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Department of Biochemistry, Molecular Biology and Cell Biology Northwestern University Evanston, Illinois 60208
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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N, in which part of the
N-terminal domain between the putative signal sequence and the first
transmembrane domain was deleted, and GHRHC, which was truncated
downstream of the first intracellular loop, were generated. Both the
receptors were deficient in ligand binding, indicating that neither the
N-terminal extracellular domain (N terminus) nor the membrane-spanning
domains with the associated extracellular loops (C terminus) are alone
sufficient for interaction with GHRH. In subsequent studies, chimeric
proteins between the receptors for GHRH and vasoactive intestinal
peptide (VIP) or secretin were generated, using the predicted start of
the first transmembrane domain as the junction for the exchange of the
N terminus between receptors. The chimeras having the N terminus of the
GHRH receptor and the C terminus of either the VIP or secretin receptor
(GNVC and
GNSC) did not bind GHRH
or activate adenylate cyclase after GHRH treatment. The reciprocal
chimeras having the N terminus of either the VIP or secretin receptors
and the C terminus of the GHRH receptor
(VNGC and
SNGC) bound GHRH and
stimulated cAMP accumulation after GHRH treatment. These results
suggest that although the N-terminal extracellular domain is essential
for ligand binding, the transmembrane domains and associated
extracellular loop regions of the GHRH receptor provide critical
information necessary for specific interaction with GHRH.
(Molecular Endocrinology 12: 750–765, 1998) |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The GHRH receptor is a G protein-coupled receptor (GPCR) with seven potential membrane-spanning domains and belongs to family B, group III (B-III) of the GPCR superfamily (13). It is highly homologous to the receptors for secretin (SEC) (14), vasoactive intestinal peptide (VIP) (15), pituitary adenylate cyclase-activating peptide (PACAP) (16), glucagon (17), glucagon-like peptide-1 (GLP-1) (18), and gastric inhibitory peptide (GIP) (19), which are also grouped in family B-III (13). These receptors are related to the receptors for PTH (20), PTH-related peptide (21), CRH (22), and calcitonin (23), which have a longer N-terminal extracellular domain but share many common amino acids and a similar overall structure to the receptors of family B-III (24).
The receptors of family B-III have several conserved residues in the
N-terminal extracellular domain, including six cysteines, an aspartate,
a tryptophan, and a glycine at positions corresponding to amino acids
60, 65, and 100 in the GHRH receptor (24). Mutation of the conserved
cysteines in the VIP receptor (25), the aspartate in the GHRH (7, 8, 26), VIP (27), and glucagon (28) receptors, and the tryptophan or
glycine in the VIP receptor (27) results in a loss of hormone binding
and hormone-stimulated cAMP accumulation in cells expressing these
mutant receptors. The N-terminal domains of the receptors of this
family are predicted to have a similar structure, presumably maintained
by disulfide bonds between the conserved cysteines, and are thought to
be involved in ligand binding (29). An 
-helical region within this
N-terminal domain is hypothesized to be involved in a coiled-coil
interaction with an -helical region in the respective hormone (30),
again suggesting a role for this domain in ligand binding.
The superfamily of GPCRs bind to, and are activated by, a diverse group of ligands ranging from ions and small peptides to large glycoproteins, and different receptors exhibit considerable variation in the structural determinants of ligand recognition (31, 32, 33, 34, 35, 36). Ligand binding by several receptors in family B has been studied using mutagenesis and chimeric receptor approaches. These studies reveal that the large N-terminal extracellular domains of the GLP-1 (37), VIP (38, 39), and PACAP (40) receptors are sufficient for low levels of specific ligand binding. However, other studies indicate that the presence of one or more extracellular loop regions and residues within the transmembrane domains is required for high-affinity ligand binding by the receptors for secretin, VIP, glucagon, calcitonin, PTH, GIP, and CRH (38, 39, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52), indicating that family B receptors are likely to exhibit multiple determinants of ligand interaction.
Other than the known importance of the conserved aspartate at position 60, which is mutated in the little mouse, there is no information about the role of specific domains or residues of the GHRH receptor in ligand binding and signaling. As an initial step toward understanding the molecular mechanism of agonist-dependent activation of the GHRH receptor, we designed experiments to identify domains of the GHRH receptor required for high-affinity GHRH binding. We generated two truncation mutants of the human GHRH receptor and four chimeras between the human GHRH receptor and the receptors for VIP or secretin and studied their ligand-binding and signaling properties.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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),
and it was observed that the membranes of cells expressing the
wild-type and HA-tagged GHRH receptors showed a similar dose-dependent
competition of GHRH binding, with ED50 values for
competition of 5.2 and 3.2 nM, respectively. In contrast,
membranes of cells expressing the FLAG-tagged GHRH receptor construct
did not bind GHRH. From Scatchard analysis of saturation-binding data,
the apparent dissociation constants (KD) for GHRH binding
by the wild-type and HA-tagged GHRH receptors were determined to be 36
and 31.6 pM, respectively, with maximal binding
(BMAX) values of 50.9 and 34.2 pmol/mg protein (data not
shown). In response to GHRH stimulation, cells expressing the wild-type
and HA-tagged forms of the GHRH receptor showed a similar
dose-dependent accumulation of cAMP (Fig. 2B) with ED50
values of 5.7 and 4.4 nM, respectively. Cells expressing
the FLAG-tagged GHRH receptor did not accumulate cAMP, consistent with
the inability of this receptor to bind GHRH. Receptor constructs with
the FLAG epitope tag were therefore used only for comparing the
cellular localization of various receptor proteins in intact cells. In
studies not shown, the presence of the C-terminal HA epitope tag was
found not to affect the properties of the epitope-tagged VIP (VIPR.HA)
and secretin (SECR.HA) receptors compared with their wild-type
counterparts.
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N by
removing sequences between the two AflII sites, and the
amino acids downstream of the first intracellular loop were deleted in
the receptor GHRHC by removing sequences between the two
KpnI sites (Fig. 1
). Immunoprecipitation of the
metabolically labeled HA-tagged receptor proteins (Fig. 3A) shows that the two truncated
receptors, GHRHN and GHRHC, were expressed at or above levels of
the full-length receptor. The apparent molecular masses were
approximately 40 kDa and 20 kDa, respectively; however, the
immunoprecipitated protein from cells expressing GHRHN showed the
presence of two products, differing in size by 4 kDa. The cellular
localization of the full-length and truncated epitope-tagged receptors
was determined by immunofluorescence analysis using confocal microscopy
with intact (for the FLAG-tagged receptors) or permeabilized (for the
FLAG- and HA-tagged receptors) cells (Fig. 3B). The full-length (GHRHR)
and C-terminally truncated (GHRHC) receptors were localized
similarly; receptor protein was distributed both on the cell surface
and intracellularly, with the latter presumably representing receptor
protein being transported to, or internalized from, the cell surface.
For these two receptors, similar localization patterns were observed
using antibodies against either the N-terminal FLAG tag or the
C-terminal HA tag in permeabilized cells. In contrast, cells expressing
the N-terminally truncated receptor (GHRHN) appeared to accumulate
protein preferentially in intracellular membranes, with a smaller
proportion of the total receptor expressed on the cell surface. A
perinuclear localization of this receptor protein was observed using
both the FLAG and HA antibodies in permeabilized cells (Fig. 3B).
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The truncated receptors GHRHN and GHRHC did not bind GHRH when
expressed by themselves or when coexpressed. In addition, neither
truncated receptor decreased the ligand-binding ability of the
full-length GHRH receptor when coexpressed with it. GHRH treatment did
not stimulate cAMP accumulation in cells expressing the truncated
receptor GHRHN, expressed either alone or together with the receptor
GHRHC (Fig. 4B), consistent with the results of the binding studies.
Coexpression of either GHRHN or GHRHC with the full-length GHRH
receptor did not diminish activation of adenylate cyclase by the
full-length receptor (Fig. 4B).
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Expression of Chimeric GHRH and VIP or Secretin Receptors
As described in Table 1, the
chimeras between the GHRH and VIP or secretin receptors were generated
by the exchange of the N termini of the HA-tagged receptors, with the
fusions made at the AflII site immediately before the first
transmembrane domain. The GHRH-VIP receptor chimeras were
GNVC, which consists of the N-terminal 127
residues of the GHRH receptor and residues 144–459 of the VIP
receptor, and VNGC, generated by replacement of
the 127 residues constituting the N terminus of the GHRH receptor with
the equivalent 143 residues forming the N terminus of the VIP receptor.
The GHRH-secretin receptor chimeras were GNSC,
consisting of residues 1–127 of the GHRH receptor and residues
143–449 of the secretin receptor, and SNGC,
consisting of residues 1–142 of the secretin receptor and residues
128–423 of the GHRH receptor. The expression of the wild-type and
chimeric receptor proteins was assessed by immunoprecipitation and, as
shown in Fig. 5A, the proteins produced
had the expected molecular sizes, and the chimeric receptors were
expressed at levels comparable to, or above, those of the wild-type
receptors. The chimeric receptors were glycosylated as determined by
treatment with peptide-N-glycosidase F (data not shown), which reduced
the observed size of the receptor proteins in accord with the number of
potential glycosylation sites in the N-terminal extracellular domain of
each (one in GNVC, three in
VNGC, one in GNSC, and
four in SNGC).
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, the wild-type and chimeric receptors are localized
in a similar manner within the cell. All receptor proteins are
distributed both on the cell surface and intracellularly, with the
latter presumably representing receptor protein that is being
transported to, or internalized from, the cell surface.
Analysis of Chimeric GHRH and VIP Receptors
The ligand-binding properties of the chimeras between the GHRH and
VIP receptors were analyzed by measurement of binding to both ligands
using competition assays (Fig. 6). The
chimera GNVC bound GHRH and VIP at extremely
low levels, while the chimera VNGC bound GHRH
at levels comparable to membranes of cells expressing the wild-type
GHRH receptor (Fig. 6A) but bound VIP at much lower levels than
membranes of cells expressing the wild-type VIP receptor (Fig. 6B).
Scatchard analysis of saturation-binding data for binding of GHRH and
VIP to the chimera VNGC was performed, and the
KD values were determined to be 46 pM and 156
pM, respectively. Dose-dependent accumulation of cAMP in
cells transfected with the wild-type and chimeric receptors in response
to treatment with GHRH and VIP is shown in Fig. 7. Cells expressing the chimera
GNVC show an increase in intracellular cAMP in
response to GHRH and VIP only at high hormone concentrations
(>10-7 M). However, cells expressing the
chimera VNGC showed a robust accumulation of
cAMP in response to treatment with both GHRH and VIP, with
ED50 values of 1.75 and 7.95 nM, respectively,
consistent with the ability of this receptor to bind GHRH, and more
weakly, VIP. Interestingly, the chimera VNGC
mediates a much stronger cAMP response to GHRH than does the wild-type
GHRH receptor, although the two receptors bound GHRH at comparable
levels. The binding and signaling properties of the wild-type and
chimeric GHRH and VIP receptors are summarized in Table 2.
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). The chimera
GNSC did not show any significant binding of
GHRH while the reciprocal chimera SNGC bound
GHRH at lower levels than the wild-type GHRH receptor, although it was
expressed at higher levels than the wild-type receptor. The
ED50 for competition of GHRH binding by the chimera
SNGC was 1.15 nM, and the
KD value determined by Scatchard analysis of
saturation-binding data was 259.3 pM. We were unable to
iodinate secretin to high specific activity and therefore could not
directly examine binding of secretin to these chimeras, but we studied
the interaction of these chimeras with secretin by quantifying the
intracellular cAMP levels in response to secretin stimulation. After
treatment with GHRH, adenylate cyclase was not activated in cells
expressing the chimera GNSC. In contrast, cells
expressing the reciprocal chimera SNGC
accumulated cAMP in a dose-dependent manner, although there was a small
rightward shift in the response curve (Fig. 9A). Cells expressing either of the two
chimeras GNSC or SNGC
did not accumulate cAMP after treatment with secretin (Fig. 9B). The
binding and signaling properties of the wild-type and chimeric GHRH and
secretin receptors are summarized in Table 3.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Studies on the GHRH receptor of the little mouse, in which
mutation of the conserved aspartate at position 60 results in a loss of
the ability of the receptor to bind ligand (26) and activate adenylate
cyclase (7, 8), provided the first indication that the integrity of the
N terminus of the GHRH receptor is essential for ligand binding. In
this study, we found that the insertion of a FLAG epitope tag at
position 37 in the N terminus of the receptor resulted in inactivation
of the receptor. Although this effect could be due in part to altered
expressed or localization of the FLAG-tagged receptor, our data are
consistent with this insertion directly affecting the ligand-binding
properties of the receptor. From computational structural studies on
the receptors of family B, it has been proposed that an -helical
region in the N terminus of these receptors interacts with an
-helical region in the C terminus of the bioactive region of the
corresponding peptide hormone to form a coiled-coil motif (30). For the
GHRH receptor, this -helical region is predicted to extend from
residues 26–40 and to interact with an -helix formed by residues
13–23 of the hormone. Inactivation of the receptor by the introduction
of the FLAG epitope tag at position 37 might therefore result from the
interruption of this -helical domain.
Deletion of the N terminus affected transport of the receptor GHRHN
to the cell surface and resulted in accumulation of the receptor
protein in intracellular membranes, despite inclusion of the signal
peptide in this construct. This abnormal localization of GHRHN could
be a consequence of the absence of the site for N-linked glycosylation
or of defective transport due to the proximity of the signal peptide
and the first transmembrane domain. The larger of the two bands seen in
immunoprecipitation analysis of extracts from cells expressing GHRHN
is likely to be receptor protein in which cleavage of the signal
peptide has not occurred, potentially due to structural constraints
resulting from the adjacency of the first transmembrane domain. The
receptor GHRHN did not bind GHRH in a competition assay using total
cell membranes, nor did it activate adenylate cyclase upon hormone
treatment, implying that the N terminus is essential for GHRH binding.
The deletion of the N termini of the receptors for VIP and glucagon
also resulted in proteins that did not bind to their respective ligands
(27, 48), suggesting that the N terminus plays an important role in the
formation of the binding site of receptors of family B-III.
The receptor truncated in the first intracellular loop, GHRHC, was
localized within cells in a manner similar to the wild-type GHRH
receptor but did not bind GHRH, indicating that although essential for
ligand binding, the N terminus alone is not sufficient for interaction
with GHRH. Our results with GHRHC are similar to those obtained with
a C-terminally truncated glucagon receptor (48) but are distinct from
those obtained with a similarly truncated PACAP receptor that
specifically bound PACAP, although with significantly lower affinity
than the wild-type PACAP receptor (40).
Although coexpression of N- and C-terminal domains of receptors can
result in partially functional receptors (32, 54, 55), suggesting
reconstitution of receptors by domain-domain interaction, coexpression
of the two truncated forms of the GHRH receptor, GHRHN and GHRHC,
did not reconstitute ligand-binding activity. This could be due to poor
colocalization of the receptor fragments or due to a lack of
interaction of the two truncated proteins resulting from disruption of
the complex by the presence of the first transmembrane domain in both
receptor fragments. Additionally, we coexpressed the truncated and
wild-type GHRH receptors and found that there was no decrease in
hormone binding or signaling by the wild-type receptor, implying that
the truncated receptors GHRHN and GHRHC do not act as functional
dominant negative mutants.
The generation of truncation constructs of GPCRs has been useful for the identification of structural domains involved in ligand binding in several receptors (40, 54, 55, 56, 57, 58). However, this approach has disadvantages, in that deletion of domains or truncations can result in structural alterations and mislocalization of the receptor proteins (48, 58). The generation of chimeras between two similar but distinct receptors can be used to correlate gain or loss of function with the exchanged domain, often with minimal effects on the structure of the receptor. Chimeric receptors generated by the exchange of homologous domains have proven useful in localizing the ligand-binding domains of receptors for biogenic amines (31, 32), glycoprotein hormones (33), and small peptides (34, 35, 36), and also of several receptors in family B including the VIP, secretin, glucagon, GIP, GLP-1, CRH, and PTH receptors (38, 39, 44, 46, 47, 49, 51, 52). We therefore applied this approach to characterize the ligand-binding domains of the GHRH receptor.
The wild-type GHRH, VIP, and secretin receptors show a high degree of
specificity in their interaction with their respective ligands,
although some ability of GHRH to activate the VIP receptor and of VIP
to activate the GHRH receptor at high ligand concentrations has been
observed (3, 59). Both the VIP and secretin receptors require the
N-terminal extracellular domain as well as the first extracellular loop
for high-affinity ligand recognition (29, 38, 39, 41, 42, 43, 44). Exchange of
the N-terminus of the VIP or secretin receptors with the corresponding
region of the GHRH receptor resulted in chimeras
GNVC, which showed a very weak response to both
GHRH and VIP, and GNSC, which was unresponsive
to both GHRH and secretin, consistent with studies of chimeras between
the VIP and secretin receptors (38, 39). However, the chimeric
receptors VNGC and
SNGC, having the N terminus of the VIP or
secretin receptors and the C terminus of the GHRH receptor, bound GHRH
and stimulated adenylate cyclase in a dose-dependent manner in response
to GHRH. The chimera SNGC bound GHRH at lower
levels but had a similar cAMP response compared with the wild-type
receptor, while the chimera VNGC showed similar
binding but a higher signaling response to GHRH than the wild-type
receptor. Our observation that the chimera VNGC
bound VIP and activated adenylate cyclase in response to VIP can be
explained by the fact that the aspartate residue at position 196 in the
first extracellular loop of the VIP receptor, which is predicted to be
important in interaction with VIP (43), is also present in the
corresponding region of the GHRH receptor. The VIP receptor and the
chimeric receptor GNVC mediate cAMP
accumulation in response to very high concentrations of GHRH,
consistent with a low-affinity interaction of the VIP receptor with
GHRH. The differences between the binding and signaling responses in
the chimeric receptors SNGC and
VNGC suggest that some of the determinants
required for GHRH binding are present in the N terminus of the VIP
receptor but not the secretin receptor. Alignment of the N-terminal
regions of the three receptors (3, 24) indicates that among the
residues predicted to form the -helix suggested to be involved in
the coiled-coil interaction with the ligand (30), there is a higher
homology between the GHRH and VIP receptors than between the GHRH and
secretin receptors. Additionally, comparison of the amino acids at
positions 13–23 in the hormones GHRH, VIP, and secretin, which are
predicted to form the -helices that interact with the receptors
(30), shows that this region of GHRH is more homologous to VIP than to
secretin. These differences between VIP and secretin, and between their
receptors, are likely to account for the differences in the
GHRH-binding and signaling responses of the chimeras
VNGC and SNGC.
Our data from the truncated receptors demonstrate that neither the N
terminus nor the C terminus of the GHRH receptor can bind ligand in the
absence of the other domain. Our studies of the chimeric receptors
suggest that one or more of the extracellular loops, in conjunction
with residues in the transmembrane domains, function as the
determinants of binding specificity and activation for the GHRH
receptor. Although the N terminus is essential for ligand binding,
substitution of this domain with the corresponding domain of a related
family B-III receptor reconstitutes functional GHRH binding. It has
been hypothesized that all the receptors in this family have a similar
structure in the N-terminal domain as a result of the conserved pattern
of cysteine residues forming conserved disulfide linkages (29), and an
-helix in this region is predicted to be involved in the primary
interaction with the ligand (30). From our results and those obtained
from studies on other receptors in this family (38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 49, 50, 51, 52, 60), it
is conceivable that the N terminus of these receptors plays a primary
structural role in the initial interaction with the ligand, while
residues in the extracellular loops and the transmembrane domains are
involved in secondary interactions with the ligand that determine the
specificity of ligand binding. Our results provide a basis for future
studies to identify specific receptor domains and residues involved in
recognition and binding of GHRH. Chimeras in which individual
extracellular loops of the receptor are substituted will enable the
localization of binding determinants in a more precise manner, allowing
site-directed mutagenesis to be used to define the key residues
involved in specific ligand interaction. Identification of domains that
are structurally important for the interaction of GHRH with its
receptor will facilitate the design of specific receptor agonists and
antagonists that might have therapeutic use in the diagnosis or
treatment of diseases of the GH axis.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Generation of Epitope-Tagged Receptor Constructs
Oligonucleotides having 20–22 nucleotides complementary to the
C-terminal end of the GHRH, VIP, or secretin receptors and encoding the
influenza virus HA epitope (61) were 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. These primers, together with upstream primers
within each of the receptor cDNAs, were used to amplify the C-terminal
fragment of each receptor. The PCR products were cloned in context with
the respective receptor into the expression vector pcDNA-3 to
generate full-length clones for the epitope-tagged receptors (GHRHR.HA,
VIPR.HA, and SECR.HA).
The amino acids for the FLAG epitope tag (62) were inserted at the N-terminal domain of the GHRH receptor downstream of the putative signal peptide at residue 37 using gene splicing by overlap extension (63). An oligonucleotide primer encoding the FLAG epitope tag with a site for the enzyme EcoRV, 3' of the tag and having 22 nucleotides complementary to the GHRH receptor was synthesized. This primer and a downstream primer were used to amplify a fragment corresponding to amino acids 130–580 of the receptor. A primer complementary to the sense primer for the FLAG epitope and the T7 promoter primer were used to amplify a fragment corresponding to amino acids 1–140 of the receptor. The two fragments were annealed and amplified using the two external primers, and the product was cloned in context with the C-terminal region of the receptor to generate the FLAG-tagged GHRH receptor GHRHR.F.
Generation of Truncation and Chimeric Constructs of the GHRH
Receptor
Using site-directed mutagenesis (64), the recognition site for
the enzyme AflII (CTTAAG) was engineered in the tagged GHRH
receptors as shown in Fig. 1. The site was introduced at nucleotides
100–106 and 385–390 (corresponding to amino acids 34–35 and
129–130), respectively, in the HA-tagged receptor and at nucleotides
385–390 in the FLAG-tagged receptor. This resulted in changes in amino
acids 35 and 129 (Arg to Lys and Val to Leu, respectively).
Removal of the 285-bp AflII-AflII fragment
resulted in the deletion of the extracellular domain between amino
acids 35 and 129 to give the N-terminally truncated HA-tagged construct
(GHRHN.HA). Deletion of the 278-bp EcoRV-AflII
fragment of the FLAG-tagged GHRH receptor, followed by filling-in with
Klenow polymerase and ligation, generated the N-terminally truncated
FLAG-tagged construct (GHRHN.F).
An oligonucleotide complementary to the first intracellular loop of the
GHRH receptor and with nucleotides 467–472 altered to form a
recognition site for the enzyme KpnI, together with the T7
promoter primer, was used to amplify a 480-bp N-terminal fragment of
the GHRH receptor. The PCR product was digested with HindIII
and KpnI and ligated with the large fragment obtained by
digestion of the full-length HA-tagged GHRH receptor with the same
enzymes, to generate the construct GHRHC.HA, truncated within the
first intracellular loop. The same primers were used to amplify a
516-bp fragment using the FLAG-tagged GHRH receptor as a template, and
the product was ligated into pcDNA-3 to generate the clone
GHRHC.F.
The chimeras were constructed by the exchange of N-terminal
extracellular domains between the HA-tagged receptors with the
predicted start of the first transmembrane domain of the receptors as
the junction (Table 1). The recognition site for AflII was
engineered at nucleotide positions 385–390, 427–432, and 424–439
(amino acids 129–130, 143–144, and 142–143) in the cDNAs for the
GHRH, VIP, and secretin receptors, respectively, and the wild-type
receptors having the epitope tag and AflII site were used
for all subsequent studies. The N-terminal fragments that were
exchanged between the receptors were generated by digestion of the
cDNAs for the wild-type receptors with HindIII and
AflII.
Expression of Receptor Constructs in HeLa T4 cells Using the
Vaccinia-T7 RNA Polymerase Hybrid Expression System
All experiments were performed using cells transfected with
various receptor constructs using the Vaccinia Virus-T7 polymerase
hybrid expression system (65). Subconfluent monolayers of HeLa T4 cells
cultured in DMEM (Sigma Chemical Co., St. Louis, MO) supplemented with
5% FBS (GIBCO BRL, Grand Island, NY) were infected with Vaccinia virus
vTF7.3 expressing the bacteriophage T7 RNA polymerase (obtained under
license from Dr. Bernard Moss, NIH, Bethesda, MD), at a multiplicity of
infection of 10, for 30 min in PBS/0.1% BSA. The various plasmid DNAs
were incubated with liposomes (66) at the ratio of 4–5 µg of lipid
per µg DNA, in Opti-MEM I medium (GIBCO BRL), for 20–30 min at room
temperature. Virus was aspirated from the cells, and the DNA/liposome
mixture was added to the cells and incubated at 37 C in 5%
CO2 for 15–16 h. The amount of DNA used for transfection
varied with the size of the plates.
Metabolic Labeling of Transfected Cells and Immunoprecipitation
of Epitope-Tagged Receptors
Cells grown in 35-mm dishes and transfected with 5 µg DNA per
plate were starved in cysteine/methionine-deficient DMEM (ICN
Biomedical Inc., Irvine, CA) for 30 min and labeled with 50 µCi/plate
ProMix (Amersham) in the same medium for 2 h at 37 C in 5%
CO2. The cells were washed with PBS, harvested, and
subjected to one cycle of freeze-thaw. The cell pellets were
resuspended in 400 µl RIPA buffer [150 mM NaCl, 50
mM Tris-HCl (pH 7.5), 1% Nonidet P-40, 0.5% deoxycholic
acid, and 0.1% SDS] containing 0.1 mM
phenylmethylsulfonyl fluoride and 1 µg/ml leupeptin, and the
membranes were solubilized by incubation on ice for 1 h with
vortexing. The lysate was clarified by centrifugation and incubated
with 0.5 µg of the HA-specific 12CA5 ascites fluid (a gift from Dr.
Robert A. Lamb, Northwestern University) for 3–4 h at 4 C on a
hematology mixer. At the end of this period, 30 µl of a 50%
suspension of protein A-Sepharose beads (Pharmacia, Piscataway, NJ) in
PBS were added to the tubes, and the incubation was continued for 30
min. The beads were washed four times with 500 µl cold RIPA buffer
and once with cold wash buffer (50 mM Tris-HCl/pH 7.5, 150
mM NaCl, 5 mM EDTA) and resuspended in 30 µl
of 2x SDS-PAGE sample buffer (50 mM Tris-HCl/pH 6.8, 2%
SDS, 10% glycerol, 5% ß-mercaptoethanol, 0.1% bromophenol blue).
The samples were boiled for 5 min before separation 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 Company, Rochester, NY). For
glycosylation analysis, immunoprecipitated proteins were digested for
4 h at 37 C with 0.2 U of peptide-N-glycosidase F
(Boehringer Mannheim Corp., Indianapolis, IN) in a buffer containing 20
mM Na2HPO4 (pH 8.0), 20
mM EDTA, 1% NP40, 1 µg/ml leupeptin, 0.1 µg/ml
pepstatin A, and 1 µg/ml aprotinin.
Immunofluorescence Localization of Epitope-Tagged Receptors
HeLa T4 cells cultured on glass coverslips in 35-mm plates were
transfected as described. For permeabilized cells expressing the
FLAG-tagged constructs, the coverslips were washed twice in PBS and
incubated for 2 h at 4 C with 3 µg/ml of the anti-FLAG M2
monoclonal antibody (Kodak IBI, New Haven, CT) in PBS containing 0.1%
saponin. After extensive washing, the coverslips were incubated at 4 C
for 30 min with 2 µg/ml of fluorescein isothiocyanate
(FITC)-conjugated goat anti-mouse secondary antibody (Jackson
ImmunoResearch Laboratories, West Grove, PA) in PBS containing 0.1%
saponin and 0.2% whole goat serum, and after extensive washing with
PBS, the coverslips were mounted in FITC-Guard (Testog Inc., Chicago,
IL). For nonpermeabilized cells, incubation was carried out under
similar conditions using primary and secondary antibodies diluted in
PBS (without saponin). For cells expressing the HA-tagged receptor
constructs, the coverslips were washed twice in PBS, fixed with 1%
(wt/vol) paraformaldehyde in PBS for 10 min, and immunofluorescense
analysis was performed as described, using 1 µg/ml of the HA-specific
12CA5 ascites fluid. The images of cells expressing the FLAG-tagged and
HA-tagged receptors in Fig. 3B were optical sections obtained using
confocal laser scanning microscopy with a Bio-Rad MRC 600 (Bio-Rad
Laboratories, Richmond, CA) connected to a Nikon microscope using a
40x objective. All samples were scanned under the same contrast
settings for equivalent times, and optical sections were taken through
the central plane of the cell. Photomicroscopy of cells expressing the
wild-type and chimeric receptors in Fig. 5B was performed using a 63x
objective on a Zeiss Axiophot microscope (Carl Zeiss Inc., Oberkochen,
Germany) with equivalent exposure times.
Measurement of Ligand Binding
For binding assays, cells grown in 10-cm dishes were
transfected with 10 µg DNA per plate. Binding assays were performed
using approximately 50 µg of membrane protein per reaction, prepared
from transfected cells as described (3). The reactions were carried out
at 25 C for 1 h in a 300-µl reaction volume and were terminated
by centrifugation at 4 C for 10 min. The membrane pellets were washed
once with binding buffer, and the amount of bound radioligand was
measured using a 
-counter. The assays were performed with duplicate
samples, and the means were used for all further calculations. For
competition studies, the membrane proteins were incubated with
either
(3-[125I]iodotyrosyl10)GHRH(1–44)-amide or
(3-[125I] iodotyrosyl10)VIP present at a
concentration of 70 pM (Amersham), in the absence or
presence of increasing concentrations of the unlabeled hormone. The
nonspecific binding, determined as the percent of input counts bound in
the presence of 1 µM unlabeled hormone, was approximately
13% and 7% for GHRH and VIP, respectively, and was subtracted from
all raw data to give the specific bound counts. The percentage of the
maximum specific bound counts for each data point was calculated using
the specific counts bound by the wild-type receptor in the absence of
competitor as 100%. Saturation-binding assays are representative of at
least two independent experiments. The concentration of the radioligand
ranging from 1.37 pM to 700 pM and the
corresponding unlabeled hormone was present at 1 µM. The
binding-competition data were fit to a one-site competition equation to
determine ED50, and Scatchard analysis of
saturation-binding data was used to determine KD and
BMAX values using the program GraphPad Prism (GraphPad
Software Inc., San Diego, CA).
Measurement of Intracellular cAMP Levels
For the measurement of cAMP responses, cells were transfected in
12-well plates using 2.5 µg plasmid DNA per well. The cells were
treated with hormones as described (3) for 20 min at 37 C, lysed in 150
µl of cold 0.1 M HCl, and harvested, and the lysates were
neutralized with an equal volume of 150 mM Tris-HCl (pH 8)
containing 4 mM EDTA. The protein was removed by
centrifugation for 10 min at 4 C, and the supernatants were used to
assay cAMP by a competitive protein-binding assay (67) using 4.5
nM [8-3H]cAMP (Amersham), as a tracer. The
protein-bound [8-3H]cAMP in the supernatant was measured
by liquid scintillation counting using CytoScint (ICN), and a linear
standard curve was performed in each assay. The assays were performed
with duplicate samples, and the means were used for all further
calculations. The percentage of the maximum cAMP accumulated for each
data point was calculated using the cAMP accumulated in cells
expressing the wild-type receptor, and treated with 1 µM
hormone, as 100%. The dose-response curves were fit to a sigmoidal
dose-response equation, and ED50 values were determined
using the program GraphPad Prism (GraphPad Software Inc.).
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by NIH Grant DK-48071 (to K.E.M.).
Received for publication October 13, 1997. Revision received January 15, 1998. Accepted for publication January 15, 1998.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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) represents the putative
signal-peptide cleavage site, the (
) represents the consensus site
for N-linked glycosylation, the (
) represents
aspartate residue 60, which is mutated in the little
mouse, and the (
) represents the cysteines that are conserved within
family B-III receptors. The location of the AflII and
KpnI sites used to make truncations and fusions are
indicated by arrows. The location and sequences of the
FLAG and HA epitope-tags are also shown.
