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RECEPTORS |
Institute for Hormone and Fertility Research at the University of Hamburg (D.M., J.O., A.K.M.), 22529 Hamburg, Germany; and Institute of Anatomy (R.M.), University of Hamburg, 20246 Hamburg, Germany
Address all correspondence and requests for reprints to: Dr. Dieter Müller, Institute for Hormone and Fertility Research at the University of Hamburg, Grandweg 64, D-22529 Hamburg, Germany. E-mail: mueller{at}ihf.de
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Introduction |
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The diverse biological activities of ANP (7) and the widespread tissue distribution of its GC-linked receptor (8) suggest the existence of mechanisms capable of locally regulating receptor activity. In fact, phosphorylation/dephosphorylation of GC-A has been established as an important regulatory mechanism responsible for sensitization and desensitization processes (9, 10). Another posttranslational modification that may affect receptor properties in a tissue-specific manner is glycosylation. Evidence for functional variants of GC-A, based on differences in glycosylation, has been provided earlier by studies in vitro with transfected cell lines. Results obtained suggested a correlation between phosphorylation and complete glycosylation (11) and that the degree of N-linked glycosylation of the GC-A extracellular domain influences the ability to bind ANP (12). The latter was consistent with the identification of glycosylation sites close to the ANP-binding domain (13). Furthermore, the elimination of potential glycosylation sites in the related receptor, GC-B, was found to inhibit ligand binding (14). In contrast, recent experiments performed with the recombinant extracellular domain of GC-A, expressed in COS-1 cells, failed to detect any influence of oligosaccharide moieties on ANP binding, suggesting functions of glycosylation in folding and/or transport of the receptor to the plasma membrane (15). Thus, the role of glycosylation for GC-A signaling still seems to be unclear. In addition, a certain limitation of most of these studies was that effects of glycosylation have been assessed with receptor molecules recombinantly expressed in cell lines, a situation where the extent of gycosylation and the type of oligosaccharides linked to GC-A may differ considerably from an expression under physiological conditions.
The aim of the present study was to examine whether there are structural (size) variants of GC-A in vivo that may reflect tissue-specific differences in posttranslational modification. The results obtained demonstrate a central nervous system (CNS)-specific GC-A glycoform in two species (rat and bovine) and provide evidence for an alteration in oligosaccharide processing during brain maturation.
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Materials and Methods |
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Tissues
Tissues from male Wistar rats were dissected after decapitation
of the animals, immediately frozen in liquid nitrogen, and stored at
-80 C. Bovine tissues from 14- to 20-month-old heifers were obtained
from a local slaughterhouse, transported on dry ice, and then stored at
-80 C.
Membrane preparations
Tissues were pulverized, under liquid nitrogen, in a mortar;
suspended in 20 ml/g tissue of ice-cold homogenization buffer (50
mM Tris-HCl, pH 7.5, containing 1 mM EDTA, 1
mM dithiothreitol, and 0.1 mM
phenylmethylsulfonylfluoride); and homogenized, by 4 strokes, in a
Potter-Elvehjem homogenizer. After centrifugation at 3,000 x
g for 8 min, to remove cell debris and nuclei, the
supernatant fractions were centrifuged for 30 min at 100,000 x
g. The resulting pellets were washed once each in
homogenization buffer supplemented with 0.6 M
KCl, in salt-free homogenization buffer, and were finally resuspended
in 50 mM Tris-HCl buffer, pH 7.5. The membrane
suspensions (1–7 mg protein/ml) were frozen in liquid nitrogen and
stored at -80 C. Protein concentrations were determined by using a kit
from Bio-Rad Laboratories, Inc. (Munich, Germany), with
BSA (Sigma, fraction V) as standard.
Photoaffinity labeling protocol
Reactions were performed in 40 µl of 20-mM HEPES
buffer, pH 7.5, containing 5 mM
MgCl2, 125 mM NaCl, and the protease
inhibitors parahydroxymercury benzoate (60 µg/ml), bacitracin (1
mg/ml), bestatin (50 µg/ml), phosphoramidon (50 µg/ml), and
1,10-phenanthroline (1 mM). Membranes were preincubated in
this buffer for 5 min at 4 C before the addition of
125I-ANP (final concentration,0.5
nM). After incubation for 15 min at 22 C, the samples (in
1.5-ml Eppendorf polystyrene tubes) were irradiated (peak
wave-length, 302 nm) on a UV table (UV transilluminator model TM-36,
UVP Inc., San Gabriel, CA), in the dark, for 10 min, at room
temperature. Reactions were terminated by chilling and immediate
addition of 20 µl of 3x SDS-PAGE sample buffer consisting of 0.3
M Tris-HCl (pH 6.8), 200 mM dithiothreitol,
30% (vol/vol) glycerin, 15% (wt/vol) SDS, and 0.06% (wt/vol)
bromophenol blue. Samples were boiled for 3 min before analysis by
SDS-PAGE, under reducing condition, in 6–7.5% polyacrylamide
separation gels. Gels were dried after staining and then exposed to
XAR-5 films (Eastman Kodak Co., Rochester, NY),
between intensifying screens, at -70 C.
To confirm the identity of GC-A, reactions were also performed in the presence of an excess (1 µM) of the unlabeled peptides ANP, CNP, or C-ANF, respectively. Labeling of the 122- and 130-kDa proteins was completely blocked by ANP but not by CNP or the natriuretic peptide clearance receptor-specific ligand, C-ANF (16).
Chemical cross-linking
Membranes (40 µg protein) from rat olfactory bulb or adrenal
gland were incubated with 125I-ANP as described
above. Instead of UV light irradiation, cross-linking was induced here
by addition of disuccinimidyl suberate (Pierce Chemical Co., Rockford, IL), to a final concentration of 0.5
mM, and samples were incubated for 15 min at 20 C.
Analogous assays were performed with rat liver or olfactory bulb
membranes (50 µg of protein each) in the presence of
125I-insulin (0.2 nM) instead of
radiolabeled ANP. Reactions were terminated and analyzed in the same
way as stated above.
Deglycosylation
125I-ANP was cross-linked to membranes,
essentially according to the protocol given above but at increased
reaction volumes (80 µl) and protein amounts (adrenal, 100 µg;
olfactory bulb, 130 µg). After UV irradiation, membranes were
pelleted by centrifugation at 12,000 x g for 5 min.
The pellets were resuspended in 15 µl of 50-mM
HEPES (pH 7.5), 0.5% SDS, 6 mM
1,10-phenanthroline and were heated at 100 C for 2 min. The membrane
suspensions were quenched on ice and diluted to 80 µl by adding 15
µl H2O and 50 µl of
100-mM HEPES buffer (pH 7.5) containing 10
mM 1,10-phenanthroline, 2
mM phenylmethylsulfonylfluoride, 100 µg/µl
leupeptin, 1.2% Triton X-100, and 2 mM
ß-mercaptoethanol. Samples were incubated at 37 C for 1 h,
either without (negative control) or with the addition of 4 U (20 µl)
N-glycosidase F (Roche Molecular Biochemicals, Mannheim,
Germany; 903337). Reactions were stopped by mixing with 0.5 vol
of 3x SDS-PAGE sample buffer and analyzed via SDS-PAGE and
autoradiography.
Statistical validity of the cross-linking experiments
All cross-linking experiments have been performed at least three
times with the same membrane preparations. Quantitative assessments, by
gamma-counting of excised radiolabeled receptor bands, revealed
variations of less than 15%. Moreover, the results (see
Figs. 1–6
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are representative for assays carried out with membranes derived from
(three bovine or at least four rat) different animals. For calculation
of 50% inhibitory concentration (IC50)
values (see Fig. 4), the relative intensities of autoradiographic
signals were scanned with a Hoefer densitometer GS 300
(Hoefer Scientific Instruments, San Francisco, CA).
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), revealed a size heterogeneity of the
high-molecular-weight ANP receptor, GC-A. As estimated in relation to
stained reference proteins, the receptors from CNS-derived tissues
(olfactory bulb, cerebellum, pons, and spinal cord) had a lower
apparent molecular mass (122 kDa) than those expressed in peripheral
organs (130 kDa). A corresponding size difference was also evident
between bands visible at approximately 205 kDa in the upper part of the
autoradiogram, which presumably represent receptor dimers (18, 19). In additional experiments (data not shown), analyses of ANP
receptors from liver, spleen (130 kDa each), and thalamus (122 kDa)
confirmed these findings. It is noteworthy that the pineal gland, one
of the brain’s circumventricular organs, generates the 130-kDa ANP
receptor form (Fig. 1). The smaller size of GC-A in CNS tissues was not
attributable to proteolysis during the membrane preparation procedure,
because mixing adrenal with olfactory bulb homogenates before membrane
preparation and affinity cross-linking did not reduce the amount of
radioactivity associated with the 130-kDa receptor subtype.
Furthermore, the same distinct apparent molecular masses were
determined when the chemical agent disuccinimidyl suberate was used,
instead of UV irradiation, to generate cross-links between
125I-ANP and GC-A (see Fig. 3).
To examine whether differences in glycosylation could explain the
observed GC-A size heterogeneity, we analyzed the effects of
glycosidase treatments of receptors from either the adrenal or
olfactory bulb. After labeling with 125I-ANP and
subsequent incubation with N-glycosidase F, the apparent molecular
weights of both receptors were reduced to the same value of
approximately 116,000 (Fig. 2),
indicating a greater extent of N-linked glycosylation in peripheral
than in CNS tissues. Similar phenomena have been reported for other
glycoproteins, including the insulin receptor (20). To
compare directly the differential glycosylation between these two
proteins, ANP and insulin receptors from peripheral and CNS tissues
each were cross-linked to their radiolabeled ligands and
size-fractionated on the same SDS-polyacrylamide gel (Fig. 3). The hormone-coupled insulin receptor
-subunit and GC-A derived from olfactory bulb have similar apparent
molecular masses (approximately 122 kDa), whereas the peripherally
expressed polypeptides (137 and 130 kDa, respectively) significantly
differ in size. Thus, the variance between the CNS-specific and the
peripheral glycoforms is smaller in the case of the ANP receptor (8
kDa), compared with the insulin receptor -subunit (15 kDa).
Because glycosylation sites have been shown to reside in close vicinity
of the peptide-binding domain of GC-A (13, 21) and
based on findings that less glycosylated forms of the receptor are
characterized by an apparent loss of ligand binding capability
(12, 22), it was of interest to investigate whether the
two glycoforms of GC-A differ in their binding affinities to the three
natriuretic peptides. Fig. 4 shows an
analysis of the potencies of unlabeled ANP, BNP, and CNP to compete
with 125I-ANP-binding to receptors from either
peripheral (testis, adrenal) or central (olfactory bulb) tissues. The
same rank order (ANP > BNP >> CNP), as expected for GC-A
(23), and the same effective peptide concentrations were
determined with all three membrane preparations. More detailed studies
revealed equal IC50 values of 2.5, 8, and 200
nM for ANP, BNP, and CNP, respectively.
Membranes from the olfactory bulb and testis were used for a
comparative analysis between basal and ANP-stimulated GC activities in
CNS- and peripheral tissues. These organs were selected based on
similar receptor band intensities after radioligand cross-linking (Fig. 1), suggesting comparable concentrations of GC-A per milligram of
membrane protein. In the presence of MgCl2/ATP,
both membranes produced similar amounts of cGMP (Table 1); and coincubations with the
GC-A ligand, ANP, resulted in 4-fold increases each (Table 1).
Consistent with findings that hormone-sensitivity of GC-A requires
ATP-binding (8), only a slight stimulation (1.5- and
1.8-fold, respectively) of GC by ANP was detectable when incubations
were performed in the presence of MnCl2 instead
of MgCl2/ATP. Under these conditions, however,
the basal GC activities in the absence of ligand differed strongly
between the two membrane preparations. Compared with cGMP generation in
the presence of MgCl2/ATP, replacement of these
agents by MnCl2 induced a 23-fold increase in
basal GC activity in olfactory bulb but only a 4-fold increase in
testis membranes (Table 1).
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). Evidently, adrenal receptors
from rat and bovine are similar in size, and both species produce a
subtype of lower molecular weight in CNS tissues. As in rat, the
peripheral (130-kDa) receptor form is expressed in the bovine pineal
gland.
The occurrence of a CNS-specific GC-A variant in adult animals raised
the question of whether the same glycoform is expressed also in
premature brain tissue. To address this point, rat brain membranes from
early postnatal developmental stages (d 1 and 7) were analyzed (Fig. 6). Obviously, receptors expressed during
this period are similar in size to those in peripheral tissues of adult
animals, indicating an alteration in the oligosaccharide processing of
GC-A in brain during postnatal development. This study also revealed
markedly (6-fold) higher levels of GC-A in the adult than in the early
postnatal animals, suggesting that ANP plays a more important role for
the mature than for the developing brain.
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Discussion |
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As indicated by the amount of radioactivity associated with the
receptor bands (see Fig. 1), GC-A is highly expressed in the peripheral
tissues examined, but its detection in the olfactory bulb, cerebellum,
pons, and spinal cord also refers to a broad distribution of the
receptor within the CNS. Among the latter structures, an exceptionally
high concentration of GC-A is evident in the olfactory bulb. Whether
this particular accumulation has a functional role in the
neurochemistry of olfaction, however, is not yet clear
(24). The first molecular demonstration of GC-A in the
spinal cord, as shown here, is consistent with recently published
cGMP-immunocytochemical data, indicating local ANP-mediated synthesis
of the second messenger (25). Together with previous
reports on ANP immunoreactivity (26) and gene expression
(27) in the spinal cord, these findings further support an
implication of the peptide in modulation of nervous system function.
Based on our findings of relatively low expression levels of GC-A in
brain at stages of early postnatal development, ANP, however, may play
only a minor role during the differentiation and maturation of this
organ.
In contrast to other circumventricular organs, such as the subfornical organ and area postrema (which are characterized by relatively high levels of ANP receptors) (28), there is only a poor expression of GC-A in the pineal gland. It is to be noted, however, that this organ is characterized by exceptionally high levels of the CNP receptor, GC-B (17).
Oligosaccharide heterogeneity of GC-A
The data presented here clearly demonstrate a structural
heterogeneity of GC-A in vivo that is attributable to
differential glycosylation. As indicated by the analysis of various rat
and bovine tissues, native GC-A size heterogeneity seems to be
explained essentially by a distinct difference in N-linked receptor
glycosylation between CNS and peripheral tissues. Analogous phenomena
have been reported previously for other membrane proteins, such as
insulin (20, 29) and IGF-I (30) and -II
(31) receptors. As examined in this study (Fig. 3), the
difference in the extent of glycosylation is smaller in the case of
GC-A, compared with the insulin receptor -subunit. Because cDNA
analyses (32) revealed that the specific glycosylation
properties of brain insulin receptors do not stem from an altered
primary structure, tissue-specific posttranslational processing was
thought to account exclusively for this effect. Likewise, there is no
evidence, in the literature, for differences in amino acid composition
of the two GC-A glycoforms investigated here.
The question of whether the two receptor forms may differ in their phosphorylation states has not been examined in this study. However, it seems unlikely that variations in the degree of glycosylation (within the extracellularly-located receptor moiety) that apparently do not affect ligand binding should have any influence on kinase/phosphatase activities directed against intracellularly-located phosphorylation sites.
Glycosylation of GC-A has been addressed in a number of previous studies. By immunoblotting, two receptor size variants (135 and 125 kDa), representing differentially glycosylated proteins, were detected after stable expression of human GC-A in the embryonic kidney cell line 293 (12). In contrast to the 135-kDa species, the less glycosylated 125-kDa form did not bind to or cross-link ANP, suggesting an essential role of receptor glycosylation for ANP-binding activity. A heterogeneous glycosylation of GC-A, resulting in apparent molecular masses of approximately 122 and 135 kDa, respectively, was also found after transient expression of the receptor in COS-7 cells, and analyses of site-specific mutations located in the kinase homology domain of GC-A revealed a correlation between complete glycosylation and phosphorylation of the receptor (11). In another study (33), cells expressing recombinant GC-A were grown in the absence or presence of the specific glycosylation inhibitor, tunicamycin. As assessed by determinations of GC activities in the corresponding cell membrane preparations, the capability of ANP to stimulate cGMP production was found to be unaffected by tunicamycin treatment, suggesting, in contrast to the conclusions drawn by Lowe and Fendley (12), that glycosylation does not influence ANP binding. An apparent drawback of this study, however, is that the report fails to demonstrate convincingly that tunicamycin treatment, in fact, has altered the apparent molecular mass of the ANP receptor. Miyagi et al. (15) have used the recombinant extracellular domain of GC-A, expressed in COS-1 cells, to examine the role of glycosylation for ANP-binding. After enzymatic deglycosylation, the protein was found to retain the normal affinity for ANP, and the same results were obtained with the recombinant full-length receptor. In contrast, Marquis et al. (22) provided evidence for an important role of the degree of GC-A glycosylation for ligand-binding. By immunoblotting, the authors found several molecular forms of GC-A, probably attributable to differences in glycosylation, in rat kidney and bovine adrenal tissue, but only the apparently most-glycosylated species could be cross-linked to ANP. Thus, based on these reports, there is still considerable uncertainty concerning the role of glycosylation for GC-A properties. Moreover, with respect to physiological consequences, enzymatic deglycosylation of GC-A in vitro and the production of partially glycosylated forms of the protein in transfected cell lines may result in receptor molecules that do not occur in vivo.
In this study, we demonstrate that the two glycoforms of GC-A expressed in rat tissues have equal affinities for the natriuretic peptides, ANP, BNP, and CNP. In addition, we couldn’t recognize any difference between the receptor variants, with respect to ANP-induced stimulation of GC activity in the presence of MgCl2/ATP. Under these conditions, which involve allosteric binding of ATP to the receptor’s intracellular kinase homology domain, GC-A is thought to respond most effectively to ANP (8). We also examined possible effects of a differential receptor glycosylation on GC activities in the presence of Mn2+. Consistent with previous studies (cited in Ref. 34), this treatment resulted in strongly enhanced basal enzyme activities and a concomitant loss of ANP effects. Low increases (1.5- and 1.8-fold, respectively) in cGMP levels elicited by ANP in olfactory bulb and testis membrane preparations under these conditions were similar to those reported recently for lung membranes (34).The only marked difference observed between the two membranes relates to the basal GC activities, assessed in the presence of MgCl2/ATP vs. that of Mn2+. There is a much higher increase in basal GC activity induced by Mn2+ in the olfactory bulb than in the testis membranes. This phenomenon might be explained either by the less glycosylated (olfactory bulb) GC-A form being more sensitive to Mn2+-induced effects than the peripheral GC-A form or by the presence in olfactory bulb membranes (but not in testis) of other GCs that are highly sensitive to Mn2+.
The observed equal affinities of the two GC-A glycoforms to ANP might be explained most convincingly by the idea that the close receptor/ligand contact sites implicated in high-affinity binding are not influenced sterically by any of the carbohydrate moieties that differ between the two receptors. On the other hand, the residues responsible for the generation of covalent linkages between the receptor and ANP are thought to be (or even must be) located within this area of close contact sites, suggesting that the efficiency of cross-linking might also not be influenced by the differential glycosylation. In agreement with this, comparative Western blot experiments (data not shown) demonstrated a correlation between levels of 125I-ANP attached to the receptors after cross-linking and levels of GC-A protein detectable on immunoblots.
The expression of the 122-kDa receptor form in olfactory bulb, cerebellum, cerebral cortex, spinal cord, and thalamus and of the 130-kDa type in the pineal gland is indicative of a CNS-specific (rather than a brain-specific) oligosaccharide processing. Common properties of all the organs expressing the 122-kDa receptor subtype are: 1) that they contain nervous tissue, belonging to the CNS; and 2) that they are separated from the peripheral circulation by the blood-brain barrier. A significant role of the latter would be consistent with the observed (peripheral-like) receptor glycoform in brain at d 1 and 7 after birth, a period before the blood-brain barrier is established completely (35).
In conclusion, the occurrence of a specific, less glycosylated GC-A
subtype in the rat brain reflects an alteration of the glycosylation
processing that is developmentally regulated and apparently restricted
to CNS tissues separated by the blood-brain barrier. Our findings that
the two receptor glycoforms do not differ in ligand binding properties
are in agreement with data obtained on recombinantly-expressed
receptors (15), indicating that oligosaccharide structures
on GC-A are not implicated directly in ANP-binding. Furthermore,
ligand-induced stimulation of GC-A seems to be independent of the
degree or extent of receptor glycosylation. The appearance of similar
amounts of radiolabeled receptor dimers after affinity cross-linking
(see Fig. 1) suggests that the two GC-A glycoforms, in addition, may
not differ essentially in properties relevant for receptor
dimerization. Possible functional consequences of the observed
differential glycosylation that still remain to be examined include
effects on receptor internalization/desensitization processes.
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Acknowledgments |
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Footnotes |
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Abbreviations: ANP, Atrial natriuretic peptide; BNP, brain natriuretic peptide; cGMP, cyclic guanosine 3',5'-monophosphate; CNP, C-type natriuretic peptide; CNS, central nervous system; GC, guanylyl cyclase; IC50, 50% inhibitory concentration.
Received July 23, 2001.
Accepted for publication September 25, 2001.
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