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Department of Physiology (G.P.F., L.-R.T., H.M.G.), University of Massachusetts Medical School, Worcester, Massachusetts 01655; and Molecular and Cellular Biology Group (W.R.B.), American Cyanamid, Princeton, New Jersey 08540
Address all correspondence and requests for reprints to: H. Maurice Goodman, Ph.D., Department of Physiology, University of Massachusetts Medical School, 55 Lake Avenue, North, Worcester, Massachusetts 01655-0127.
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Abstract |
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The half life of GHBP in serum was estimated to be 2.4 h in rats treated with cycloheximide, whereas that of GHRS was 20 min in liver and 8.5 h in fat. These results suggest that GHRS is synthesized in liver 8 times faster than it is released into serum, whereas synthesis in fat is less than 30% of the rate at which it is released into serum by all tissues. Therefore, liver appears to be the major source of GHBP in serum. Although secretion into the circulatory system accounts for little or perhaps none of its turnover in some tissues, GHRS pools in tissues do appear to be regulated, suggesting that GHRS may function primarily in the cells in which it is synthesized.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Earlier studies in this laboratory indicated that despite the absence of a transmembrane domain, adipocytes retain substantially more GHRS than GHRL. In cell free extracts, GHRS is almost exclusively associated with the particulate fraction (11). GHRS appears to reside largely intracellularly because, in contrast to GHRL, only a small fraction of GHRS was sensitive to mild digestion of adipocytes with trypsin (11). Immunohistochemical studies also indicate an association of GHRS with intracellular membranes and the nucleus (12, 13). Newly synthesized GHRS in adipocytes consists of a pair of proteins with electrophoretic mobilities of 38 and 42 kDa (14), which is smaller than the 44- and 52-kDa circulating GHBP (5). These findings prompted us to study the relationship of the circulating GHBP to GHRS in tissues.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Western blot analysis
Whole tissue extracts were prepared by a procedure designed to
recover the entire tissue pool of GHRS regardless of its
subcellular distribution. Pieces of freshly excised liver, quadriceps
muscle, or adipose tissue weighing approximately 50 mg were sonicated
for 5 seconds with a Branson sonicator equipped with a microtip in 500
µl sample buffer (60 mM Tris, pH 8.8, 20% glycerol, 5%
ß-mercaptoethanol, and 2% SDS) and heated to 100° for 5 min
Detergent extracts of the particulate fraction were prepared by homogenizing tissues in 0.25 M sucrose containing 1 mM EDTA (10 ml/g muscle or liver, 5 ml/ml adipocytes) using a Tissuemizer for muscle and liver or a Dounce homogenizer for adipocytes. The homogenates were sedimented at 16,000 x g for 10 min and the resulting supernatants were then sedimented at 250,000 x g for 1 h. The 250,000 x g pellets were resuspended in extraction buffer (10 mM Tris, pH 8.0, 0.14 M sodium chloride, 0.025% sodium azide, 0.1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 10 µg/ml aprotinin, and 10 µg/ml leupeptin) and clarified by centrifugation at 16,000 x g for 10 min. Portions of the supernatant were mixed with an equal volume of 2x sample buffer and stored at -20 C for Western blot analysis, or assayed to determine the abundance of GH receptor isoforms.
Samples were analyzed on 7.5 or 10% polyacrylamide gels and
transferred electrophoretically to nitrocellulose membranes. The
membranes were blocked for 16 h at 23 C in PBS containing blocking
protein (3% BSA or 5% nonfat dry milk) and 0.02% sodium azide and
then incubated for 3 h with the primary antiserum. After four
washes with PBS plus 0.3% Tween 20, the membranes were incubated for
1 h in washing buffer plus blocking protein and donkey 
-rabbit
serum coupled to horseradish peroxidase (Amersham, Arlington Heights,
IL). The membranes were again washed and proteins were
detected using enhanced chemiluminescence.
Assay for GHRS and GHRL
Detergent extracts of tissues were prepared by homogenizing
0.2 g of liver, 0.4 g of muscle, or 0.4 g of fat in 5 ml
buffer (10 mM Tris, pH 8.0, 0.14 M NaCl, 1%
Triton X-100, 5 mM EDTA, 1 mM
phenylmethylsulfonyl fluoride, 0.02% sodium azide) and clarified by
centrifugation at 16,000 x g for 10 min. Samples
(50–100 µl) were incubated overnight at 23 C in 1.5 ml plastic
centrifuge tubes containing 1 ml of 10 mM Tris, pH 8, 0.14
M sodium chloride, 0.1% Triton X-100, 0.1% hemoglobin,
0.02% sodium azide, 20 ng/ml [125I]-hGH, and
-GHRS diluted 5000-fold or -GHRL diluted
1000-fold. Immune complexes were recovered by adding 25 µl of a 1:3
suspension of protein A-agarose beads (Sigma, St. Louis, MO) and gently
agitating the sampes on a rocker platform for 2 h at 23 C. The
beads were collected by centrifugation at 16,000 x g
for 1 min and washed twice with 1 ml of Tris saline plus 0.1% Triton
X-100 before counting. To calculate receptor levels from the amount of
bound GH, a 1:1 complex was assumed (17).
Antibodies
-GHRS was obtained from rabbits immunized with a
synthetic peptide (GPKYNSQHPHQEIDNHL), which differs from the 17 amino
acids at the unique carboxyl end of GHRS only by the
susbstitution of tyrosine for phenylalanine, and has been described
previously (14). -GHRL was obtained from a rabbit
immunized with the intracellular domain of GHRL fused to
the maltose binding protein and has been described previously (14).
-GHRS and -GHRL are both highly specific
and do not cross-react with the other GHR isoform. -GHBP was
obtained from rabbits immunized with recombinant rat GHBP and has been
previously described (5). It can detect both GHR isoforms as well as
GHBP on Western blots. Affinity purified antisera were prepared using
the respective antigens cross-linked to agarose beads. Nonspecific
antibodies were removed by washing the beads with 10 mM
Tris containing 0.5 M sodium chloride and specific
antibodies were then eluted with 0.1 M glycine, pH 2.5, and
collected in tubes containing 2 M Tris, pH 9 (final pH
8).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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, two bands were detected
both in serum and in cell extracts, but the electrophoretic mobilities
of the serum and adipocyte proteins differed. Whereas serum contained
immunoreactive proteins with apparent sizes of 44 and 52 kDa, the
corresponding proteins in adipocyte extracts were only 38 and 42 kDa.
The GHBP isoforms in serum appear to be identical to those reported by
Sadeghi et al. (5), which were degraded to a single 31 kDa
polypeptide by endoglycosidase F, demonstrating that they reflect
heterogeneous glycosylation of the same protein. The higher
electrophoretic mobilities of the bands corresponding to
GHRS recovered from adipocytes suggests that this tissue
pool is not the immediate precursor of the secreted GHBP.
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). Bands corresponding to the 44- and
52-kDa proteins in serum were not detected in extracts of any tissue,
suggesting that these three tissues all retain forms of
GHRS that differ from the GHBP released into the
circulatory system. Because GHBP levels are higher in female than in
male serum (5), and still higher levels are reported in pregnant rats
(8), we analyzed tissue extracts from male, virgin female, and pregnant
rats to determine whether GHRS pools that comigrate with
GHBP might accumulate in these tissues.
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were much weaker than those shown in
Fig. 1 despite loading the gel with protein samples large enough to
cause slight distortion of some bands, consistent with the expected
greater enrichment of GHRS in the particulate fraction than
in the whole cell extract. Consequently, we sought to enrich for
GHRS by homogenizing tissues in isotonic sucrose and
isolating a particulate fraction from each tissue. Western blots of
these samples are shown in Fig. 3a. In
serum samples from virgin female rats, the 44-and 52-kDa bands were
stronger than in samples from male rats, and these proteins appeared to
be most abundant in samples from pregnant rats. A similar pattern was
also seen for the 38- and 42-kDa bands in liver extracts. Furthermore
the intensity of 35- and 50-kDa bands that appeared to be insignificant
in extracts from male rat liver increased in proportion to the
intensities of the 38- and 42-kDa bands and may reflect additional
heterogeneity in the glycosylation of the polypeptide. The 35-kDa band
might also reflect partial degradation of one of the larger bands.
Finally, in pregnant liver extract a band at 110 kDa that corresponds
to the expected size of GHRL was also apparent. The
abundance of GHRS in muscle or fat extracts of virgin
female and pregnant rats appeared to be similar to that of males.
Prolonged exposure of the Western blot shows that adipose tissue and
muscle extracts, like those of liver, also contain traces of the 35-
and 50-kDa proteins (Fig. 3b).
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),
indicating that these bands all contain the same polypeptide chain. No
change in the electrophoretic mobility of the 38 and 42 kDa bands was
seen when a liver sample was degraded with neuraminidase (Fig. 4b),
suggesting that these glycoproteins lack terminal sialic acid residues
that are accessible to the enzyme. In contrast, neuraminidase degraded
both the 44- and 52-kDa bands of the serum sample to products with
apparent sizes of 40 and 45 kDa, respectively, indicating that these
serum proteins contain sialic acid residues that are accessible to the
enzyme. Endoglycosidase H, which specifically cleaves the high mannose
carbohydrates often found on immature glycoproteins, failed to degrade
the 44- and 52-kDa bands of GHBP in a serum sample (Fig. 4c), but it
degraded the 38- and 42-kDa bands of GHRS from liver to a
31-kDa band indistinguishable from that produced by endoglycosidase F.
These results suggest that the 38- and 42-kDa bands retained by tissues
may represent immature, high mannose forms of GHRS. The
incomplete digestion of GHBP by Endo F in Fig. 4c may reflect partial
renaturation of the sample during the overnight digestion with EndoF,
which works best on denatured glycoproteins.
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, tissue levels of GHRS were
generally more than 10 times higher in liver than in fat or muscle,
when normalized to tissue weight. Serum levels of GHBP were higher in
females than in males, and were higher in pregnant than in virgin
females. Tissue levels of GHRS tended to parallel GHBP
levels in serum, although differences between male and female or
between pregnant and virgin female were not always statistically
significant.
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To gain further insight into the relationship between GHRS
in tissues and GHBP in serum, we estimated the turnover of
GHRS, GHRL, and GHBP in groups of rats that
were given cycloheximide by iv injection and killed at various times
thereafter. After inhibition of protein synthesis, GHRS
levels in tissues of cycloheximide-treated rats initially declined at
rates that appeared to be linear when plotted on a semilogarithmic
scale (Fig. 6a). GHRS
declined much more quickly in liver than in adipose tissue or serum,
with initial half lives of 20 min, 8.5 h, and 2.4 h,
respectively. For comparison, we also estimated the half-life of
GHRL in liver and adipose tissue. As shown in Fig. 6b, GHRL declined rapidly in both tissues, with initial half
lives of 20 and 31 min, respectively. The initial rapid declines in
GHRS and GHRL were not sustained, suggesting
that these isoforms may be present in more than one tissue pool or that
the inhibitory effect of cycloheximide on protein synthesis may
diminish at later times. The results suggest that turnover and
therefore synthesis of GHRS and GHRL are
regulated independently and in a tissue specific manner. Estimates for
the rates of biosynthesis of GHRS and GHBP in male rat
tissues are shown in Table 1. The
relatively slow synthesis of GHRS in adipose tissue, and
its low concentration in lymph (18) suggest that this tissue may
secrete little or none of the GHBP in serum. The relatively high rate
of GHRS turnover in liver compared with that of GHBP in
serum suggests that secretion of GHBP accounts for only a small
fraction of GHRS turnover in the liver.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Sialylated GHRS does not appear to be retained in tissues, suggesting that when it is translocated to the Golgi, where sialylation is thought to occur, it is then rapidly secreted. The tissue pool of GHRS is therefore not the immediate precursor of the secreted GHBP and appears to remain in a high mannose form that is sensitive to digestion with endoglycosidase H. Therefore it might be expected to be confined to the endoplasmic reticulum. Alternatively, GHRS may reside in vesicles that do not fuse with the trans-Golgi network in which sialylation occurs. In contrast, the cellular GHRL pool appears to be sialylated, as indicated by its sensitivity to neuraminidase in cross-linking studies (19). The absence of sialic acid in the GHRS retained in tissues is consistent with the suggestion that GHRS is largely intracellular, as indicated by the earlier finding that it is less sensitive than GHRL to mild digestion with trypsin (11).
The glycosylation state of the cellular GHRS pool is consistent with the possibility that it is an intermediate in the GHBP secretory pathway that is retained within the endoplasmic reticulum. The relatively large size of the pool in rat liver and its high turnover relative to that of GHBP indicate that most of the GHRS is never secreted, and it is likely that little or none of the GHRS formed in other tissues is secreted. It is possible that intracellular GHRS pools represent biosynthesis in excess of that needed for secretion and are therefore simply degraded. However, the consistent relationship between the amounts of GHRS and GHRL in various tissues suggests that the pool sizes and subcellular locations of both isoforms may be regulated. The molecular features of GHR isoforms by which the cell distinguishes GHRS and GHRL are unknown, but the amino acid sequences diverge near the carboxyl end of the WSXWS motif, which is common to all cytokine receptors and known to influence their subcellular distribution. A large fraction of the homologous erythropoietin receptor is retained within cells, where it too appears to be incompletely glycosylated (20). Amino acid substitutions in its WSXWS motif alter translocation of the erythropoietin receptor to the plasma membrane (21), and a mutation in this motif that reduces transport to the cell surface has been reported for the GH receptor from the sex-linked dwarf chicken (22). Therefore it has been suggested that the WSXWS motif is an important determinant of the structural stability of cytokine receptors that influences their subcellular distribution. By interacting with this adjacent feature, the carboxyl end of GHRS might influence its subcellular distribution. Strong interaction of its carboxyl end with an integral membrane protein might also account for the recovery of GHRS in the particulate fraction of cell extracts. Additional studies are needed to determine how GHR isoforms become segregated within the cell and whether this segregation depends upon or causes the difference with respect to sialylation.
Localization of GH and GHBP to the cell nucleus (12, 13) suggests that these proteins may act within cells to effect GH responses. Indeed, addition of GHBP to cultured human hepatoma cells modulates expression of the GH receptor gene (23, 24). These findings raise the possibility that intracellular GHRS may act directly within the cells in which it is synthesized.
Present estimates (Fig. 5) for the abundance of both GHRS
and GHRL in whole tissues from male rats are substantially
larger than our earlier estimate for isolated adipocytes (11) and
indicate that in adipose tissue the ratio of
GHRS/GHRL is closer to 2 than 6. The previous
estimates were based on extracts of the particulate fraction prepared
from isolated adipocytes, and may have underestimated the tissue
contents due to incomplete recovery of both isoforms in the particulate
fraction. In addition, GHRL may be lost selectively during
the preparation of adipocytes due to its location on the cell surface
and its sensitivity to proteases that are present in collagenase
preparations. Furthermore, cells other than adipocytes that are present
in fat, such as endothelial cells, macrophages, or fibroblasts that may
have lower GHRS/GHRL ratios than adipocytes
could account for some of the difference in
GHRS/GHRL ratios in these two studies.
The serum GHBP values reported for male and female rats in Fig. 5 are
somewhat lower than estimates made by RIA (5) or ligand
immunofunctional assay (25). The differences between current and
previous estimates (5, 25) are greater for females than for males, and
may reflect differences in the age or strain of the animals rather than
inadequacies of the techniques. It is also possible that endogenous GH
might compete with [125I]GH and therefore lower our
estimates for GHBP. This factor was minimized by use of a large excess
of [125I]GH for our immunoprecipitations.
Adipocytes lost GHRS with a t1/2 of 40 min when they were incubated in vitro (11), more than 10 times as rapidly as the in vivo turnover reported here. In contrast, GH binding to adipocytes (15) suggested that GHRL turnover in isolated cells equalled the turnover rate in vivo. Therefore GHRS degradation by adipocytes in vivo is only a fraction of the maximum potential rate and may be inhibited by hormonal signals that are lost when tissues are removed from the rat. Inhibition of GHRS degradation might be expected to increase the size of tissue pools. At steady state, GHRS and GHRL appear to be synthesized at similar rates in liver because the tissue pools and turnover rates of both isoforms are similar. However, for the same reason, GHRS must be synthesized at less than 10% of the rate of GHRL in adipocytes. These findings suggest that tissue-specific factors may regulate both the synthesis and degradation of GHRS in adipocytes. Because Northern blots (3, 6, 7, 8, 9, 10) and RNA protection assays (26) indicate that the transcripts that encode GHRS are more abundant in fat, liver, and other tissues than those that encode GHRL, translation of GHRS may be regulated in a tissue specific manner. Despite synthesis and degradation of GHRS in fat at rates that are more than 10 times slower than GHRL, liver and fat tissue both maintain substantial pools of GHRS, suggesting that synthesis and degradation may be regulated to maintain tissue pools of both isoforms. Increased turnover of GHRS in liver may reflect the need for faster synthesis to provide for secretion of circulating GHBP.
Soluble isoforms of hormone receptors, particularly those of cytokine receptors, are frequently found in serum (27). Although these proteins interfere with hormone signaling by competing for ligands that could otherwise bind to membrane bound receptors, they can also enhance hormone action by slowing ligand clearance, and some can generate intracellular signals by interacting with signaling proteins such as gp130 (28, 29). Evidence suggesting all these modes of action has been reported for GHBP (23, 24, 30, 31, 32, 33). The size, turnover rates, and distinctive glycosylation of the GHRS pools in tissues raise the possibility that these or other modalities may also be implemented within the cells that synthesize GHRS.
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Footnotes |
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Received November 24, 1997.
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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