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J Biol Chem, Vol. 274, Issue 9, 5620-5625, February 26, 1999
From the Parathyroid hormone (PTH) is predominantly
cleared from the circulation by glomerular filtration and degradation
in the renal proximal tubules. Here, we demonstrate that megalin, a
multifunctional endocytic receptor in the proximal tubular epithelium,
mediates the uptake and degradation of PTH. Megalin was purified from
kidney membranes as the major PTH-binding protein and shown in BIAcore analysis to specifically bind full-length PTH and amino-terminal PTH
fragments (Kd 0.5 µM). Absence of the
receptor in megalin knockout mice resulted in 4-fold increased levels
of amino-terminal PTH fragments in the urine. In F9 cells expressing
both megalin and the PTH/PTH-related peptide receptor (PTH/PTHrP
receptor), uptake and lysosomal degradation of the hormone was mediated
through megalin. Blocking megalin-mediated clearance of PTH resulted in 3-fold increased stimulation of the PTH/PTHrP receptor. These data
provide evidence that megalin is involved in the renal catabolism of
PTH and potentially antagonizes PTH/PTHrP receptor activity in the
proximal tubular epithelium.
Calcium concentrations in plasma and extracellular body fluids
have to be kept at approximately 2.5 mM to assure normal
body functions. Mammalian organisms therefore have developed an
elaborate endocrine system to regulate the systemic calcium
homeostasis. The parathyroid hormone
(PTH)1 is the principal fast
acting regulator of the calcium balance. It is produced in the
parathyroid glands and released into circulation within minutes after
the decline of extracellular calcium concentrations. There, PTH acts on
several target tissues to mobilize calcium and to counterregulate
hypocalcemia: 1) it induces osteoclastic breakdown of bone; 2) it
increases tubular re-absorption of calcium in the kidney; and 3) it
stimulates renal conversion of 25-OH vitamin D3 to
1,25-(OH)2 vitamin D3, which in turn increases
intestinal absorption of dietary calcium (reviewed in Refs. 1 and
2).
In the circulation, some PTH molecules are proteolytically cleaved
between amino acids 33 and 39 to generate amino-terminal and
carboxyl-terminal fragments of the hormone. Whereas the
carboxyl-terminal fragments are functionally inactive, the
amino-terminal fragments exhibit biological properties similar to the
full-length hormone (reviewed in Ref. 3). Both PTH 1-84 and
amino-terminal fragments bind to a heptahelical receptor expressed in
PTH target organs, the PTH/PTH-related peptide receptor (PTH/PTHrP
receptor). This 60-kDa protein was cloned initially from proximal
tubular cells and osteoblasts. It belongs to a novel family of G
protein-coupled receptors characterized by seven transmembrane domains
and at least two conserved N-linked glycosylation motifs (4,
5). Upon binding of PTH, the PTH/PTHrP receptor transduces its signal through activation of adenylate cyclase and phospholipase C.
The kidney not only is the prime target organ of PTH activity but it
also plays an active role in the removal of the hormone from the
circulation. This is suggested by several lines of experimental evidence. First, in patients with renal failure the plasma half-life of
the hormone is significantly increased (6, 7). Furthermore, in organ
perfusion studies, PTH was shown to be lost from the plasma mainly by
glomerular filtration followed by re-absorption and degradation in the
proximal tubules (8, 9). Finally, cellular uptake and degradation of
PTH was demonstrated directly in cultured proximal tubular cells. In
these cells, the PTH/PTHrP receptor was suggested to mediate the uptake
and degradation of the hormone (10, 11). Whether this receptor is also
responsible for systemic clearance of PTH or whether alternative uptake
mechanisms exist in vivo remained unclear.
Recently, a major endocytic receptor pathway for uptake of ligands from
the glomerular filtrate has been elucidated in the proximal tubules.
Megalin is a multifunctional clearance receptor expressed on the apical
surface of the proximal tubular epithelium (12). Cloning of the rat
cDNA revealed that megalin is a member of the low density
lipoprotein (LDL) receptor gene family (13). Like other members of this
gene family, megalin exhibits a broad ligand specificity and mediates
the uptake and lysosomal degradation of numerous macromolecules.
Ligands internalized by megalin in the proximal tubules include albumin
and trans-cobalamin/vitamin B12 complexes (14, 15). The
significance of megalin for tubular function is underscored by the
finding that the receptor resorbs aminoglycosides from the glomerular
filtrate and is therefore responsible for the nephrotoxicity associated
with these compounds (16). The central role of megalin in the tubular
resorption processes was further confirmed in mice genetically
deficient for the receptor. Most megalin-deficient newborns die
perinatally from holoprosencephaly, a developmental defect of the
forebrain (17). The causes underlying this defect have not been
elucidated with certainty, but the severity of the phenotype varies
among individual animals and 1 in 50 of the megalin The current study was conducted to investigate a potential role of
megalin in the renal catabolism of PTH. We show that megalin mediates
the cellular uptake and degradation of PTH and that receptor-deficient mice exhibit impaired renal catabolism of the hormone. Furthermore, we
demonstrate that megalin-mediated degradation of PTH antagonizes activation of the PTH/PTHrP receptor.
Materials and General Methods--
Mature bovine PTH as well as
bovine and human PTH fragments were obtained from Sigma (Deisenhofen).
Rabbit anti-human PTH antibody was purchased from Accurate Chemicals
(Westbury, NY). Sheep anti-rat megalin antibody was a gift from P. Verroust (INSERM, Paris). Polyclonal antibodies directed against rat
PTH/PTHrP receptor were obtained from BAbCO (Richmond, CA) or kindly
provided by A. Abou-Samra (MGH, Boston). Proteins were radiolabeled
with 125I using the IODO-GEN method (19).
Cloning and Purification of Mature Bovine Parathyroid
Hormone--
The coding region of amino acids 1 to 84 of mature PTH
present on exon 3 of the bovine PTH gene (20) was amplified by
polymerase chain reaction from genomic calf liver DNA using primers:
5'-GATCCTCATCATATGGCTGTGAGTGAAATACAGTTT-3' and
5'-GGTGGGATCCCTATTACTGGGGTTTAGCTTTAATTAATA-3'. NdeI
and BamHI restriction sites in the polymerase chain reaction
primers were used to clone the amplified PTH gene fragment into vector
pET-14B (Novagen, Madison, WI). Expression of this vector results in a recombinant PTH molecule carrying an amino-terminal extension of 6 histidine residues and a thrombin cleavage site (recPTH). Integrity of
the PTH gene sequence was confirmed by DNA sequence analysis. For
purification, the expression plasmid was introduced into
Escherichia coli strain BL-21pLys (Novagen). RecPTH was
recovered from bacterial lysate by standard nickel-affinity
chromatography (Qiagen, Hilden).
Purification of PTH-binding Proteins by PTH Affinity
Chromatography--
5 mg of purified recPTH was coupled to 2 ml of
CNBr-activated-Sepharose 4B (Pharmacia, Uppsala) according to
manufacturer's recommendations. Crude rat kidney membrane extracts
were prepared as described previously (17). 80 mg of membrane extracts
were diluted in 100 ml Tris-buffered saline, 0.5% Triton X-100, and circulated over the PTH column for 16 h at 4 °C. The column was washed with 100 ml Tris-buffered saline, 0.5% CHAPS, and bound proteins were eluted in 100 mM glycine, 0.5% CHAPS, pH
2.8.
Biosensor Measurements--
Binding of PTH fragments to megalin
was quantified by BIAcore (Biosensor, Sweden). Briefly, rabbit megalin
immobilized on CM5 sensor chip (34 fmol/receptor/mm2) was
incubated with PTH peptides (10-50 µg/ml) in 10 mM
HEPES, 150 mM NaCl, 1.5 mM CaCl2, 1 mM EGTA, 0.005% surfactant P20, pH 7.4, and the relative
increase in response between megalin and control flow channels was
determined. Kinetic parameters were determined by using the BIA
evaluation 3.0 software. Number of ligands bound per immobilized
receptor molecule was estimated by dividing the ratio
RUligand/massligand with
RUreceptor/massreceptor.
Cell Culture and Degradation Experiments--
Mouse F9 embryonal
carcinoma cells (ATCC, CRL-1720, Manassas, VA) were cultured in
standard medium (DMEM, 10% fetal calf serum) and induced with 1 µM trans-retinoic acid and 1 µM
dibutyryl cAMP (21, 22). Cellular degradation of the
125I-ligands added to the culture medium was determined by
standard protocols (23) and expressed as nanograms of
125I-labeled trichloroacetic acid-soluble (noniodide)
material released into the culture medium per mg of total cell protein.
Determination of PTH and cAMP Levels--
For urine collection,
mice were placed in metabolic cages for 16 h and given 10%
sucrose in drinking water. Urine samples obtained were qualitatively
indistinguishable from samples collected without sucrose load. Urine
volume per hour and creatinine levels were identical in
megalin In initial experiments, we intended to identify proteins in the
kidney that interact with PTH and potentially play a role in the renal
catabolism of the hormone. To do so, a fusion protein of mature bovine
PTH with an amino-terminal hexahistidine epitope (recPTH) was produced
in bacteria and coupled to Sepharose resin. Total membrane extracts
from rat kidneys were applied to this recPTH affinity column. The
column was washed extensively and bound proteins were eluted in low pH
buffer. Reproducibly, two major protein bands were identified that
bound specifically to the PTH column (Fig.
1, lane 2). No binding was
observed in Sepharose columns lacking PTH (data not shown). The 60-kDa
protein eluted from the PTH column was identified as the renal
PTH/PTHrP receptor using antibodies against rat PTH/PTHrP receptor
(lane 3). A second protein of high molecular weight also
bound to the PTH column (lane 2). This protein was
identified as megalin by Western blot analysis (lane 4).
The ability of megalin to interact with PTH was investigated in more
detail using plasmon surface resonance (BIAcore) analysis. As shown in
Fig. 2A, megalin immobilized
on the BIA sensor chip bound recPTH reversibly. Ligand binding was
dependent on calcium and abolished by the addition of EDTA, a
characteristic feature of ligand binding to megalin. The intensity of
the binding response signal indicated two PTH binding sites per
receptor molecule. To further map the receptor binding epitope on PTH,
binding of PTH fragments to megalin was analyzed (Fig. 2B).
PTH 1-34 bound specifically to the receptor, eliciting a response
signal equal to half that of the full-length hormone (300 versus 600 response units). Because PTH 1-34 exhibits half
the molecular weight of recPTH, this result indicated that the same
number of receptor binding sites were occupied by PTH 1-34 and recPTH.
In the same assay, no binding was observed for PTH 39-84. The exact
binding affinities of recPTH, native bovine PTH, and various PTH
fragments were calculated from the BIAcore analysis data (Table
I). Bovine PTH, recPTH, recPTH in which
the His6 epitope was removed by thrombin cleavage
(recPTH*), and the amino-terminal fragment PTH 1-44 bound with similar
affinities (~0.4-3 µM). In contrast, carboxyl-terminal PTH fragments did not bind at all. Binding of PTH 1-34 was weaker than
that of PTH 1-44 (26 versus 3.2 µM) suggesting
that amino acids 35 to 44 may contribute to receptor binding. In
conclusion, these studies demonstrated that megalin recognizes the
amino-terminal region of PTH which has already been shown to harbor the
PTH/PTHrP receptor recognition site (4).
It is well established that ligands bound to megalin are subjected to
cellular uptake and lysosomal degradation. To test whether the receptor
also mediates the degradation of recPTH, we investigated the catabolism
of the hormone in F9 mouse embryonic carcinoma cells. Upon stimulation
with trans-retinoic acid and dibutyryl cAMP, these cells
express high levels of megalin and the PTH/PTHrP receptor (Fig.
3, inset) (21, 22). Expression
of other receptors of the LDL receptor gene family is either not
increased (LDL receptor) or even reduced (LDL receptor-related protein)
upon stimulation (Fig. 3, inset). Induction of megalin and
PTH/PTHrP receptor expression in F9 cells correlated with a 3-fold
increase in 125I-recPTH degradation as compared with
noninduced control cells (Fig. 3; p < 0.009). To
dissect the contribution of different pathways for PTH degradation, we
investigated the cellular catabolism of 125I-recPTH in the
presence of various inhibitors (Fig. 4).
The receptor-associated protein (RAP) is a cellular chaperone which
blocks binding of all known ligands to megalin (25). These antagonistic
properties of RAP can be exploited to specifically interfere with
megalin-mediated ligand uptake into cells (16, 26). When purified
glutathione S-transferase (GST)-RAP fusion protein was added
to the culture medium of F9 cells, the amount of PTH degraded was
reduced by 50% as compared with control cells that received GST only.
(Fig. 4A, columns 1 and 2,
p < 0.006). PTH degradation was blocked to the same
extend by chloroquine, an inhibitor of lysosomal degradation (Fig.
4A, column 3, p < 0.001). A
combination of GST-RAP and chloroquine had no additive inhibitory
effect (Fig. 4A, column 4), suggesting that
lysosomal degradation of PTH is mediated through a RAP-sensitive receptor. Because only 50% of PTH degradation was mediated through lysosomal pathways, we reasoned that extracellular proteolytic activity
might be responsible for the residual catabolism of the hormone. In
particular, extracellular processing of 125I-recPTH into
amino-terminal and carboxyl-terminal fragments would result in the
generation of labeled PTH peptides that are trichloroacetic acid-soluble and indistinguishable from lysosomal degradation products
in our assay. This hypothesis was confirmed by application of
chymostatin, a protease inhibitor that blocks extracellular processing
of PTH (27). Addition of chymostatin reduced the amount of
125I-degradation products by 50% (Fig. 4A,
column 5, p < 0.001), and the combined
application of chymostatin and RAP inhibited the degradation of the
hormone almost completely (Fig. 4A, column 6,
p < 0.009). This indicated that a RAP-binding receptor
was primarily responsible for lysosomal degradation of full-length PTH
in these cells. To test whether the PTH/PTHrP receptor might be
affected by RAP, we tested binding of 125I-RAP to F9 cell
membrane preparations separated by SDS-PAGE and transferred to
nitrocellulose filters. In this ligand binding assay,
125I-RAP bound strongly to a high molecular weight protein
co-migrating with megalin (Fig. 4B). No significant binding
to other membrane proteins including the PTH/PTHrP receptor was
observed (Fig. 4B).
Megalin Antagonizes Activation of the Parathyroid Hormone
Receptor*
,
,,,, and Franz-Volhard-Clinic, Cell Biology, University of Aarhus, 8000 Aarhus, Denmark
ABSTRACT
Top
Abstract
Introduction
References
INTRODUCTION
Top
Abstract
Introduction
References
/
mice survives to adulthood. Absence of the receptor in the kidneys of
these mice results in tubular resorption deficiency and excretion of
low molecular weight plasma proteins in the
urine.2
EXPERIMENTAL PROCEDURES
/ and in control mice (data not shown). PTH
fragments in urine were measured by radioimmunoassays developed for rat
PTH fragment 1-38 (Immundiagnostik, Bensheim) or human PTH fragment
35-84 (Diagnostic Systems, Sinsheim), and shown to recognize mouse PTH.
Concentrations of cAMP in cells and in the culture medium was
determined as described (24). Student's t test was used to
determine the statistical significance of data points.
RESULTS
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Fig. 1.
Purification of PTH-binding proteins from rat
kidney membranes by PTH affinity chromatography. 80 mg of crude
rat kidney membranes were subjected to PTH affinity chromatography
using 5 mg of recPTH coupled to CNBr-activated-Sepharose 4B resin as
described under "Experimental Procedures." Proteins bound to the
PTH column were eluted and 20 µl of the eluate (total of 1 ml) were
subjected to 4-15% SDS-PAGE (lanes 2-4). Parallel samples
were characterized by staining with silver nitrate (lane 2)
or immunoblot analysis using 5 µg/ml anti-rat PTH/PTHrP receptor
(PTH-R, lane 3) and anti-rat megalin antisera (lane
4). For comparison, 2.5 µg of total rat kidney extract were
analyzed by staining with silver nitrate in lane 1. The
arrows indicate two proteins specifically eluted from the
PTH column in lane 2.
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Fig. 2.
BIAcore analysis of binding of PTH and PTH
fragments to immobilized megalin. RecPTH (amino acids 1-84) was
incubated with immobilized megalin in the absence or presence of 20 mM EDTA (A). Binding to the receptor was
detected by surface plasmon resonance signal (BIAcore, see Experimental
Procedures) and is indicated in response units. Binding of recPTH
(1-84), amino-terminal bovine PTH fragment (1-34), and
carboxyl-terminal human PTH fragment (39-84) to immobilized megalin
(B).
Affinities of binding of PTH and PTH fragments to megalin
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Fig. 3.
Degradation of 125I-recPTH by
induced and noninduced F9 cells. F9 cells were cultured in the
absence (0; noninduced) or presence (+; induced) of 1 µM
dibutyryl cAMP and 1 µM trans-retinoic acid
for 7 days. Subsequently, the medium was changed to 1 ml of DMEM
(without glutamine) containing 0.2% (w/v) bovine serum albumin and 32 ng/ml 125I-recPTH (3,220 cpm/ng). After incubation at
37 °C for the indicated periods of time, the amount of
125I-labeled recPTH degradation products (trichloroacetic
acid-soluble material) released into the culture medium was determined
(23). Where no error bar (mean ± S.E.) is shown, the size of the
bar is smaller than the respective symbol. Inset, analysis
of receptor expression in F9 cells. Cell extracts from noninduced (0)
and induced (+) F9 cells were subjected to 4-15% SDS-PAGE and
immunoblot analysis using antisera against rat megalin, rat LDL
receptor-related protein (LRP), bovine LDL receptor
(LDL-R), and rat PTH/PTHrP receptor
(PTH-R). Bound IgG was visualized by ECL.
View larger version (24K):
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Fig. 4.
Inhibition by GST-RAP, chloroquine and
chymostatin of 125I-recPTH degradation in F9 cells.
A, induced F9 cells received 1 ml of DMEM (without
glutamine) containing 0.2% bovine serum albumin and 27 ng/ml
125I-recPTH (3,720 cpm/ng). Where indicated, the medium
included 90 µg/ml GST (columns 1, 3, and
5), 90 µg/ml GST-RAP (columns 2, 4,
and 6), 200 µM chloroquine (columns
3 and 4) and 50 µg/ml chymostatin (columns
5 and 6). The amount of 125I-recPTH
degradation products secreted into the medium was determined after
incubation for 120 min at 37 °C. B, F9 cell membrane
extracts were separated by 4-15% SDS-PAGE (20 µg of protein/lane)
followed by transfer to nitrocellulose membrane. The nitrocellulose
strip on the left was preincubated in Tris-buffered saline
containing 5% (w/v) bovine serum albumin for 1 h at room
temperature. Then, 67 ng/ml 125I-GST-RAP (1,480 cpm/ng)
were added to the incubation buffer for 1 h. Bound
125I-GST-RAP was detected by autoradiography for 16 h
at 
70 °C. Replicate nitrocellulose strips were subjected to
immunoblot analysis using 5 µg/ml anti-rat megalin antibody
(middle) or 5 µg/ml anti-rat PTH/PTHrP receptor antibody
(right).
Previously, we have generated mice genetically deficient for megalin (17). These animals were used to evaluate the consequence of megalin deficiency for the renal catabolism of PTH. Consistent with a role of the receptor in the tubular uptake of biologically active PTH peptides, levels of amino-terminal PTH fragments in urine of knockout mice were increased 4-fold as compared with control litter mates (374 ± 48 versus 87.5 ± 29 pmol/liter; p < 0.002). In contrast, levels of carboxyl-terminal PTH fragments were unchanged (354.6 ± 76 versus 345.4 ± 45.2 pmol/liter). At the same time, the megalin gene defect did not affect the total amount or subcellular localization of the PTH/PTHrP receptor in proximal tubular cells as shown by immunohistochemistry (Fig. 5) and Western blot analysis (data not shown). Finally, the significance of megalin for tubular catabolism of PTH was confirmed by microinfusion of 125I-labeled recPTH into rat proximal tubules in vivo (with method described in Ref. 16). In these micropuncture experiments, 50.6 ± 0.9% of labeled recPTH was taken up into the tubules. The uptake of 125I-recPTH was significantly impaired when RAP was co-infused into the tubules (41.1 ± 2.8%; p < 0.025). Similar effects have been observed for other receptor ligands previously (16).
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The results obtained so far have established a dual receptor system in
the renal proximal tubules that recognizes full-length and
amino-terminal PTH fragments. This receptor system consists of the
PTH/PTHrP receptor that transduces PTH signals and of the endocytic
receptor megalin that mediates the catabolism of the hormone. It was of
particular interest to learn about the significance of this dual
receptor system for PTH activity. So far, the limited number
(n = 8) of megalin/ animals available
and their complex phenotype confounded the detailed analysis of their
PTH metabolism. We therefore focused on PTH-mediated signal
transduction in F9 cells. First, the ability of recPTH to stimulate the
PTH/PTHrP receptor was tested (Fig. 6A). When recPTH was added to
F9 cells, cAMP levels did not rise above the level in untreated control
cells (columns 1 and 2). However, when recPTH was applied together with
an inhibitor of phosphodiesterase activity (isobutylmethylxanthine) a
significant increase in cAMP levels was observed (columns 3 and 4, p < 0.014). To study the influence of megalin on
PTH/PTHrP receptor activity, we next determined PTH-induced signal
transduction in the presence of the megalin inhibitor RAP (Fig.
6B). As already shown in Fig. 6A, recPTH alone
did not increase steady state cAMP levels (column 3). In contrast,
addition of recPTH and RAP caused a 3-fold elevation of cellular cAMP
concentrations (column 4) even in the absence of isobutylmethylxanthine
(p < 0.012). GST or GST-RAP had no direct effect on
cAMP levels (columns 1 and 2). These results demonstrated that
megalin-mediated degradation of PTH antagonizes activation of the
PTH/PTHrP receptor.
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DISCUSSION |
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We have identified megalin as an endocytic receptor for the
cellular uptake and degradation of PTH. The receptor was purified from
kidney membranes as a major PTH-binding protein and shown in BIAcore
analysis to bind full-length PTH and amino-terminal PTH fragments. In
F9 cells and in microinfused proximal tubules, uptake and lysosomal
degradation of the hormone was mediated through megalin. Expression of
the receptor in the proximal tubular epithelium and enhanced excretion
of amino-terminal but not carboxyl-terminal PTH fragments in urine of
megalin/ mice suggests that the receptor also plays an
important role in the renal catabolism of the hormone in
vivo.
A renal endocytic pathway for uptake and degradation of PTH has been anticipated by findings that the kidney is largely responsible for removal of the hormone from the circulation. In isolated perfused kidneys, PTH and PTH fragments are mainly cleared through glomerular filtration and re-absorbtion in the proximal tubules (8, 9). In both patients and laboratory animals, plasma PTH levels are directly correlated with glomerular filtration rates and clearance of PTH is blocked in nonfiltering kidneys (6, 28). Previously, two types of PTH binding sites have been identified in renal membrane preparations that could be involved in the tubular catabolism of the hormone: high affinity (Kd ~ 10 nM) and low affinity binding sites (Kd ~ 1 µM) (29, 30). The high affinity binding sites are coupled to G proteins and most likely represent the PTH/PTHrP receptor (31). In contrast, the low affinity binding sites are not linked to G proteins (30) and potentially represent the endocytic receptor megalin. This is indicated by the fact that megalin is a major PTH-binding protein in the kidney (Fig. 1) and exhibits PTH binding affinities in the micromolar range (Table I). Although megalin binds recPTH in vitro with an affinity significantly below the circulating levels of the hormone, this does not exclude a role of the receptor in the renal uptake of PTH in vivo. Megalin is one of the most abundant membrane proteins on the brush border surface of the proximal tubules, and its abundance could compensate for low affinity in ligand uptake. For example, polybasic drugs such as aminoglycosides, aprotinin, and polymyxin B are efficiently internalized by megalin in the proximal tubules. The affinity of the receptor for these compounds is similar to the one for PTH (16).
The degradation of PTH was investigated in a number of established proximal tubular cell lines. In opossum kidney cells most PTH was subjected to endocytosis and lysosomal degradation, whereas a minor fraction of the hormone was metabolized by extracellular chymotrypsin-like endopeptidases (10, 11, 27). It has been suggested that in opossum kidney cells the PTH/PTHrP receptor is responsible for the endocytic uptake of the hormone. However, no specific antagonist other than PTH peptides have been available to specifically block the PTH/PTHrP receptor and to investigate the potential contribution of various receptor pathways to PTH clearance. We have analyzed PTH degradation in F9 embryonic carcinoma cells where expression of megalin and the PTH/PTHrP receptor can be co-induced by trans-retinoic acid and dibutyryl cAMP. Although not of tubular origin, high levels of PTH/PTHrP receptor and megalin expression mimics well the situation in the proximal tubules in vivo. Similar to opossum kidney cells, F9 cells metabolized PTH both by extracellular proteolysis and by lysosomal degradation (Fig. 4A). The latter pathway most likely involves megalin, because lysosomal degradation was blocked by RAP. This inhibitor does not bind to the PTH/PTHrP receptor (Fig. 4B) or affect its function directly (Fig. 6B). In addition, we have investigated the degradation of PTH in porcine proximal tubular cells (LLC-PK1). These cells express megalin but not the PTH/PTHrP receptor (32).3 Again, more than 80% of PTH degradation in these cells was blocked by RAP (data not shown). Finally, we analyzed the PTH catabolism in mice genetically deficient for megalin. These animals specifically excreted increased levels of amino-terminal but not carboxyl-terminal PTH fragments.
Endocytic mechanisms are known to modulate signal transduction pathways. Upon binding of signaling molecules, a number of signaling receptors undergo rapid sequestration and internalization. Consequently, the total number of receptor molecules on the cell surface is reduced, and target cells are protected from overstimulation by excess ligands (down-regulation). The PTH/PTHrP receptor also has been shown to undergo endocytosis after PTH binding (33, 34). Both positive and negative regulatory elements controlling endocytosis have been identified in the carboxyl-terminal tail of this receptor (35).
An alternative endocytic mechanism regulating signal transduction has been identified in the insulin-like growth factor II (IGF-II) signaling pathway. IGF-II is a growth factor that controls mitogenic processes during embryonic development via binding to the IGF-I receptor. Loss of IGF-I receptor or IGF-II expression in knockout mice results in a growth-deficiency phenotype (36, 37). IGF-II also binds to the IGF-II/cation-independent mannose 6-phosphate receptor, which mediates internalization and lysosomal degradation of the growth factor. Gene inactivation of cation-independent mannose 6-phosphate receptor results in increased circulating levels of IGF-II and, as a consequence, in overgrowth of embryonic tissues. These findings demonstrate that cation-independent mannose 6-phosphate receptor is essential to reduce circulating levels of IGF-II and to protect the IGF-I receptor from overstimulation (18, 38). On the basis of our findings, it is intriguing to speculate that megalin might play a similar role in regulation of the PTH/PTHrP receptor activity. According to this model, endocytosis of the PTH/PTHrP receptor predominantly regulates the availability of signaling receptors on the cell surface, whereas megalin removes excessive ligands from the extracellular space. In F9 cells, such an antagonistic property of megalin was demonstrated (Fig. 6B). Additional support for this hypothesis comes from the fact that megalin only recognizes full-length and amino-terminal PTH fragments that are able to stimulate the PTH/PTHrP receptor. Biologically inactive fragments of the carboxyl-terminal region of the hormone are not bound (Fig. 2 and Table I). Furthermore, megalin and the PTH/PTHrP receptor are co-localized on the brush border surface of the proximal tubular epithelium (Fig. 5) where megalin could regulate PTH concentrations locally.
At present, the limited number of megalin knockout animals available
and their complex phenotype makes it difficult to test this concept
in vivo. The generation of mouse models with kidney-specific megalin gene defect will be helpful in answering this important question.
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ACKNOWLEDGEMENTS |
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We are indebted to Hannelore Schulz, Karin Karczewski, Gabriele Born, and Hanne Sidelmann for expert technical assistance and to Dr. Armbruster, Immundiagnostik Bensheim for PTH (1-38) measurements. We are grateful to Dr. A. Abou-Samra (Massachusetts General Hospital, Boston) for providing anti-rat PTH/PTHrP receptor antibody G48.
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FOOTNOTES |
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* This work was supported in part by grant Wi 1158/3-1 and a Heisenberg fellowship (to T. E. W.) from the Deutsche Forschungsgemeinschaft and by the Danish Medical Research Council and the Novo Nordic Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
** To whom correspondence should be addressed: Max-Delbrueck Center, R.-Roessle-Strasse 10, D-13125 Berlin, Germany. Tel.: 49-30-9406-2569; Fax: 49-30-9406-2110; E-mail: willnow{at}mdc-berlin.de.
2 A. Nykjaer, H. Vorum, D. Dragun, D. Walther, C. Jacobsen, J. Herz, F. Melsen, E. I. Christensen, and T. E. Willnow, manuscript submitted for publication.
3 J. Hilpert, A. Nykjaer, C. Jacobsen, G. Wallukat, R. Nielsen, S. K. Moestrup, H. Haller, F. C. Luft, E. I. Christensen, and T. E. Willnow, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are: PTH, parathyroid hormone; LDL, low density lipoprotein; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; DMEM, Dulbecco's modified Eagle's medium; RAP, receptor-associated protein; GST, glutathione S-transferase; PAGE, polyacrylamide gel electrophoresis; IGF, insulin-like growth factor.
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Abstract
Introduction
References
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Copyright © 1999 by The American Society for Biochemistry and Molecular Biology, Inc.
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