turns are
indicated in boldface italics. I, II, III, the regions of potential endocytic signals. TR2,
and TR11, the positions of C-terminal truncations (TR plus the
number of remaining tail residues).
16-22, marks
the internal deletion with indicated positions of the first and the
last deleted residues. B, peptide sequence covering the second
potential internalization signal with indicated amino acid
substitutions. The mutated residues are shaded and underlined. The peptide sequences predicted to form tight turn
structures are in boldface italics. The code names of the
mutants are given at left. PLA
R, wild-type peptide sequence. C, amino
acid substitutions at the distal part of the M-type sPLA
receptor cytoplasmic domain. R/L34G,
/L34G,
substitution of Leu-34 by a glycine in the full-length and
16-22 receptors, respectively. S36A, S36D,
substitution of Ser-36 by an alanine and an aspartic acid,
respectively, at the putative casein kinase II phosphorylation site. D, comparison of the cytoplasmic sequences of the M-type
sPLA
receptors from different mammalian
species(13, 17, 18, 19) . The
identical amino acid residues are shaded. The cytoplasmic
sequences of the rabbit, human, and bovine M-type sPLA
receptors are given in full. *, the cytoplasmic portion of the
mouse receptor has 15 additional C-terminal residues that are not
shown.
Three mutants
were first designed for a preliminary mapping of endocytic signals (Fig. 3A). The mutant TR2 lacks all but two residues of
the cytoplasmic portion. The extramembrane dipeptide was left in the
sequence to ensure the anchoring of the expressed protein in the lipid
bilayer. In the mutant TR11, the carboxyl-terminal truncation was
introduced three amino acid residues downstream from the putative
internalization signal NKGFF. The deletion mutant 16-22
lacks the peptide sequence NSYYPTT of the receptor tail.
Wild-type
and mutant M-type sPLA receptors were transiently expressed
in COS cells. Cell surface expression of each construct was analyzed by
indirect immunofluorescence microscopy (Fig. 4). All constructs
exhibited an evenly dispersed pattern of staining with high
fluorescence intensity at cell borders characteristic for a plasma
membrane-delivered protein. Western blotting analysis of membrane
proteins (Fig. 5A) revealed similar levels of receptor
expression for all constructs. The receptors were recognized by
antibodies as single protein bands of the expected electrophoretic
mobilities (apparent molecular mass, 170-180 kDa). No sign for
proteolytic degradation of the expressed mutants was detected. The
expression level of receptor constructs (B
)
estimated on total cell lysates was typically 1-2 pmol/mg of
protein, while control COS cells had no OS
binding sites
(not shown). The affinity of
I-OS
for the
different mutant receptors was essentially the same as that of the
wild-type receptor (K
= 40 ± 10
pM) and of the endogenous receptor in rabbit skeletal muscle
cells. Taken together, all of these results indicate that deletions in
the receptor cytoplasmic domain do not affect the plasma membrane
delivery and the binding properties of the M-type sPLA
receptor.
Figure 4:
Cell
surface expression of the wild-type sPLA receptor (A),
16-22 (B), TR11 (C), and TR2 (D) mutants in transfected COS cells. The transfected cells
were fixed with paraformaldehyde after 72 h of expression and incubated
with polyclonal anti-sPLA
receptor antibodies followed by
fluorescein isothiocyanate-conjugated goat anti-guinea pig IgG.
Immunocomplexes were visualized by fluorescence microscopy at a
1000-fold magnification.
Figure 5:
Expression and internalization activity of
deletion mutants of the M-type sPLA receptor. A, western blotting analysis of the wild-type (lane 1) and
mutant sPLA
receptors (lanes 2-4). Aliquots
of COS cell membrane fractions (10 µg of total protein) were
subjected to SDS-PAGE (6.5% polyacrylamide) followed by Western
blotting using anti-sPLA
receptor antibodies. Indications
correspond to those in Fig. 4. Lane 5, control
mock-transfected cells. The position of the molecular weight standards
is shown at left. B,
I-OS
internalization in transfected COS cells. The cells expressing
the wild-type and mutant sPLA
receptors were incubated with
I-OS
(60 pM) for 3-15 min at
37 °C. Internalization rate constants (k
) were determined as described under
``Experimental Procedures.'' The results are the mean values
of three independent experiments, each performed in
triplicate.
Fig. 5B represents a diagram of specific
internalization rate constants (k) for the wild-type
and mutant M-type sPLA
receptors. The
I-OS
internalization by the wild-type
receptor was 0.02 ± 0.003 min
. The
16-22 mutant internalized the ligand with a 33% efficiency
as compared with the wild-type receptor (k
= 0.006 ± 0.002 min
). The TR11
and TR2 receptor mutants were essentially internalization deficient (k
= 0.001 ± 0.001 min
and 0.0003 ± 0.0002 min
, respectively).
The significantly lowered internalization rate constant of the
16-22 mutant indicated receptor endocytosis to be mainly
dependent on the deleted NSYYPTT sequence.
A series of point amino
acid substitutions in the putative signaling region was then introduced
to define more precisely the determinants required for efficient M-type
sPLA receptor endocytosis. The list of residues mutated in
the region NSYYPTT is shown in Fig. 3B. Western
blotting and immunofluorescence analyses revealed that all mutants were
expressed, processed, and delivered to the plasma membrane (not shown).
The affinity of all the mutants for
I-OS
was
essentially the same as that of the wild-type receptor. A diagrammatic
representation of internalization rate constants for the wild-type and
mutant receptors is shown in Fig. 6. Substitution of any of the
two tyrosine residues in the NSYYPTT sequence by an alanine (mutants
Y18A and Y19A) dramatically impaired receptor internalization.
Internalization rate constants (k
= 0.0054
± 0.0005 min
and 0.002 ± 0.001
min
, respectively) were at most 27 and 10% of those
of the wild-type receptor. Substitution of Pro-20 by an alanine (P20A)
had little effect on internalization (k
=
0.018 ± 0.003 min
). Mutation of Ser-17 to a
proline (S17P) increased
I-OS
internalization
by 25% (k
= 0.025 ± 0.001
min
).
Figure 6:
Internalization activity of mutants with
point amino acid substitutions. All mutants were transiently expressed
in COS cells. The rate constants were calculated as described under
``Experimental Procedures.'' A, comparison of
internalization rate constants of the wild-type (PLAR) and mutant receptors. B,
I-OS
internalization by the mutant
receptors tentatively mimicking putative phosphorylated (S36D) and
dephosphorylated (S36A) receptor states.
The 16-22 mutant was still
capable of relatively high internalization (Fig. 6). To verify
that the endocytosis of the mutant remained mediated by clathrin-coated
pits, transfected COS cells were incubated in the hypertonic sucrose
medium 30 min prior to internalization experiments. A total blockade of
I-OS
internalization into these cells
(results not shown) proved mutant internalization via clathrin-coated
pits and suggested an implication of some other additional regions
(presumably, LI motif) in the process. The role of the LI motif was
assessed by substitution of Leu-34 by a glycine in the sequence of the
wild-type and
16-22 receptors (mutants R/L34G and
/L34G, respectively; Fig. 3C). The
I-OS
internalization by the
/L34G
receptor was practically abolished and represented only 1% of the
wild-type efficiency (Fig. 6). The same amino acid substitution
in the full-length receptor (R/L34G mutant) did not affect
internalization (k
= 0.019 ± 0.002
min
). These results indicate an implication of the
LI motif in internalization of the
16-22 mutant receptor but
argued against an analogous role of this motif in the context of the
full-length receptor.
The internalization rate constant of the
M-type sPLA receptor was at least 4-fold higher in rabbit
skeletal muscle cells than in transfected COS cells (see above),
suggesting that the molecular principles and/or signaling structures
governing receptor internalization in these cell lines could differ. To
verify that the observed differences in the internalization efficiency
of the mutants were not restricted to one particular cell line, the
experiments were reproduced in HEK 293 cells stably transfected with
the wild-type,
16-22, and TR11 receptors. The HEK 293 cell
line, similar to COS cells, lacks endogenous M-type sPLA
receptor. Internalization experiments were performed on two
independent cell clones for each receptor construct, expressing
0.5-2 pmol of M-type receptor/mg of total protein. Fig. 7shows a diagram of internalization rate constants of the
wild-type and mutant receptors in HEK 293 cells. The full-length
receptor internalized
I-OS
as efficiently
(k
= 0.067 ± 0.003 min
) as
in rabbit skeletal muscle cells. The rate constant of the
16-22 mutant was also relatively high (k
= 0.03 ± 0.005 min-
), representing 45%
of the wild-type receptor efficiency. The TR11 mutant was
internalization deficient (k
= 0.001 ± 0.002
min
). All these experiments confirmed the results
obtained in the corresponding studies with COS cells, although the
latter have significantly lower internalization efficiency.
Figure 7:
Internalization rate constants of the
wild-type (PLAR),
16-22, and TR11 receptors stably expressed in HEK 293 cells.
The rate constants were calculated as described under
``Experimental Procedures.''
Figure 8:
Molecular organization and internalization
efficiency of the chimeric sPLA/mannose receptor. A, schematic representation of the molecular organization of the
rabbit M-type sPLA
and human macrophage mannose receptors (PLA
R and MAN R,
respectively). The structural organization of the chimera is boxed. The external (OUT), transmembrane (TM), and cytoplasmic (IN) domains are shown,
although not to scale. Numbers indicate the location of the first and
last cytoplasmic residues in the polypeptide chain of the receptors. B,
I-OS
internalization by the
chimeric (PLA
/MAN) and the
wild-type (PLA
R)
receptor.
This paper describes the endocytic properties of the M-type
sPLA receptor and characterizes its structurally encoded
internalization signal(s). The endocytic receptors studied to date can
be subdivided into two main groups. Receptors of the first group
recycle constitutively and function as shuttles delivering their
ligands to the cell interior. Their internalization is independent on
ligand binding. It can be rather fast, i.e. 10-60%/min
(corresponding to a k
of 0.1-0.6
min
) for the LDL, asialoglycoprotein(38) ,
or transferrin receptors (30) or slow, i.e. 1-2%/min for the Fc and fibronectin
receptors(37, 39) . Internalization of the other group
of receptors (such as hormone and growth factor receptors) is clearly
ligand-dependent. It is negligible in the absence of their ligands and
can be increased more than 50-fold (k
of
0.3-0.6 min
) in the presence of corresponding
ligands(40, 41) . In many cell types, a long term
exposure of this latter group of receptors to their ligands leads to
rapid intracellular sequestration and degradation (down-regulation) of
the receptors.
The calculated internalization rate constant of the
M-type sPLA receptor in a primary culture of rabbit
skeletal muscle cells (k
= 0.1
min
) was that of efficiently internalized receptors
but was significantly lower than that reported for the structurally
homologous mannose receptor (k
= 4.12
min
) in sinusoidal endothelial cells from rat
liver(24) . The blockade of
I-OS
uptake observed under hypertonic conditions, which causes a
disassembly of plasma membrane-associated clathrin
lattices(33) , proved receptor internalization via
clathrin-coated pits (Fig. 2A). Long term exposure to a
high concentration of a possible endogenous ligand, pancreatic-type
sPLA
, had no effect on the number of the cell
surface-exposed
I-OS
binding sites arguing
against ligand-induced sequestration and/or down-regulation of the
sPLA
receptor in rabbit skeletal muscle cells. In the
absence of the added ligand, monensin, a classical inhibitor of the
trans-Golgi endosomal trafficking(31, 34) , produced a
large, more than 57% reduction in the number of cell surface
OS
-binding sites, indicating the rapid uptake and recycling
of the ligand-free sPLA
receptor. Similar extent of the
monensin-induced protein trapping within the cells was reported earlier
for the constitutively recycling transferrin receptor(34) .
Taken together, the above results suggest that internalization of the
M-type sPLA
receptor in rabbit skeletal muscle cells is
rapid, clathrin-coated pit-mediated, and ligand-independent. These
properties allowed us to classify the M-type sPLA
receptor
to the group of constitutively recycling receptors, the same as for its
structural homologue, the mannose
receptor(20, 21, 23) . The classification of
the M-type receptor into this group is in contrast with previously
suggested role of the sPLA
receptor in cell proliferation (5) and eicosanoid production(16) . Indeed, such
biological effects are generally supposed to be mediated by receptors
of the other group, i.e. by ligand-activated receptors. This
apparent inconsistency is presently difficult to explain. Noteworthy,
the mannose receptor, which also belongs to the group of constitutively
recycling receptors, was shown to be involved in cell proliferation (42) .
Internalization of the membrane receptors is
dependent on their cytoplasmic signal sequences selectively recognized
by adaptor proteins of clathrin-coated pits (for a recent review, see (37) ). The internalization signals identified to date fall
into three main groups. The first group is represented by four to
six-amino acid sequences containing an aromatic residue, usually a
tyrosine, in a tight -turn
structure(43, 44, 45, 46, 47) .
This type of signal is found in the vast majority of endocytic
receptors, a typical example being a consensus NPXY motif (36) , first detected in the structure of the LDL
receptor(43) . The dileucine (or leucine-isoleucine)
dipeptide(48, 49, 50, 51, 52, 53) and
the recently identified KK(X/F)(X/F) sequence (54) represent two other groups of internalization signals.
Importantly, analogous motives were shown to be involved in other
intracellular sorting events, such as lysosomal
targeting(48, 55, 56, 57, 58, 59) ,
transcytosis, and basolateral sorting in polarized
cells(53, 60, 61, 62) , and in the
endoplasmic reticulum protein
retrieval(54, 63, 64) .
The cytoplasmic
portion of the rabbit sPLA receptor contains three
stretches of potential signal sequences (Fig. 3): (i) the tight
turn-forming region NSYY; (ii) the LI dipeptide in the distal part of
the tail; and (iii) the juxtamembrane NKGFF tight turn region, which
resembles the internalization signal QQGFF of the epidermal growth
factor receptor(35) . The latter motif, being poorly conserved
in mammalian sPLA
receptors (Fig. 3D), was a priori considered the least plausible candidate for an
internalization signal. Indeed, the receptor mutants TR11 and
16-22, although retaining this peptide sequence, exhibited
dramatically impaired internalization (Fig. 5). Conversely, the
internalization defect of the
16-22 mutant strongly
suggested the NSYY motif to be the endocytic signal of the rabbit
M-type sPLA
receptor (Fig. 6).
The NSYY motif,
although obviously related to the NPXY signal of the LDL receptor, has
some peculiar features (Fig. 3D). The sequence contains
two evolutionary conserved tyrosine residues instead of one, and in the
rabbit and mouse M-type sPLA receptors, the canonical
proline residue is substituted by a serine. Alanine scanning of the
region has proved Tyr-19 to be the key residue of the signal sequence.
This feature clearly distinguishes the M-type sPLA
receptor
from the structurally homologous macrophage mannose receptor, where an
alanine substitution of the single cytoplasmic tyrosine impaired
internalization only partially (about 50% of the wild-type
efficiency)(65) . The internalization defect of the Y18A mutant
(25% of the wild-type rate) suggested that in the signal sequence motif
of the rabbit M-type sPLA
receptor, the amino acid residue
occupying the consensus X position can be a subject for
certain structural restraints. Substitution of Ser-17 by a proline was
made to reconstitute the canonical NPXY motif. The mutation increased
internalization, but only slightly (25%), indicating that both serine
and proline residues are permissible at this position. Indeed, both
residues are known to be turn-promoting(66) . However, the
M-type sPLA
receptor is to our knowledge the first reported
example of an endocytic receptor in which this substitution has
naturally occurred. Alanine substitution of the highly conserved Pro-20
immediately adjacent to the critical tyrosine residues (Fig. 3D) was of no significant effect on endocytosis,
suggesting that this residue is not important for receptor
internalization.
Interestingly, the internalization of the
16-22 mutant, although significantly impaired, was still
clathrin-coated pit-mediated, and considerably higher than that of the
C-terminally truncated mutants TR11 and TR2 (Fig. 5).
Substitution of Leu-34 by a glycine (
/L34G) abolished
internalization and proved the endocytosis of the
16-22
mutant to be dependent on the LI motif (residues 34-35). In the
full-length receptor, however, the same glycine substitution had no
effect on internalization (R/L34G mutant, Fig. 6). These results
suggest that the LI motif, although recognized as an internalization
signal in the receptor mutant with the deleted NSYY region, does not
play the same role in the context of the full-length sPLA
receptor. The data on the presence of ``cryptic''
internalization (sorting) signals in the cytoplasmic portions of the
proteins are not unprecedented. Analogous findings were made for the
lysosomal acid phosphatase, the interleukin 6 signal transducer gp130,
and the cation-independent mannose 6-phosphate/insulin-like growth
factor II receptor(51, 57, 67) .
The
negatively charged region surrounding the LI motif of the sPLA receptor is highly conserved from mouse to human (Fig. 3D) and contains a consensus casein kinase II
phosphorylation site, with Ser-36 as a putative target
residue(13) . Similar structures were found in the cytoplasmic
domains of many recycling receptors (68) and are supposed to be
implicated in intracellular sorting events, such as basolateral
targeting(60) , sorting into Golgi-derived clathrin-coated
pits(57) , and/or to regulate the efficiency of receptor
internalization(69) . Amino acid substitutions at Ser-36
mimicking putative phosphorylated and dephosphorylated sPLA
receptor states (Fig. 6B) were of no significant
effect on the rate of endocytosis, arguing against implication of the
region in internalization of the M-type sPLA
receptor.
The M-type sPLA receptor and the macrophage mannose
receptor are predicted to share the same structural
organization(13) , although the amino acid sequence identity of
these two receptors is quite low (28% identity in the extracellular
region and only 17% in the cytoplasmic tail). The cytoplasmic NTLY
motif of the macrophage mannose receptor resembles the NSYY signal
sequence of the rabbit M-type sPLA
receptor and was
previously shown to be implicated in internalization(65) . It
was, thus, interesting to analyze the internalization properties of a
chimera composed of the predicted extracellular and transmembrane
domains of the rabbit M-type sPLA
receptor and of the
cytoplasmic portion of the human macrophage mannose receptor. The
chimeric receptor was properly delivered to the plasma membrane and
displayed the normal binding affinity for
I-OS
, proving the correct assignment of the
topological domains. The internalization efficiency of the chimera was
only 50% less efficient than that of the wild-type sPLA
receptor, suggesting that the cytoplasmic domains and the encoded
internalization signals of these two proteins are interchangeable.
In conclusion, this work has revealed the mechanism of endocytosis
of the M-type sPLA receptor and has identified the
structural regions implicated in its internalization. The role of the
M-type sPLA
receptor internalization process remains,
however, obscure. Internalization of the M-type sPLA
receptor could first be an important component of the signal
transduction system coupled to the binding of the sPLA
to
its specific receptor. It could serve to terminate the signals produced
by sPLA
on target cells. Alternatively, the endosomal
vesicles could serve as a vehicle delivering sPLA
to
specific intracellular compartments, where the ligand, before being
degraded in lysosomes, could manifest its enzymatic activity. Another
possibility is that the internalization of the M-type sPLA
receptor could serve a clearance function selectively withdrawing
sPLA
from the extracellular fluid. This could be of a
crucial importance at various inflammatory disease states (such as
rheumatoid arthritis, acute peritonitis, septic shock) when high levels
of inflammatory type II sPLA
are produced and
secreted(8) . Further investigations on the properties of the
M-type sPLA
receptor in various tissues and species should
provide more evidence on its function and the role of internalization
process, in particular.