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Department of Medicine (B.D.G., C.E.L., M.O.T.), University of Virginia, Charlottesville, Virginia 22908; and Department of Biochemistry, Molecular Biology & Cell Biology (V.I.D., K.C.W., K.E.M.), Northwestern University, Evanston, Illinois 60208
Address all correspondence and requests for reprints to: Bruce D. Gaylinn, Box 511, Department of Medicine, University of Virginia, Charlottesville, Virginia 22908. E-mail: bg2g{at}virginia.edu
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The little mouse is a dwarf strain with an autosomal recessive defect characterized by substantially reduced levels of circulating GH and GH messenger RNA (mRNA) and anterior pituitary hypoplasia, with fewer pituitary somatotroph cells and sparse granulation (3, 4, 5, 6, 7). Cultured somatotroph cells from these animals do not release GH upon GHRH treatment but secrete GH upon treatment with cAMP or agents that increase cAMP levels, indicating that the defect is in the GHRH signaling pathway (5, 6). Genetic mapping and cloning studies have localized this defect to a point mutation in the N-terminal extracellular domain of the GHRH receptor (8, 9), where an aspartic acid residue at position 60 is mutated to glycine. Consistent with the dwarf phenotype, the mutant receptor is inactive, and cells expressing the mutant receptor do not accumulate cAMP in response to GHRH (8, 9). Mutations in the GHRH receptor have recently been identified in heritable isolated GH deficiencies in humans (10, 11, 12, 13), and thus, establishment of the molecular and cellular basis of these inactivating mutations promises to enhance our understanding not only of normal regulation of the GH axis but also of diseases of GH secretion.
The GHRH receptor belongs to family B-III of the G protein-coupled receptor (GPCR) superfamily (14), whose members include the receptors for secretin, vasoactive intestinal peptide (VIP), pituitary adenylate cyclase-activating peptide (PACAP), GLP-1, glucagon, and glucose-dependent insulinotropic polypeptide (GIP, also known as gastric inhibitory peptide). All these receptors share a high degree of homology and share several conserved residues, including the aspartic acid residue that is mutated in the little mouse. In all receptors of family B-III, the N-terminus has been shown to be important for ligand binding (15, 16, 17, 18, 19, 20, 21, 22, 23, 24). Studies on the GHRH receptor have indicated that the N-terminus is essential for ligand binding (24, 25, 26) but that the N-terminus from VIP or secretin receptors can substitute for the GHRH receptor N-terminus, resulting in chimeric receptors that bind GHRH (24). This demonstrates that residues of the transmembrane domains and/or the connecting extracellular loops are important in determining the specificity of ligand binding.
There are multiple levels at which the mutation of the aspartate 60 to glycine in the GHRH receptor might impact receptor function. It could influence the folding of the receptor protein, resulting in protein instability and thus decreased levels of receptor expression. It could affect transport of the receptor through the endoplasmic reticulum-Golgi network, resulting in lower cell surface expression of the receptor protein. The mutation could also alter receptor glycosylation, because it is only 10 residues from the single site for N-linked glycosylation at amino acid 50. Alternatively, the GHRH receptor protein might be normally expressed, but the little mutation might impact the ability of the receptor to bind its ligand, GHRH. Finally, the mutation might interfere with an appropriate hormone-induced conformational change affecting G protein coupling and signaling. To characterize the defect in the GHRH receptor of the little mouse, we used protein blot analysis and immunoprecipitation to compare expression and glycosylation of the wild-type and mutant receptor proteins and immunofluorescence analysis to compare the subcellular localization of the 2 receptors. We also developed a homologous binding assay for the mouse GHRH receptor and compared the ability of the wild-type and mutant GHRH receptors to bind GHRH and to activate adenylyl cyclase. Our results point to a selective defect in GHRH binding by the little mutant GHRH receptor.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Generation of antibodies to the GHRH receptor and Western
blotting
The synthetic peptide Ac-HMHPEADFITQLREDESAALQAAC-COOH, modeled
after the proposed N-terminus of the mature human GHRH receptor after
signal peptide cleavage (28) and with two internal cysteines replaced
with alanines, was prepared by solid-phase synthesis and purified by
HPLC, and its identity was confirmed by mass spectrometry (University
of Virginia Biomolecular Research Facility). This peptide was then
coupled through a C-terminal cysteine to maleimide-activated keyhole
limpet hemocyanin (Pierce Chemical Co., Rockford, IL) and
used to raise antiserum in rabbits (HRP Antiserum Services, Denver,
PA). This crude antiserum was used directly for the detection of mouse
or human GHRH receptor by Western analysis.
Crude membranes from HEK293 cells expressing the various receptors were extracted in 5 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) and 11.4 µg of total protein, as measured using the bicinchoninic acid assay (Pierce Chemical Co.), was electrophoresed on SDS-polyacrylamide gels with prestained low-range molecular weight standards (Life Technologies, Inc.), as previously described (29). Deglycosylation was performed by treatment with peptide-N-glycosidase F (PNGase F) (Roche Molecular Biochemicals, Indianapolis, IN), as described (29). The separated proteins were then electroblotted to nitrocellulose membrane (Protran, Schleicher & Schuell, Inc., Keene, NH), and electrophoretic transfer to nitrocellulose was confirmed by ponceau S staining of the blot. Crude rabbit sera, before (preimmune) or after immunization, were used at a 1:10,000 dilution and incubated with blots at 4 C overnight. The antibody-antigen complexes were detected by incubation with a 1:10,000 dilution of horseradish peroxidase-conjugated goat antirabbit antibody and visualized using the enhanced chemiluminescence method (Amersham Pharmacia Biotech, Arlington Heights, IL).
Generation and expression of epitope-tagged receptor
constructs
An oligonucleotide primer complementary to the 3' end of the
mouse GHRH receptor and encoding the sequence for the hemagglutinin
(HA) epitope (30) was synthesized. Recognition sites for the enzymes
KpnI and XbaI were engineered on either side of
the HA epitope with the stop codon within the XbaI site.
This primer, together with an upstream primer within the third
intracellular loop of the receptor cDNA was used to amplify a 700-bp
fragment from the 3' region of the receptor. The PCR product was cloned
in context with the 5' end of the wild-type or mutant receptor into the
expression vector pcDNA-3 (Invitrogen, San Diego, CA) to
generate full-length constructs for the wild-type and mutant receptor,
having the HA epitope at the C-terminus [mGHRHRwt.HA (wild-type mouse
GHRH receptor) and mGHRHRlit.HA (little mouse
GHRH receptor)].
An oligonucleotide primer complementary to the 5' end of the mouse GHRH receptor and encoding the sequence for the FLAG epitope (31) was synthesized and used along with a downstream primer in the third transmembrane domain of the receptor, to amplify a 700-bp fragment from the 5' end of the cDNAs of the wild-type and little mouse GHRH receptors. The PCR products from the wild-type and little mouse receptor were cloned in context with the 3' end of the receptor into the expression vector pcDNA-3, to generate full-length constructs for the respective receptors having the FLAG epitope at the N-terminus (mGHRHRwt.FLAG and mGHRHRlit.FLAG).
To assess the activity of the epitope-tagged wild-type mouse GHRH receptors, ligand binding and signaling were measured, and it was observed that the presence of the HA tag did not affect the activity of the receptor. The introduction of the FLAG tag at the N-terminus of the wild-type mouse GHRH receptor disrupted hormone binding in a manner similar to the FLAG-tagged human GHRH receptor (24); however, the receptor was expressed at levels comparable with the HA-tagged wild-type receptor and was present at the cell surface of intact cells.
The epitope-tagged constructs of the wild-type and mutant mouse GHRH receptors were expressed in HeLa T4 cells, as described (24), using the Vaccinia virus-T7 RNA polymerase system (obtained under license from Dr. Bernard Moss, National Institutes of Health, Bethesda, MD) (32). Cells were transfected using plasmid DNAs that were complexed with liposomes at a ratio of 4–5 µg lipid/µg DNA (33).
Metabolic labeling of transfected cells and immunoprecipitation of
epitope-tagged receptors
Transfected cells expressing the epitope-tagged receptor were
labeled with Pro-mix in vivo cell labeling mix
(L-[35S]Methionine and L-[35S]Cysteine,
Amersham Pharmacia Biotech) and immunoprecipitated, as
described (24), using the anti-HA monoclonal antibody 12CA5 (a gift
from Dr. R. A. Lamb, Northwestern University). The samples were
separated by SDS-PAGE using a Tris-glycine buffer with See-Blue
Pre-Stained Standard (Novex, San Diego, CA) as size
markers. The gels were fixed in 20% methanol/7% acetic acid,
saturated with glacial acetic acid, impregnated with 22% wt/vol of
2,5-diphenyl-oxazole in acetic acid, dried, and exposed to
Kodak X-OMAT AR film (Eastman Kodak Co.,
Rochester, NY).
Immunofluorescence localization of the wild-type and mutant
epitope-tagged receptors
Subconfluent monolayers of HeLa T4 cells, cultured on glass
coverslips, were transfected, the coverslips were washed twice in PBS,
and immunofluorescense analysis was performed as described (24). For
permeabilized cells, the coverslips were incubated for 2 h at 4 C
with 3 µg/ml of the anti-FLAG M2 monoclonal antibody
(Kodak IBI Division, New Haven, CT) or
1 µg/ml of the HA specific 12CA5 ascites fluid in PBS containing
0.1% saponin. After washing, the coverslips were incubated at 4 C for
30 min with 2 µg/ml fluorescein isothiocyanate-conjugated goat
antimouse secondary antibody (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA) in PBS containing 0.1%
saponin and 0.2% whole goat serum, washed with PBS, and mounted using
FITC-Guard (Testog Inc., Chicago, IL). For nonpermeabilized cells,
incubation was carried out using similar conditions with primary and
secondary antibodies diluted in PBS (without saponin). The images of
cells are optical sections obtained using confocal laser scanning
microscopy with a Bio-Rad Laboratories, Inc. MRC 600
connected to a Nikon microscope using a 40x objective.
All samples were scanned using the same contrast settings for
equivalent times, and optical sections were taken through the central
plane of the cell.
Iodination of GHRH analogs and assay of GHRH binding and cAMP
accumulation
Mouse GHRH and [His1, Nle27]human GHRH
(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)NH2 were purchased from Peninsula Laboratories, Inc. (Belmont, CA). These were iodinated to low
stoichiometry using the Iodobead method (Pierce Chemical Co.) and purified by reverse-phase HPLC, as previously described
(29).
GHRH binding was measured in crude membrane pellets permeabilized with
alamethicin, as described (29). Each tube contained approximately
100,000 cpm of probe corresponding to approximately 3.5 x
10-11 M 125I-GHRH and was
incubated for 1 h at room temperature. The crude membranes were
pelleted, detergent extracted with 5 mM CHAPS, and free
GHRH was removed with charcoal-dextran. Soluble, receptor-bound GHRH
was then assayed using a 
-counter (29). The binding data were
analyzed by nonlinear least-squares fitting to model curves using the
computer program Ligand (34). Intracellular cAMP was assayed by RIA of
extracts from cells grown in 24-well cluster plates and pretreated with
isobutylmethylxanthine, as described (28).
Photoaffinity cross-linking
The UV-activatable heterobifunctional cross-linking reagent
ANB-NOS (Pierce Chemical Co.) was coupled at low
stoichiometry to the lysines of [His1,
Nle27]human GHRH (1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32)NH2, and the crude
products were iodinated as above. The iodinated photoprobe was then
purified from this mix by HPLC, and used in the GHRH binding protocol,
as described above. After 60-min binding, the GHRH-receptor complexes
were cross-linked with long-wave UV, SDS denatured, analyzed by gel
electrophoresis as described for Western blots, and then
autoradiographed as described (29). Endoglycosidase H was obtained from
Roche Molecular Biochemicals (Indianapolis, IN)
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Results |
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The specificity of the antiserum for the GHRH receptor was confirmed by
using it to probe Western blots of CHAPS-extracted crude membranes from
nontransfected HEK293 cells and from cells transfected with the mouse,
little mouse, and human GHRH receptors (Fig. 1
). Consistent
with previous GHRH cross-linking studies (29), a band of approximately
52 kDa was detected in cell lines transfected with the human GHRH
receptor cDNA (Fig. 1D, lane 2). A
similar band of slightly greater mobility was seen in cell lines
transfected with either wild-type mouse or little mouse GHRH
receptors (Fig. 1, B and C). This band was not observed in control
HEK293 cells, and it was not detected by preimmune serum in the control
or receptor-expressing cell lines. As a further proof of specificity,
inclusion of excess of the synthetic peptide immunogen blocked the
labeling of this band by GHRH receptor antibody. After treatment with
PNGase F (Fig. 1, lane 3), the receptor-specific band shifted to a
greater apparent mobility, as expected from our previous observations
of receptor glycosylation (29). Because deglycosylation with
endoglycosidase H (not shown) gave the same results as the PNGase F
treatments in Fig. 1, this suggests that the glycosylation of both
wild-type and little GHRH receptors seen in these Western
blots is of the high mannose, core-glycosylated type.
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). To
better resolve receptor glycosylation forms, these gels were run in
10% polyacrylamide vs. 7.5% polyacrylamide in Fig. 1
. In
Fig. 2, lane 2 demonstrates three distinct receptor glycosylation
forms, each of which bind GHRH, because they are detected by
cross-linking of the labeled ligand. Lane 3 shows that all of these
forms can be deglycosylated to a higher mobility form by PNGase F. Lane
4 shows that endoglycosidase H, which can cleave high mannose (core
glycosylated) but not complex carbohydrates, shifts the lowest of the
glycosylated forms to the deglycosylated position but does not shift
the upper 2 bands. This cross-linking data demonstrates that both core
glycosylated and complex glycosylated forms of the wild-type GHRH
receptor are accessible in our permeabilized binding assay and bind
GHRH. Thus, complex glycosylation is not required for GHRH binding. It
is not clear why the complex glycosylated receptor was not detected by
Western analysis.
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), using anti-HA monoclonal antibody
12CA5, confirmed that the wild-type and mutant receptor proteins were
produced in similar abundance.
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). In intact cells expressing the
FLAG-tagged receptors, it was observed that the mutant GHRH receptor
was localized on the cell surface in a manner indistinguishable from
that of the wild-type receptor, whereas in permeabilized cells
expressing either the FLAG- or HA-tagged receptors, the receptors were
present both on the cell surface and intracellularly. Again, no
differences in the cellular distribution of the wild-type and
little receptors were observed. Thus, aspartate 60 is not
required for receptor protein expression, core glycosylation, or
appropriate transport to the cell surface, although it may be required
for attaining a receptor conformation necessary for high-affinity GHRH
binding and subsequent activation of GS 
. To test this
possibility, we developed a species homologous ligand-binding assay for
the mouse GHRH receptor.
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),
with the receptor protein expressed at 210 and 18.9 fmol/mg protein,
respectively. Competition binding of both mouse and human GHRH to
membranes from cell lines expressing the mouse or human receptor was
performed, and the data were normalized to the amount of receptor
expressed. As shown in Fig. 6, each
receptor bound better to the species-homologous ligand. Under these
assay conditions, the specific binding of human GHRH to the mouse GHRH
receptor was less than 10% of that seen with mouse GHRH in the same
membrane preparation, explaining our previous inability to detect
specific binding of human GHRH to the mouse receptor.
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). Specific binding of GHRH to the
wild-type receptor was competed by GHRH and decreased by the G protein
uncoupling agent guanosine 5'-O-(3-thiotriphosphate) (GTPS). Because
some types of receptor mutations can cause inactivation resulting from
intracellular accumulation of protein (35), this binding assay employed
homogenized and permeabilized cell membranes to detect binding by both
cell surface and intracellular membranes. Though binding to wild-type
receptor could be detected on intact live cells, more apparent binding
sites were seen in permeabilized membrane preparations, consistent with
the presence of functional receptor at both surface and internal sites.
GHRH binding to the little mouse receptor containing
permeabilized membranes was no greater than to membranes from
nontransfected cells, indicating that the mutant receptor was unable to
bind GHRH.
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),
confirming our previous findings in a different cell background
(8).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Several inherited human diseases have been attributed to single
amino acid substitutions in membrane proteins that either affect
protein folding, resulting in the degradation of the protein with a
reduction in the level of mature protein (37, 38, 39); or affect
glycosylation, processing, and transport, resulting in a decrease in
the functional protein at the cell surface (35, 38, 40, 41, 42). The most
striking example of such a phenomenon is the 
F508 mutant of the
cystic fibrosis transmembrane conductance regulator. The mutant protein
is misfolded, contains only core carbohydrates, and is rapidly degraded
within a pre-Golgi compartment, resulting in a decrease in the level of
mature protein in the cell (37, 38, 39). In another example, a point
mutation in the extracellular domain of the LH and FSH receptors
resulted in core-glycosylated receptors that are retained in the
endoplasmic reticulum of transfected cells (35). The mutant LH receptor
was unresponsive to its ligand because of its localization but bound
its ligand in a permeabilized membrane binding assay. The analogous
mutation in the closely related FSH receptor resulted in both retention
in the endoplasmic reticulum and loss of ability to bind ligand, even
in permeabilized cells (35). In several other diseases, single-point
mutations in GPCRs have been shown to abolish the ligand-binding
ability of the receptor (40, 43, 44). In the type-2 vasopressin
receptor, a point mutation in the first extracellular loop decreases
the cell surface expression of the receptor as well as the affinity of
the receptor for its ligand (40), whereas the mutation of a conserved
proline residue in the N-terminus of the PTH receptor affects the
binding and, consequently, the signaling ability of the receptor,
without affecting the cell surface localization of the receptor
(44).
To investigate the expression, glycosylation, and cellular localization
of the GHRH receptor protein, and to determine whether differences in
these processes might explain the loss of function of the
little mutant receptor, we generated a receptor-specific
polyclonal serum and used it to detect GHRH receptor protein in stably
transfected HEK293 cells. Western blots of cells stably transfected
with the wild-type and mutant GHRH receptor constructs indicate that
both proteins are expressed and are of a comparable size and that the
little mutation, which is only 10 residues away from the
site of N-linked glycosylation of the receptor, does not inhibit core
glycosylation of the receptor protein (Fig. 1). This core glycosylation
is comparable in both the wild-type and mutant receptors and is
therefore unlikely to be a consequence of the mutation. Complex
glycosylation could only be seen by cross-linking (Fig. 2), which only
shows receptors functional in binding and so could give no information
about the little mutant.
Using the alternative detection strategy of epitope-tagging the receptor proteins, we observed that using equivalent amounts of wild-type and little DNA in transient transfection experiments resulted in equivalent levels of expressed receptors, suggesting that the little mutant protein does not have a significantly reduced stability. Interestingly, both Western protein blot analysis of the native receptors and immunoprecipitation of the epitope-tagged receptors indicate that the human GHRH receptor migrates more slowly than the mouse GHRH receptor, although the cDNAs from both species encode an identical number of amino acids and the proteins are predicted to have similar masses. This difference persists, even after complete deglycosylation with PNGase F, suggesting that the difference in mobilities between the two receptors is attributable to a higher negative charge in the human receptor (six more negative charges are expected, based on differences in protein sequence).
We were able to use the epitope-tagged GHRH receptors for immunofluorescence experiments designed to investigate cellular localization of the receptor proteins. Our studies, using both intact and permeabilized cells expressing the wild-type and mutant forms of the epitope-tagged mouse GHRH receptor, indicate that both receptors have a similar cellular distribution. Both receptors could be visualized on the cell surface in intact cells using confocal microscopy. Importantly, the mutant GHRH receptor does not seem to be trapped intracellularly to any extent. Overall, these results indicate that the wild-type and mutant GHRH receptor proteins have similar expression levels, glycosylation pattern, and cellular distribution, implying that the defect is at the level of binding or signaling.
To determine whether the little mouse GHRH receptor is defective in ligand binding, we found it necessary to develop a species homologous assay using iodinated mouse GHRH. Binding of human GHRH to the human, ovine, bovine, porcine, rat, and mouse receptors has been previously examined, and the mouse receptor was found to be the only one that did not bind human GHRH at appreciable levels. GHRH binding to mouse pituitary membranes displayed the same species-specificity as was seen with the recombinant mouse receptor (data not shown). This is most likely because, of all the characterized mammalian GHRH peptides, mouse GHRH has the least sequence similarity to human GHRH, with 11 substitutions or deletions in the biologically active region of the peptide, which includes residues 1–29 (45, 46, 47). The decreased ability of the mouse GHRH receptor to bind human GHRH, combined with the relatively high nonspecific binding of GHRH to HEK293 cells, makes specific binding of iodinated human GHRH to the mouse receptor difficult to detect and explains previous difficulties in studying GHRH binding by the mouse receptor using the human peptide (48).
We observed a low level of GHRH-displaceable binding (Fig. 7) and
GHRH-induced stimulation of cAMP (Fig. 8) in nontransfected
HEK293 cells, even though GHRH receptor mRNA was not detectable in
Northern blots of these cells. This trace binding to nontransfected
cells could be significantly displaced by 50 µM GTPS
(Fig. 7) or 10 nM VIP (data not shown). The GTPS effect
suggests that the observed binding is to a GPCR that can be uncoupled
by the nonhydrolyzable GTP analog. As in our assay, 10 nM
VIP does not displace GHRH binding to GHRH receptors (not shown); this
suggests that the trace GHRH binding is caused by the ability of GHRH
to cross-react with some closely related endogenously expressed GPCR,
such as that for VIP or PACAP (49, 50). Consistent with this idea,
RT-PCR analysis, using degenerate primers designed to detect related
receptors of this family, demonstrate that HEK293 cells express the
type I PACAP receptor but not the GHRH receptor (unpublished
results).
Our data indicate that the GHRH receptor bearing the little mutation is defective at the level of GHRH binding. When the homologous conserved aspartic acid residue was mutated in the related receptors for glucagon (51) and VIP (17), the mutant receptors did not bind their respective ligands. This aspartic acid is conserved in nearly all known members of GPCR family B. Because these receptors bind diverse nonhomologous ligands (14) it is unlikely that it plays a role in the determination of ligand specificity. It is not anticipated that this aspartate interacts directly with GHRH; instead, this residue probably provides an acidic charge crucial for the structure or conformation required for hormone binding in all receptors of this family. Studies on the GHRH receptor have indicated that the N-terminus is essential for ligand binding (24, 25, 26) and deletion of this domain or alterations, such as the introduction of an epitope-tag into this domain, can result in inactivation of the receptor (24). Our current study demonstrates that the conserved aspartic acid at position 60 in the N-terminus of the GHRH receptor is essential for the interaction with the ligand, further supporting the role of the N-terminal domain in ligand recognition.
The discovery that the little mutation in mice is in the gene encoding the GHRH receptor (8, 9) provides support for the importance of the GHRH signaling system in normal growth. Our findings provide a cellular and molecular basis for understanding the defect in GHRH signal transduction in the little mouse and provide a foundation for further studies on the interaction of GHRH with its receptor.
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Acknowledgments |
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Footnotes |
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Received March 29, 1999.
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