(Received for publication, January 2, 1996)
From the
Receptor-mediated endocytosis is the cellular mechanism by which
type C receptors of natriuretic peptides exert their clearance
function. In the present work, performed in recombinant Chinese hamster
ovary cells stably transfected with wild type or mutated human kidney C
receptors, we determined net endocytic rates (ER) of C receptor-ligand
complexes, lysosomal hydrolysis of ligand (I-labeled
native atrial natriuretic factor, ANF
), and
receptor recycling. Equilibrium ligand binding, immunocytochemistry,
and immunoprecipitation were performed to characterize the transfected
receptors. The net ER of recombinant wild type C receptors was
6%
of occupied receptors internalized per min, and C receptor-mediated
lysosomal hydrolysis of ligand amounted to
250% of specifically
bound
I-ANF
/h, with efficient
recycling of internalized C receptors to the cell surface. Hypertonic
sucrose reduced net ER and lysosomal hydrolysis of
I-ANF
more than 10-fold, indicating
that endocytosis occurred via clathrin-coated pits. Total deletion of
the cytoplasmic domain also reduced net ER and lysosomal hydrolysis of
I-ANF
by almost 10-fold, whereas
deletion of the terminal 28 amino acids of the cytoplasmic tail led to
a 4-fold reduction in these parameters. Replacement of cytoplasmic
domain Tyr
by Ala, or Tyr
and Phe
by Ala, reduced net endocytosis and lysosomal hydrolysis of
I-ANF
by 40-50%. Replacement
of extracellular domain Cys
by Ala impeded the
constitutive formation of homodimers and reduced by
50% the net ER
and lysosomal hydrolysis of
I-ANF
.
These results demonstrate that the cytoplasmic domain of C receptors,
Tyr
within this domain, and constitutive receptor
dimerization are the major molecular determinants of the clearance
function of C receptors.
Natriuretic peptides, particularly atrial natriuretic factor
(ANF), ()play an important role in volume-pressure
homeostasis(1) . There are two functionally and biochemically
distinct classes of receptors for natriuretic peptides. Guanylyl
cyclase receptors (subtypes A and B) are signaling receptors that
mediate the biological effects of natriuretic peptides via the
generation of cGMP. Clearance (C) receptors play an important role in
the removal of natriuretic peptides from the circulation, thus
contributing to their plasma homeostasis(1, 2) .
C receptors have a very high affinity for all members of the natriuretic peptide family, including ANF, type B natriuretic peptide, and type C natriuretic peptide, as well as truncated forms of natriuretic peptides containing as few as five amino acids from their ring structure(2, 3, 4) . C receptors are more abundant than guanylyl cyclase receptors and are localized in tissues and cells that receive a large fraction of the cardiac output, including vascular endothelial and smooth muscle cells and the renal cortex(2) . Blockade of C receptors by specific ligands decreases the metabolic clearance of ANF and consequently increases its plasma levels in mammals(3, 4) . Thus, a major function of C receptors is to serve as a hormone buffer system to impede inappropriate alterations in plasma levels of natriuretic peptides(3) . Although there is some controversy over whether C receptors may also function as signaling receptors, it has been well documented that they do not mediate the major known cardiovascular, adrenal, and renal effects of natriuretic peptides(2, 5) .
The C receptor is a 60-kDa
protein that is constitutively present in the cell membrane as a
homodimer of
120 kDa(2, 6) . The receptor has a
large NH
-terminal extracellular ligand binding domain, a
single transmembrane domain, and a short COOH-terminal cytoplasmic tail
of only 37 amino acids(6) . A short cytoplasmic domain is a
structural characteristic of all clearance and/or transport receptors
whose structure is known, including low density lipoprotein,
asialoglycoprotein, transferrin, cation-independent
mannose-6-phosphate-IGF type II, and polymeric immunoglobulin receptors (7, 8, 9, 10, 11, 12, 13) .
C receptors have a single tyrosine in their cytoplasmic domain
(Tyr
), an amino acid that has been shown to play a
central role in clathrin-coated pit endocytosis of
receptors(7, 8, 9, 10, 11, 12, 13) .
However, the flanking sequences of tyrosine in the C receptor do not
bear any resemblance to the degenerate sequences that contribute to
rapid internalization of other receptors (e.g. NPXY
or YXXZ, where X is any amino acid, and Z is
a large hydrophobic amino acid)(7) . Moreover, there are no
other recognized internalization motifs (e.g. dileucine) in
the cytoplasmic domain of C receptors.
Our previous studies demonstrated that native C receptors in mammalian cells undergo rapid constitutive endocytosis that is not stimulated further by ligand binding. Endocytosed receptor-ligand complexes are then dissociated intracellularly, most probably in endosomes, with a subsequent hydrolysis of the ligand in lysosomes and an efficient recycling of the internalized receptors to the cell surface(14) . In the present work we studied some of the molecular determinants of the clearance function of C receptors using cloned wild type and mutated human kidney C receptors transfected into Chinese hamster ovary (CHO) cells.
The results show that the internalization of C receptors via clathrin-coated pits depends on the integrity of their cytoplasmic domain. Within this domain, tyrosine contributes significantly to rapid endocytosis, albeit to a lesser degree than in other receptors in which it is flanked by conserved internalization motifs. Constitutive dimerization of C receptors, a property conferred by an unpaired cysteine residue in the extracellular domain, markedly enhances endocytosis, demonstrating that constitutive polymerization is an important determinant for rapid internalization of these receptors.
Figure 1:
Juxtamembrane domains and equilibrium
ligand binding parameters of cloned wild type and mutated human kidney
natriuretic peptide C receptors stably transfected into CHO cells. The left side of the figure depicts the sequences of amino acids
of wild type and mutated C receptors beginning at position 473 in the
extracellular (EC) domain and encompassing the entire
intracellular (IC) sequence flanking the putative
transmembrane (M) domain. The recombinant CHO cells are named
according to nature of the transfected C receptor (CR). The
amino acids in the wild type C receptor (CHOCRWT), whose codon was
mutated (see ``Experimental Procedures''), are numbered in superscripts. The heavy lines indicate that the
cytoplasmic tail was truncated at arginine 514 (CHOCR514t) or arginine
505 (CHOCRCYT-). The underlined alanines in bold are point mutations replacing the correspondingly numbered amino
acids in CHOCRWT (for a description of the mutagenesis and transfection
techniques, see ``Experimental Procedures''). The right side of the figure gives the values (mean ± S.E., n = 3) for the apparent density (B) of
surface receptors and their apparent ligand affinity (K
) in each of the recombinant cell
lines, as determined by equilibrium competition binding experiments
with
I-ANF
(``Experimental
Procedures'').
Figure 4:
Dynamics of endocytosis of receptor-ligand
complexes, lysosomal hydrolysis of ligand, and receptor recycling in
recombinant CHO cells stably transfected with cloned wild type
natriuretic peptide C receptors. Recombinant CHOCRWT cell monolayers
were incubated with 1 ml of binding solution containing I-ANF
and near saturating
concentrations of ANF
(1.3 nM) at 37
°C. Experiments were performed in control conditions (panel
A) or in the presence of 10 mM NH
Cl (panel B). At the end of each incubation time, cell monolayers
were processed as described under ``Experimental Procedures''
to determine membrane-bound radioactivity (M), intracellular
radioactivity (IC), and
I-ANF
degradation products in medium (DM). Results are from a
representative experiment in duplicates (for a description, see
``Results'').
Fig. 1depicts the partial sequences of wild type and
mutated C receptors, the apparent receptor-ligand (I-ANF
) equilibrium dissociation
constants (K
), and the apparent surface densities (B
) of C receptors in the recombinant cells.
Pilot experiments revealed that specific binding of
I-ANF
to nontransfected CHO-K1 cells
was negligible, a finding that was confirmed by immunocytochemistry
(see Fig. 2) and immunoprecipitation (see Fig. 3) The
recombinant cell clones used in the experiments were selected to have
an apparent density (B
) in the range of those
reported for native C receptors in mammalian cells. The apparent
affinity (K
) for ANF
in
CHOCRWT and in all cells expressing point-mutated receptors was <
0.1 nM, a value similar to that reported previously for native
C receptors in mammalian cells(2) . The apparent K
of recombinant receptors with total deletion
(CHOCRCYT-) or large truncation (CHOCR514t) of the cytoplasmic
domain was still low (
1 nM) but significantly higher than
that of the wild type and point-mutated C receptors (p <
0.01). C-ANF
(a specific ligand of C receptors)
displaced specifically bound
I-ANF
from CHOCRWT with a K
similar to that
reported previously for native C receptors (3) (data not
shown). Trypsinization-solubilization experiments in recombinant
CHOCRWT cells showed that at equilibrium, 68.4 ± 6.9% or
two-thirds of the total number receptors expressed in these cells
were localized in the cell membrane (n = 5 wells in two
independent experiments), a value similar to that found for native C
receptors in bovine aortic vascular smooth muscle cells (14) .
Figure 2: Immunofluorescence optical microscopy of cloned wild type and mutated human kidney natriuretic peptide C receptors stably transfected into CHO cells. Nontransfected (panel A) or recombinant (panels B-H) CHO cell clones fixed with formaldehyde were exposed to mouse monoclonal antibody against human kidney C receptors and visualized for optical microscopy immunofluorescence with anti-mouse IgG-fluorescein isothiocyanate conjugate as described under ``Experimental Procedures.'' High intensity specific fluorescence is seen in all recombinant CHO cell clones but not in nontransfected CHO cells. Panel A, CHO- (nontransfected); panel B, CHOCRWT; panel C, CHOCRCYT-; panel D, CHOCR514t; panel E, CHOCRA508; panel F, CHOCRA538; panel G, CHOCRA508/A538; panel H, CHOCRA473.
Figure 3:
Characterization of C receptor proteins
stably transfected in CHO cells. The lysate of nontransfected
(CHO-) and recombinant CHO cell clones prelabeled with a mixture
of S-labeled methionine and cysteine were
immunoprecipitated with monoclonal antibody against human kidney C
receptor, and their mobility in SDS-PAGE was determined under reducing (+DTT) and nonreducing (-DTT) conditions,
as described under ``Experimental Procedures.'' The
autoradiogram shows that CHO- cells do not contain detectable C
receptors, whereas intense autoradiographic bands are visible in the
immunoprecipitated extracts of all recombinant cells. Under nonreducing
conditions (-DTT), CHOCRWT and all receptors with
cytoplasmic tail mutations (CHOCRCYT-, CHOCR514t, CHOCRA508,
CHOCRA538, and CHOCRA508/A538) migrate as a
120-kDa protein, with
a smaller proportion migrating as a
60-kDa protein. As expected,
the truncated mutants (CHOCRCYT- and CHOCR514t) migrate at
correspondingly lower molecular masses. Under reducing conditions (+DTT) the
120-kDa band disappears, and a prominent
60-kDa band is seen. This confirms that CHOCRWT is constitutively
present as a homodimer and that the cytoplasmic tail mutations do not
alter the state of polymerization of the receptor. CHOCRA473 receptors,
as indicated by the arrows, migrate only as a
60-kDa
protein under reducing and nonreducing conditions, demonstrating that
the replacement of cysteine 473 by alanine impedes the constitutive
dimerization of C receptors (for a description, see
``Results'').
Fig. 2shows the immunofluorescence signal in nontransfected CHO cells and in CHO cells transfected with wild type or mutated C receptors used in the present experiments. The results show that all recombinant cells, but not nontransfected CHO cells, displayed an intense fluorescence signal. No obvious qualitative differences were observed between the morphological expression of transfected wild type and mutated receptors in the recombinant cells at the resolution of the optical microscope.
Fig. 3shows an SDS-PAGE autoradiogram of
extracts from recombinant CHO cells immunoprecipitated with a specific
monoclonal antibody against C receptors. In nontransfected cells,
immunoprecipitated bands were very faint, if at all present, confirming
that CHO-K1 cells do not express a significant density of native C
receptors. As expected, under reducing and nonreducing conditions, the
truncated forms of recombinant receptors (CHOCRCYT-, and
CHOCR514t) migrated at slightly lower molecular weights than CHOCRWT or
the point mutated C receptors. Additional bands of unknown nature
appeared in CHOCR514t under nonreducing conditions. They are probably
an artifact, because under reducing conditions a single high intensity
band was detected with this mutant. Under nonreducing conditions the
recombinant receptors, except CHOCRA473, migrated as two bands, a
higher density band of 100-120 kDa, and a lower density band
of
50-60 kDa. CHOCRA473 migrated as a single band of
60
kDa. Under reducing conditions, the migration pattern of CHOCRA473
remained unchanged, whereas in all other recombinant cells there was a
virtual disappearance of the 100-120-kDa band and a prominent
intensity of the 50-60-kDa band. Thus, all recombinant receptors,
except CRA473, were present mainly as homodimers with a small
proportion of monomers. This latter pattern is typical of native C
receptors in several cell types(2) . The finding that CRA473
was only present in monomeric form under nonreducing conditions
confirms previous observations by other investigators showing that
cysteine 473 is essential for C receptor
dimerization(24, 25) .
Fig. 4depicts the
results of a typical experiment on receptor-mediated internalization
and hydrolysis of I-ANF
in CHOCRWT
cells. Panels A and B show the time course of
specific binding of
I-ANF
to the
cell membrane, the accumulation of intracellular radioactivity, and the
appearance of
I-ANF
radioactive
metabolites in the medium in control conditions (panel A) or
in the presence of 10 mM NH
Cl (panel B).
In control conditions, specific membrane-bound
I-ANF
remained nearly constant from
10 to 60 min, indicating rapid equilibrium binding and lack of
ligand-induced down-regulation of surface receptors during the course
of incubation. Shortly after the start of the incubation, radioactivity
accumulates in the intracellular compartment, reaching a peak by 10
min, and then maintaining near steady-state levels for the remainder of
the experiment. In control conditions, there was a near linear increase
in the appearance of labeled degradation products in the medium, and by
60 min the sum of degraded radioactivity in the medium and
intracellular radioactivity far exceeds that of
I-ANF
bound to the cell surface,
demonstrating that internalized recombinant receptors are replenished
at the cell surface. The protein synthesis inhibitor cycloheximide did
not alter this pattern (not shown), indicating that the replenishment
of internalized receptors at the cell surface is due to recycling
rather than de novo synthesis of receptors. Panel B shows that NH
Cl had practically no effect on the
initial binding of radioligand to cell surface receptors or the initial
(first 5-10 min) internalization rates of receptor-ligand
complexes. At steady state, membrane-bound specific radioactivity in
NH
Cl-treated monolayers was slightly but consistently
higher than in control cells. The major effect of NH
Cl,
however, was to block almost completely the appearance of labeled
degradation products in the medium, confirming that this lysosomotropic
weak base inhibits the lysosomal hydrolysis of internalized
I-ANF
(14) . As a result,
intracellular accumulation of radioactivity in NH
Cl-treated
monolayers was approximately three times greater than in control cells
by 60 min. The dynamics of recombinant wild type C receptor-ligand
complexes in CHO cells shown in Fig. 4is very similar to that
determined previously in our laboratory for native C receptors in
bovine aortic vascular smooth muscle cells(14) .
In the
experiment shown in Fig. 4, the calculated net endocytic rate of
receptors-ligand complexes, which measures the difference between the
rates of internalization (endocytosis) and return of undissociated
receptor-ligand complexes to the cell surface
(retroendocytosis)(14) , was maximal at 5 min (9.6% of occupied
receptors internalized per min), the first time of measurement, and
then reached near steady-state levels between 10 and 60 min
(6%/min). Hereafter, all results on endocytic rates are expressed
as steady-state net internalization rates (see
``Discussion'').
Fig. 5depicts the values of
receptor-mediated lysosomal (NHCl-sensitive) hydrolysis of
I-ANF
(upper panel) and net
endocytic rate of receptor-ligand complexes (lower panel) in
recombinant CHO cells expressing wild type and cytoplasmic-truncated
forms of C receptors. Receptor-mediated lysosomal hydrolysis of
I-ANF
in CHOCRWT increased linearly
with time from 10 to 60 min (r
= 0.993) and
by 60 min amounted to 268 ± 14% of
I-ANF
specifically bound to the cell
surface. Total deletion of the cytoplasmic domain (CHOCRCYT-)
almost completely abolished lysosomal hydrolysis of radioligand,
decreasing it to 27 ± 2% of specifically bound
I-ANF
in 60 min, a 10-fold lower
value than that in CHOCRWT (p < 0.001). This low level of
lysosomal hydrolysis is similar (p > 0.05) to that obtained
when endocytosis of receptor-radioligand complexes in CHOCRWT is
blocked by hypertonic sucrose (17 ± 1% of specifically bound
I-ANF
in 60 min). Deletion of 28 of
the 37 amino acids of the cytoplasmic tail (CHOCR514t) markedly
decreased (p < 0.001) lysosomal hydrolysis of
I-ANF
to 58 ± 1% of
specifically membrane-bound radioligand in 1 h. The slope of the
regression line defining the time course of lysosomal hydrolysis of
radioligand in CHOCRWT was significantly (p < 0.001)
greater than that in CHOCRCYT- or CHOCR514t.
Figure 5:
Receptor-mediated lysosomal hydrolysis of
ANF, and net endocytic rate of receptor-radioligand complexes in
recombinant CHO cells stably transfected with wild type and cytoplasmic
truncated forms of natriuretic peptide C receptors in control
conditions or in the presence of hypertonic sucrose. Recombinant CHO
cell monolayers were incubated in six-well plates at 37 °C in 2 ml
of binding medium containing 0.3-0.5 µCi of I-ANF
for a total duration of 60 min
in the presence or absence of 10 mM NH
Cl.
Experiments were also performed in control (Ctrl) conditions
or in the presence of 0.44 M sucrose (HS). At the
times indicated on the abscissa of the upper graph,
0.1 ml of medium was removed to determine
I-ANF
hydrolytic products in medium,
as described under ``Experimental Procedures.'' At the end of
a 60-min incubation the monolayers were processed to determine
membrane-bound and intracellular radioactivity as described under
``Experimental Procedures.'' Lysosomal hydrolysis (upper
panel) was calculated by the appearance of
NH
Cl-sensitive hydrolytic products of
I-ANF
in medium (see Fig. 4)
and expressed as percent of specifically membrane-bound radioligand at
60 min. Net endocytic rates of receptor-radioligand complexes at steady
state (lower panel) were calculated as described under
``Experimental Procedures'' and are expressed as percent of
occupied receptors internalized per min, from a 20-60-min
incubation. CRWT, CHO cells transfected with wild type C
receptors; CR514t, CHO cells transfected with C receptors in
which the terminal 28 amino acids of the cytoplasmic domain were
deleted; CRCYT-, CHO cells transfected with C receptors,
in which the entire cytoplasmic domain was deleted. Results are mean
± S.E. of at least six wells in three separate experiments,
except in CR514t hypertonic sucrose, and CRCYT- hypertonic
sucrose (two wells). Hypertonic sucrose reduced lysosomal hydrolysis in
all recombinant cells to negligible values (not shown). *, p < 0.001 versus CRWT
control.
The decrease in receptor-mediated hydrolysis of radioligand in recombinant cells expressing C receptors with major or total deletion of the amino acids in the cytoplasmic tail can be entirely accounted by a decrease in receptor-mediated endocytosis rather than by a decrease in the hydrolysis of internalized radioligand or down-regulation of surface C receptors. Thus, as shown in the bottom panel of Fig. 5, the net endocytic rate of CHOCRWT under control conditions was 5.98 ± 0.31%/min, a value that decreased approximately 10-fold to 0.67 ± 0.09%/min when endocytosis was blocked by hypertonic sucrose (p < 0.001). The net endocytic rate in CHOCRCYT- was dramatically reduced to 0.79 ± 0.04%/min (p < 0.001 versus CHOCRWT), a value not significantly different (p > 0.05) from that in hypertonic sucrose-treated CHOCRWT. Hypertonic sucrose did not alter ligand binding in CHOCRWT or in CHO cells expressing the cytoplasmic truncated C receptors (not shown). The net endocytic rate in CHOCR514t (1.62 ± 0.04%/min) was markedly reduced (p < 0.001 versus CHOCRWT) but not to the same extent as in CHOCRCYT-. This residual internalization of CHOCR514t was sensitive to hypertonic sucrose, indicating that it still occurs via clathrin-coated pits.
Fig. 6summarizes the values for
receptor-mediated lysosomal hydrolysis of I-ANF
(upper panel) and the
net endocytic rate of receptor-ligand complexes (lower panel)
in recombinant cells expressing point-mutated C receptors. Replacement
of Phe
(CHOCRA538) by Ala did not significantly affect
lysosomal hydrolysis (220 ± 11% of specifically bound
I-ANF
by 60 min) or net endocytic
rate (6.55 ± 0.92% of occupied receptors internalized per min)
compared with CHOCRWT (p > 0.05). Replacement of
Tyr
(CHOCRA508) or Cys
(CHOCRA473) by Ala
significantly reduced lysosomal hydrolysis of
I-ANF
and net endocytic rates of
receptor-radioligand complexes by approximately 40-50% compared
with CHOCRWT or CHOCRA538 (p < 0.001). However, net
endocytic rates in CHOCRA508 and CHOCRA473 were still significantly
greater than in cytoplasmic-truncated receptors or in sucrose-treated
CHOCRWT (p < 0.01 versus CHOCRCYT-,
CHOCR514t, and hypertonic sucrose). In recombinant cells expressing the
double mutant C receptor (CHOCRA508/A538), net endocytic rate and
lysosomal hydrolysis of
I-ANF
were
reduced to an extent similar to that in recombinant cells expressing
the single Ala
mutant (p < 0.01 versus CHOCRWT or CHOCRA538; p > 0.05 versus CHOCRA508).
Figure 6: Receptor-mediated lysosomal hydrolysis of ANF, and net endocytic rate of receptor-radioligand complexes in recombinant CHO cells stably transfected with point-mutated natriuretic peptide C receptors in control conditions or in the presence of hypertonic sucrose. See legend of Fig. 5for description. Ctrl, control; HS, hypertonic sucrose; CRA538, CRA508, CRA508/538, and CRA473 are recombinant CHO cells transfected with C receptors in which alanine replaced phenylalanine 538, tyrosine 508, tyrosine 508, and phenylalanine 538 in the cytoplasmic domain, or cysteine 473 in the extracellular domain, respectively. Results are the mean ± S.E. of at least six wells in three separate experiments, except in the experiments with hypertonic sucrose (two wells). Hypertonic sucrose reduced lysosomal hydrolysis in all recombinant cells to negligible values (not shown). The net endocytic rate of CRA538 was not significantly different (p > 0.05) from that of CRWT in Fig. 5and was significantly faster (*, p < 0.001) than that of all other point-mutated receptors.
The slopes of the regressions line defining the
time course of the lysosomal hydrolysis of I-ANF
of all point-mutated
receptors, except CHOCRA538 (Fig. 6, upper panel), were
significantly smaller than that in CHOCRWT (p < 0.001).
However, the slopes of lysosomal hydrolysis of all point-mutated
receptors were significantly greater than those of the cytoplasmic
truncated receptors (p < 0.001 versus CHOCRCYT- or CHOCR514t). Hypertonic sucrose did not affect
ligand binding in CHOCRA538, CHOCRA508, or CHOCRA508/A538 but, for
unknown reasons, caused a time-dependent dissociation of surface
receptor-ligand complexes in CHOCRA473. Thus, sucrose-sensitive
internalization could not be investigated with this mutant. In the
other mutants, hypertonic sucrose further decreased the net endocytic
rates of receptor-ligand complexes to very low levels in all
recombinant CHO cells expressing the point-mutated receptors CRA538,
CRA508, and CRA508/A538.
The dynamic characteristics of cloned wild type C receptors
expressed in CHO cells are qualitatively and quantitatively very
similar to those determined previously in our laboratory for native C
receptors in cultured bovine aortic vascular smooth muscle
cells(14) . Recombinant wild type and native C receptors have
similar ligand binding characteristics (K < 0.1
nM) and nearly equal net rates of endocytosis (5-7% of
receptors internalized per min) and NH
Cl-sensitive
(lysosomal) hydrolysis of ligand. There is no detectable difference
between the cellular compartmentalization of recombinant CRWT in CHO
cells and native C receptors in bovine aortic vascular smooth muscle
cells (
2/3 in the membrane and 1/3 in the intracellular
compartment), and endocytosed native or recombinant wild type receptors
undergo a highly efficient process of recycling to the cell membrane.
These similarities indicate that ligand binding, receptor-ligand
endocytosis, and cellular trafficking of internalized ligand and
receptors are dependent on the molecular nature of C receptors rather
than on the cell type in which they are expressed. Thus, it is likely
that the present results on the molecular determinants of the clearance
function of cloned C receptors can be fairly extrapolated to native C
receptors in the mammalian organism.
Previous studies from our laboratory demonstrated that native C receptors are internalized constitutively with an endocytic rate similar to that of receptor-ligand complexes, a property that it also shares with other clearance and/or transport receptors(14) . In the present study, hypertonic sucrose, which causes disassembly of clathrin and in this manner disrupts receptor-mediated endocytosis(13, 23) , virtually abolished rapid endocytosis of C receptors. Thus, it is likely that constitutive internalization of C receptors, similar to other receptors, occurs via coated pits.
We chose to express our results as net endocytic rates
of receptor-ligand complexes because in steady-state conditions this is
likely to be the meaningful physiological parameter to assess the
removal of ANF or other natriuretic peptides from the circulation.
Moreover, it is not feasible to determine true unidirectional endocytic
rates of receptors without preloading the ligand at 4 °C and then
warming the cells to 37 °C to initiate the endocytic process.
However, we have shown previously that temperature transition
(4-37 °C) per se may alter the dynamics of the
constitutive internalization of C receptors(14) . Under similar
experimental conditions of steady-state and long term incubation, the
net endocytic rate of recombinant CRWT receptor-ligand complexes
(6%/min) is comparable to that determined for asialoglycoprotein
and cation-independent mannose 6 phosphate receptor-ligand complexes,
and 30-50% lower than that of low density lipoprotein receptor
and transferrin receptor-ligand
complexes(7, 8, 9, 10, 13) .
It is noteworthy that a net internalization rate of 5-7%/min for
C receptor-ligand complexes is more than sufficient for an effective
removal of natriuretic peptides from the circulation. Thus, the off
rate of ANF from surface C receptors at 37 °C is much slower than
the rate of receptor internalization, allowing for a sufficient
resident time for ligand internalization. Moreover, the high density of
surface C receptors and the rapid cycling of internalized receptors to
the cell surface contribute greatly to an efficient participation of
the C receptor in the plasma clearance and homeostasis of natriuretic
peptides in the intact organism(2, 4, 26) .
The fundamental internalization signal of C receptors, similar to
all other endocytosed receptors reported to date, resides in the
cytoplasmic domain. Only complete deletion of cytoplasmic tail brings
rapid endocytosis of C receptors to a virtual halt, i.e. the
net internalization rate of the tailless receptor is approximately
10-fold lower than that of wild type receptors and not significantly
different from that observed when endocytosis of wild type C receptors
is blocked by hypertonic sucrose. A large deletion of the cytoplasmic
domain (CHOCR514t) also markedly decreases the net endocytic rate of
receptor-ligand complexes by 75%. This decrease is not due to the
presence of a phenylalanine (Phe
) in the truncated
region, as the net endocytic rate in the mutant CHOCRA538 was not
significantly different from that in CHOCRWT. Although it cannot be
ruled out that there may be an internalization signal in the truncated
region, it is more probable that the truncation of 28 of the 37 amino
acids of the cytoplasmic domain disrupted the secondary structure of
this domain and in this manner, its interaction with clathrin adaptor
proteins.
In a strict sense, the C receptor does not contain in its
cytoplasmic tail classic internalization motifs for coated pit
internalization found in other receptors that undergo rapid endocytosis (e.g. NPXY, YXXZ, where X is any
amino acid and Z is a large hydrophobic amino acid). However,
C receptors have a cytoplasmic domain tyrosine (Tyr),
four amino acids removed from the putative single transmembrane domain.
Replacement of this tyrosine by alanine decreases C receptor
endocytosis by about 50%. This is a lower decrease than the
3-4-fold reduction reported for low density lipoprotein,
transferrin, and asialoglycoprotein receptors when the tyrosine within
the NPXY or YXXZ motifs was replaced by nonaromatic
amino acids(7, 8, 9, 13) . The
residual endocytosis of receptor-ligand complexes in CHOCRA508 cells,
as well as in all other recombinant cells, is almost completely blocked
by hypertonic sucrose, indicating that the endocytosis of mutant
receptors still occurs via clathrin-coated pits. Moreover, it is
apparent that the mutations did not alter the routing of internalized
radioligand to lysosomes or the recycling of dissociated receptors to
the cell surface.
In view of the lack of an identifiable motif
sequence for internalization of the C receptor, it is useful to compare
the amino acid sequence of its cytoplasmic domain with that of a bona
fide membrane resident protein that was mutated to contain a
cytoplasmic tail tyrosine. An example of such a protein is the
influenza virus hemagglutinin, which can be induced to internalize at a
rate of 4-5%/min, a value very similar to that of wild type C
receptors, by inserting a tyrosine in the cytoplasmic domain, five
amino acids removed from the putative transmembrane
domain(27) . The added tyrosine that confers a rapid
internalization signal to the influenza virus hemagglutinin is
separated from the putative transmembrane domain by five amino acids
(Asn-Gly-Ser-Leu-Gln), which have no homology with the three amino
acids (Arg-Lys-Lys) that separate Tyr from the putative
transmembrane domain of the C receptor. On the other hand, the
COOH-terminal sequence flanking tyrosine, namely, Arg-Ile-Cys-Ile, for
the influenza virus hemagglutinin, and Arg-Ile-Thr-Ile for the C
receptor, share a high degree of homology. However, the sequence
YRIXI does not seem to correspond to any of the postulated
internalization motifs described to date, and therefore its biological
meaning is unknown.
The present results, compared with those
obtained in mutagenesis experiments with other receptors, further
strengthen the notion that internalization motifs are degenerate,
making it difficult to assign a generic amino acid sequence for rapid
endocytosis on the basis of nature, size, hydrophobicity, or charge of
the amino acids that flank the key aromatic amino acid tyrosine or, in
some instances,
phenylalanine(7, 8, 9, 10, 11, 12, 13) .
It has been postulated that the main importance of the tyrosine in the
cytoplasmic domain of receptors is that this aromatic amino acid lies
on a -turn that confers the proper secondary structure for
interaction with clathrin adaptor proteins(7) . Recently,
however, it has been shown by direct NMR analysis of angiotensin
receptors that this may not be true for all membrane
receptors(28) . Indirect theoretical assessment of the
secondary structure of the cytoplasmic domain of C receptors predicts
that Tyr
is part of a
-turn using Garnier et al.(29) and a
-sheet using Chou and Fasman (30) algorithms. In both models, however, replacement of
Tyr
by Ala predicts the introduction of an
-helix in
place of a
-turn or a
-sheet in the cytoplasmic domain of C
receptors. In view of the low intrinsic accuracy of existing models to
predict secondary structure of proteins, direct structural analysis of
C receptors is needed to elucidate the role of Tyr
in
conferring a structural motif for the internalization of C receptors.
Native or recombinant C receptors are mostly present at the cell
surface as homodimers, with a small proportion of monomers (Fig. 3). The present results demonstrate that constitutive
dimerization plays a significant role in rapid endocytosis, as the
dimer forms of C receptors internalize at approximately twice the rate
of monomer forms in which Cys was replaced by Ala. The
duplex cytoplasmic domain in the dimer forms may interact more strongly
with adaptor proteins in clathrin-coated pit, leading to a faster
endocytic rate, or alternatively, it is possible that dimer forms are
more easily trapped in coated pits because of a lower lateral mobility
in the plane of the membrane. The present results do not permit us to
distinguish between these two possibilities. Whatever the case, the
constitutive predominance of homodimers of C receptors is likely to
increase the efficiency of receptor-mediated endocytosis of ligand by
approximately 4-fold because dimers are able to bind the double of
ligand molecules, and internalize at twice the rate of monomers. Thus,
constitutive dimerization is particularly important for the clearance
function of C receptors. To our knowledge this is the first
demonstration that constitutive polymerization markedly enhances
receptor endocytosis. It remains to be investigated whether this is
also the case for other clearance and/or transport receptors.