(Received for publication, March 29, 1995; and in revised form, June 1, 1995)
From the
In order to investigate the molecular basis for differences in
the characteristics of bombesin (Bn) and m3 muscarinic cholinergic (m3
ACh) receptors, chimeric Bn receptors possessing cytoplasmic domains
from the m3 ACh receptor were produced. The receptors were expressed in
CHO-K1 cells and binding, structural, and signal transduction
characteristics were analyzed. Cell lines bearing chimeric Bn receptors
possessing m3 ACh receptor domains in place of either the second
cytoplasmic loop (BM2L), the third cytoplasmic loop (BM3L), or both
loops (BM23L) each bound The serpentine G protein-linked receptors are so named due to
their structural characteristic of spanning the plasma membrane 7 times
and for their functional characteristic of interacting with
heterotrimeric G proteins. The best known interaction between
serpentine receptors and G proteins involves activation of G protein
Receptor internalization may also be
influenced by receptor G protein interactions. Internalization
characteristics of adrenergic and cholinergic receptors have been most
extensively characterized. After exposure to agonists, these receptors
rapidly sequester into a space that is inaccessible to hydrophilic
ligands and which likely represents receptor internalization.
Mutational studies with several G protein-coupled receptors have
produced mutants that are defective in their abilities to
sequester(3, 4, 5, 6, 7, 8) .
However, some, but not all, of these mutations interfere with G protein
activation. Therefore, the role of heterotrimeric G proteins in the
process of sequestering these receptors is unclear. Also, for bombesin
(Bn) Bn (9) and m3 ACh (10) receptors have been cloned, and
sequence analysis shows that they belong to the serpentine G
protein-linked family of receptors. These two receptors are both
expressed on pancreatic acinar cells where they each couple to
increases in phosphoinositide hydrolysis and intracellular
Ca The third
cytoplasmic loop has been shown to dictate the specificity of
receptor-G protein interactions in muscarinic and adrenergic receptors.
Studies with chimeric muscarinic receptors indicate that the proximal
16-21 amino acids of the third cytoplasmic loop are sufficient to
determine the G protein coupling profile of individual muscarinic
receptor subtypes(12, 13) . Similarly, the
juxtamembrane amino acids at the amino and carboxyl ends of the third
cytoplasmic loop of adrenergic receptors were found to be most
important for determination of G protein
specificity(14, 15) . Furthermore, substitution of
residues in the juxtamembrane portion of the third cytoplasmic domain
of several receptors has been shown to induce an constitutively active
confirmation(16) . However, other studies have indicated that
the second cytoplasmic loop is also important in receptor G protein
interactions. O'Dowd et al.(15) found that the
second cytoplasmic loop was essential for normal
Therefore, in the current study
we created chimeric bombesin receptors with the second or third or both
cytoplasmic loops substituted from the m3 ACh receptor. Cells
expressing the receptors were then analyzed for differences in agonist
affinity binding states, ligand internalization, and signal
transduction. We found that the mutant receptors bound agonist with a
single affinity state that was similar to the wild-type Bn receptor.
However, Bn receptor mutants with the third cytoplasmic loop
substituted from the m3 ACh receptor were severely diminished in their
abilities to internalize ligand and to couple with second messenger
formation.
For analysis of the effects of GTP
For
measurement of arachidonic acid release, cells grown to near confluency
in 6-well plates were incubated with 1 µCi of
[ Analysis of intracellular
[Ca
Figure 1:
Schematic
diagram of the structure of bombesin, m3 muscarinic cholinergic and
chimeric receptors. Wild-type Bn receptor (opencircles); m3 ACh receptor (filledcircles); and the BM2L, BM3L, and BM23L chimeric
constructs are shown. For BM2L, the second cytoplasmic loop of the
human m3 ACh receptor from residue 165 to residue 184 (20 amino acids)
was substituted into the region of the mouse Bn receptor between
residues 138 and 153 (16 amino acids). For BM3L, the third cytoplasmic
loop of the human m3 ACh receptor, from residue 253 to residue 492 (240
amino acids) was substituted into the region of the mouse Bn receptor
between residues 236 and 265 (30 amino acids). For BM23L, both the
second and third cytoplasmic loops were transferred between the
receptors.
To
obtain CHO-K1 cell lines with stable expression of Bn or chimeric
bombesin/m3 muscarinic receptors cells transfected with the receptor,
cDNAs cloned into the pTEJ-8 expression vector were selected with G418.
Stable expression of receptors was confirmed by
[
Figure 2:
Molecular size of wild-type Bn and
chimeric BM3L receptors revealed by cross-linking. CHO cells bearing
wild-type Bn or chimeric BM3L receptors were incubated with
[
Figure 3:
A, time-course of specific
binding and internalization of
[
To access the ability of the
receptors to internalize ligand, we utilized an acid stripping
procedure that has been previously shown to be effective at removing a
variety of cell surface-bound ligands(23) . In Bn-bearing CHO
cells, 80 ± 3%, n = 3, of
[ In order to
assess whether transfer of the cytoplasmic loops from the muscarinic
receptor to the Bn receptor would induce the formation of multiple
binding affinity states, it was necessary to examine binding of agonist
at 37 °C as multiple affinity states are not observed at 4 °C.
When binding of the agonist
[
Figure 4:
Binding characteristics of wild-type and
mutant Bn receptors. Competitive inhibition of
[
In order to more accurately estimate
receptor binding parameters, competition experiments were performed
using the bombesin antagonist [D-Tyr
Figure 5:
Effects of bombesin on total
polyphosphoinositide hydrolysis in CHO cells bearing bombesin
receptors. Confluent cells were incubated with 1.5 µCi/ml myo-[2-
Next, because of
the greater sensitivity afforded by analysis of second messengers at
the single cell level, we examined the effect of bombesin on
intracellular Ca
Figure 6:
Effects of bombesin treatment to alter
cellular calcium in CHO cells bearing Bn or BM3L receptors. Changes in
cellular calcium were assessed by determining changes in cytosolic
calcium with fura-2. A, representative tracings showing the
response of a population of CHO cells bearing either Bn or BM3L
receptors. Bombesin (10 nM) was added at the indicated time.
Data shown are representative of 4-12 experiments. B, concentration dependence of the effects of bombesin on calcium
responses in Bn or BM3L cells. CHO cells expressing Bn or BM3L cells
were superfused with increasing concentrations of bombesin, and the
number of cells that responded by increasing intracellular
[Ca
The most well understood consequence of interaction between
receptors and G proteins is the activation of G protein Chimeric Bn receptors bearing the third cytoplasmic loop
from the m3 ACh receptor were able to interact with G proteins as
indicated by their ability to display high affinity ligand binding, the
capacity of GTP The bombesin receptor has previously
been shown to rapidly and dramatically internalize agonist(6) .
We found that chimeric bombesin receptors whose third intracellular
loop had been substituted with that of the m3 ACh receptor had a
greatly reduced ability to couple to cellular effectors and to
internalize agonist. In contrast, the BM2L chimeric receptor, similar
to the wild-type Bn receptor, showed normal G protein coupling and
agonist internalization. Therefore, in this study internalization of
agonist was well correlated with the ability of the receptor to
activate G proteins. The mechanisms involved in receptor
internalization are not completely understood. Because these receptors
are internalized in an agonist-stimulated fashion, it is clear that
agonist-induced conformational changes are involved in activating the
endocytotic machinery. Thus, antagonists have been reported to be
sequestered to only a minor extent, and the ability of partial agonists
to sequester receptors is correlated with their ability to activate
receptors(26) . However, we noted that the bombesin antagonist
was internalized into an acid-resistant compartment to a small but
significant extent in cells bearing any of the Bn receptor chimeras.
Interestingly this was approximately the same level of internalization
observed with the agonist in the Bn receptor mutants possessing the
third cytoplasmic loop transferred from the m3 ACh receptor. These data
suggest that there is a small level of agonist-independent
internalization that occurs equally with each of the mutant receptors.
In contrast, the large amount of agonist-induced internalization was
only noted in receptors that were able to readily activate G proteins.
The mechanisms whereby agonist-induced conformational changes activate
endocytosis are unknown. Recently, on the basis of receptor mutants
impaired in their abilities to couple to phospholipase C and/or G
proteins, it was suggested that bombesin receptor activation of G
proteins, but not phospholipase C, is required for receptor
internalization(27) . A role for agonist-triggered changes
in second messengers in receptor internalization is unlikely, as
indicated by a variety of observations. Receptors that are uncoupled
from the generation of second messengers (28) or that show
greatly reduced ability to couple to second messengers (29, 30) that are sequestered normally have been
described. Conversely, several mutants fully able to stimulate
increases in second messengers have been found to be impaired in their
abilities to
sequester(6, 28, 31, 32) . It has
been more difficult to distinguish between conformational changes
directly activating endocytosis versus acting via
heterotrimeric G proteins. That several receptor mutants that have lost
the ability to activate G proteins do not sequester (33, 34) could be due to their inability to undergo
agonist-induced conformational changes. Similarly, the close
correlation between mutations that uncouple muscarinic receptors and
inhibit sequestration (3) or reported here for bombesin
receptors may indicate that heterotrimeric G protein activation is
required for sequestration or may indicate that the receptor domains
involved in both processes are similar or overlapping. However, normal
sequestration has been reported under a variety of circumstances where
interactions between receptors and G proteins are restricted,
including: Another approach to the question
of the role of G protein activation in sequestration is to determine
whether the two processess share the same receptor domains. A variety
of domains have previously been suggested as important in receptor
internalization. In particular, portions of the second cytoplasmic
loop(3) , third cytoplasmic loop(3, 4) , and
the carboxyl terminus (5, 6, 7, 8, 37) have been
found to influence internalization. It should be noted that none of
these sites has been disrupted in the chimeric receptors described in
the current study. Of particular interest are studies on the
receptor's carboxyl termini. Alterations of this domain have
previously been shown to not affect (33) , to
increase(38, 39) , or to decrease (6, 37) receptor sequestration. These observations
suggest that the overall confirmation of the carboxyl terminus plays an
important permissive role in receptor sequestration. However, an
internalization competent carboxyl terminus will not allow
sequestration in the absence of agonist-induced conformational changes,
as is indicated by the discussion above and the observations made in
the current study. A number of studies have shown that the
specificity of the G protein interaction and the ability to activate
the G proteins are dependent on residues in the third cytoplasmic loop.
Therefore, it was somewhat surprising in the current study that
substitution of the third cytoplasmic loop from the m3 ACh receptor
into the Bn receptor led to a receptor that was poorly coupled to G
protein activation. One possible explanation might be that the chimeric
construct lacked some juxtamembrane portion of the receptor vital to G
protein coupling. Recently the importance of a tyrosine residue
(Tyr-254) in the m3 receptor has been emphasized(40) . These
investigators have shown that the muscarinic receptor-mediated
stimulation of phosphoinositide metabolism is critically dependent on
the presence and proper positioning of an aromatic residue at the
beginning of the third cytoplasmic loop. Bn receptors also have a
tyrosine residue in the proximal portion of the third cytoplasmic loop
(Tyr-243), although it is not known whether this residue plays an
important role in this receptor. In the Bn receptor, this tyrosine is
located approximately 7 residues away from the presumed membrane
boundary, whereas, in the m3 receptor, the tyrosine is located
approximately 2 amino acids from the membrane. Although the exact
membrane boundaries are unknown, it is evident that the positioning of
the tyrosine residue in the Bn receptor is different than that in the
m3 receptor. This may account for a reduced ability of the m3 ACh third
loop to couple Bn receptor occupation with effector activation. Further
investigation will be necessary to determine the specific residues
involved in Bn receptor activation of G proteins. However, it is clear
that a simple homologous substitution from one G The chimeric bombesin receptors bearing the m3 ACh third
cytoplasmic loop did not generate a measurable increase in phospholipid
hydrolysis. However, cells bearing the BM3L receptor responded to Bn
with a small increase in intracellular Ca Another result of receptor G protein interaction is
the formation of a high-affinity binding state. Serpentine G
protein-coupled receptors often exist in two or more affinity states.
These affinity states are only apparent in agonist binding studies. For
many receptors, two binding affinity states are apparent when agonist
binding is conducted to whole cells at 37 °C and is manifest by
competition dose-response curves spanning 3 or more orders of magnitude
of agonist and by Scatchard plots showing two distinct affinity states.
These are the characteristics of the m3 ACh receptor. Other receptors,
including the Bn receptor, display only a single binding affinity. The
reason for the differences in numbers of agonist binding affinities
between receptors is not clear. However, the results from a variety of
approaches support the concept that high affinity binding requires an
interaction between receptors and G proteins that are unoccupied by
guanine nucleotides. Thus, high affinity binding for either single or
multiple affinity G protein coupled receptors is not observed in the
presence of nonhydrolyzable guanine nucleotides(42) , nor when
receptors are expressed in cells lacking the appropriate G
proteins(43) . However, the ability of serpentine receptors to
display interactions with G proteins sufficient for high affinity
binding but insufficient for efficient coupling to cellular effectors
has been reported previously (2) . Our initial hypothesis was
that transfer of the third cytoplasmic loop from the dual binding
affinity m3 ACh receptor into the single binding affinity Bn receptor
would convert the Bn receptor into a dual binding affinity receptor.
This clearly was not the case. Nor did substitution of the second, or
both the second and third cytoplasmic loops, convert the Bn receptor
into a dual binding affinity receptor. Thus, structural domains other
than the second and third cytoplasmic loops may contribute to the
interactions of the m3 ACh receptor responsible for its characteristic
dual binding affinities. Alternatively, the inability of agonist
binding to the chimeric bombesin receptor to cause a conformational
change required for G protein activation may have also prevented the
interaction required for the formation of a new affinity state. In
summary, the behavior of three chimeric receptors, one with a
substitution of the second loop, one with the third loop, and one with
both the second and third loops indicates that these domains are not
able to transfer characteristics of G protein interactions between
these receptors. Chimeric Bn receptors possessing the third cytoplasmic
loop transfered from the m3 ACh receptor were functionally uncoupled
and did not internalize. However, high affinity bombesin binding was
maintained, and the chimeric receptors interacted with G-proteins. The
correlation between the diminished ability of the mutant receptors to
couple to G protein activation and internalization of ligand suggests
that similar conformational changes are required for both processes.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
I-bombesin with a single
affinity that was approximately the same as that of the Bn receptor
(5-10 nM). However, Bn receptors possessing the m3 ACh
third cytoplasmic loop were severely affected in other respects.
Internalization of ligand in Bn and BM2L cells was rapid and extensive
(>80% of bound
I-bombesin was acid-resistant). In
contrast, internalization was dramatically reduced in BM3L and BM23L
cells (
20% of bound
I-bombesin was acid-resistant).
In Bn or BM2L cells 10 nM bombesin stimulated
10-fold
increases in phosphatidylinositol hydrolysis. Activation of Bn
receptors also induced an increase in arachidonic acid release (478
± 32% of control, n = 3) and large increases in
intracellular Ca
. In contrast, in BM3L or BM23L
cells, bombesin had no significant effect on phosphatidylinositol
hydrolysis. Furthermore, BM3L receptor activation did not increase
arachidonic acid release. However, BM3L and BM23L cells showed a small
increase in intracellular Ca
at high concentrations
of bombesin. These data indicate that the third cytoplasmic loop alone,
or together with the second cytoplasmic loop, was not sufficient to
transfer the characteristics of G protein interaction between m3 ACh
and bombesin receptors. Furthermore, for the Bn receptor, ligand
internalization does, whereas formation of the high affinity binding
state does not, appear to require activation of G proteins.
subunits and the subsequent stimulation of cellular effectors
such as phospholipases, adenylyl cyclase, or ion channels leading to
biological responses (for review, see (1) ). Less well known
and less well understood interactions between these receptors and G
proteins influence other aspects of receptor function including
receptor binding affinity and internalization. Formation of high
affinity binding states requires receptor interaction with G
proteins(1) . For
-adrenergic receptors, formation of the
high affinity state has been shown to better correlate with the ability
of receptors to interact with, and then to activate, G
proteins(2) . However, the importance of G protein activation
in the formation of high affinity binding states has not been well
studied for other receptors.
(
)and other peptide binding receptors,
internalization is assessed by determining changes in the distribution
of ligand using a technique of low pH removal of cell
surface-associated ligand. Whereas, in the case of muscarinic and
adrenergic receptors, sequestration is assessed by analyzing changes in
the distribution of the receptor determined by comparing the binding of
hydrophilic and hydrophobic ligands. Thus, it is unclear whether or not
the processes responsible for ligand internalization observed in
peptide binding receptors are equivalent to the processes responsible
for the sequestration of muscarinic and adrenergic receptors.
Consequently, little is currently known concerning the role of receptor
G protein interactions in the regulation of the internalization of
peptide binding receptors including the Bn receptor.
and activate the secretion of digestive
enzymes(11) . Both receptors are generally believed to interact
with the G
subfamily of heterotrimeric G proteins. However,
in pancreatic acinar cells, Bn and m3 ACh receptors show dramatic
differences in receptor properties, some of which may be influenced by
G protein interactions. These properties include numbers of agonist
affinity binding states (one versus two), secretory
dose-response curves (monophasic versus biphasic), and ligand
internalization (extensive versus minimal). In order to
investigate the mechanisms responsible for the differences in these
receptors, we wished to determine whether transfer of structural
domains important for G protein-receptor interaction could transfer
characteristics from one receptor to the other.
-adrenergic receptor-G
interactions.
Interactions between the second and third cytoplasmic loops were also
found to be involved in fully determining G protein selectivity in
chimeras of muscarinic and adrenergic receptors(14) . Also,
mutations in the second and third loops of rhodopsin suggested that
activation of bound transducin requires interaction with a site in each
of these two loops(17) . Collectively these data indicate that
both the second and third cytoplasmic loops are important determinants
of G protein-receptor interaction.
Materials
Radiochemicals
[I-Tyr
]Bombesin (81.4
TBq/mmol) and [
H]arachidonate (100 Ci/mmol) were
obtained from DuPont NEN. myo-[
H]Inositol (19.1 Ci/mmol) was
obtained from Amersham Corp.
[I
-D-Tyr
]Bombesin(6, 7, 8, 9, 10, 11, 12, 13) methyl
ester (2000 Ci/mmol) was prepared using iodogen and purified by high
performance liquid chromatography using a modification of the method
described by Mantey et al.(18) . Briefly, 1.0 µg
of iodogen was added to 8.0 µg of
[D-Tyr
]bombesin(6, 7, 8, 9, 10, 11, 12, 13) methyl ester with 2 mCi of Na
I in 20 µl
of 0.5 M KPO
buffer (pH 7.4). After incubation at
22 °C for 6 min, 300 µl of H
O was added. The
incubation mixture was then loaded on a Waters Associates, model 204
with a µBondapak column (0.46
25 cm). Free
I
was eluted with 0.1% trifluoroacetic acid. The radiolabeled peptide was
separated from unlabeled peptide by elution with a linear gradient of
acetonitrile in 0.1% trifluoroacetic acid (v/v) from 10 to 65%
acetonitrile in 60 min with a flow rate of 1.0 ml/min. Under these
conditions a single predominant peak of radiolabeled antagonist was
observed.
Biochemicals
Analytical grade Dowex 1-X8 (AG1-X8, 100-200 mesh) and
Bio-Rad protein assay reagent were obtained from Bio-Rad. Restriction
endonucleases were purchased from Life Technologies, Inc. Taq polymerase was obtained from Promega Corp. Oligonucleotides were
synthesized by an Applied Biosystems 380B DNA synthesizer. Bombesin was
obtained from Bachem (Torrance, CA). Trichloroacetic acid was obtained
from JT Baker (Phillipsburg, NJ). Streptolysin O (SLO) was obtained
from Wellcome Diagnostics (Greenville, NC). Soybean trypsin inhibitor
type I-S, bovine serum albumin (bovine serum albumin) fraction V, and
all nonspecified reagents were obtained from Sigma.
[D-Tyr]Bombesin (6, 7, 8, 9, 10, 11, 12, 13) methyl
ester was a kind gift from Dr. D. H. Coy (National Institutes of
Health, Bethesda, MD).
Tissue Culture Supplies
Dulbecco's modified Eagle's medium, fetal bovine
serum, penicillin, streptomycin, G418, and amphotericin B were obtained
from Life Technologies, Inc. (Grand Island, NY). Tissue culture
plasticware (24- and 6-well plates and 10-cm Petri dishes) were
obtained from Costar (Cambridge, MA). CHO-K1 cells were obtained from
the American Type Culture Collection (Rockville, MD).cDNA Clones for Bombesin and m3 Muscarinic Receptors
The plasmid containing the 1.4-kilobase pair EcoRI
DNA fragment of mouse bombesin receptor cDNA containing the entire
384-amino-acid open reading frame of the receptor was kindly provided
by Dr. J. Battey(9) . The 1.4-kilobase pair EcoRI DNA
fragment was subcloned into pBluescript SK- plasmid to obtain
pBRR plasmid. The full-length of human m3 muscarinic receptor cDNA was
a gift of Dr. E. Peralta (10) and was cloned as a 2.1-kilobase
pair EcoRI to BamHI DNA fragment into pGEM-3.Methods
Construction of Chimeric Bombesin/m3 Muscarinic
Receptors
BM3L
PCR methodology was used to construct a
chimeric receptor cDNA in which the third cytoplasmic loop of the mouse
Bn receptor was replaced by the analogous portion of the human m3
muscarinic receptor. Steps in the construction of this mutated receptor
were as follows: (a) Primers with sequences
5`-CTGTCTACTACTACTTCATTAGGATCTATAAGGAAACTGA-3` (BR-Hm3-N) and
5`-CCCACAAACACCAGTACTGTCTGGGCCGCTTTCTTCTCCT-3` (BR-Hm3-C) were used in
a 30-cycle PCR reaction containing the m3 muscarinic receptor cDNA.
This PCR reaction amplified a DNA fragment coding for the third
cytoplasmic loop of the m3 ACh receptor fused at each end to sequences
coding for the fifth and sixth transmembrane segments of the Bn
receptor. (b) The chimeric DNA fragment from (a) was
used in separate PCR reactions containing the Bn receptor with primers
in each of the flanking regions of the plasmid. These reactions
amplified a DNA fragment, which fused the third cytoplasmic loop of the
m3 ACh receptor to the fifth transmembrane segment of the Bn receptor
or the third cytoplasmic loop of m3 ACh receptor to the sixth
transmembrane segment of Bn receptor. (c) The complete
chimeric BM3L clone was formed using 20 ng each of these two PCR
products to prime a 10-cycle PCR reaction followed by addition of the
flanking primers and 30 additional PCR cycles. The final PCR reaction
product was cut with EcoRI, and then the amplified chimeric
bombesin/m3 muscarinic receptor DNA fragment was cloned into the EcoRI site of pBluescript, and the DNA sequences were verified
by sequencing.BM2L
A similar PCR protocol was used to obtain the
BM2L receptor chimera in which the entire second cytoplasmic loop of
the human m3 ACh receptor was substituted for the corresponding portion
of the Bn receptor. The two primers with sequences
5`-CACACTTACGGCACTGTCAGCTGACAGATACTTTTCATCAC-3` and
5`-AAAGCAGCTTTGAGACAGATGGCTCTCTTTGTTGTTCGTT-3` were used to amplify a
DNA fragment coding for the second cytoplasmic loop of the m3 ACh
receptor fused at each end to a sequence coding for the third and
fourth transmembrane segments of the Bn receptor.BM23L
To obtain the chimeric Bn receptor with both
second and third cytoplasmic loops replaced by the analogous portions
of the m3 ACh receptor, a 0.8-kilobase pair AccI DNA fragment
containing the N-terminal portion to the end of the fifth transmembrane
segment of BM2L was inserted into the AccI site of pBluescript
plasmid containing the C-terminal portion of BM3L chimeric receptor to
obtain the chimeric BM23L receptor. All receptors were subcloned into
pTEJ-8 (19) for expression in cell lines.Transfection of Cell Lines
CHO-K1 cells were routinely cultured in DMEM media
supplemented with 10% fetal bovine serum in a humidified atmosphere of
5% CO. For transfection, CHO-K1 cells were grown to
30-40% confluence in 60-mm dishes and transfected with 3 µg
of PvuI-linearized plasmid DNA using Lipofectin reagent (Life
Technologies, Inc.) for 6-8 h in serum-free medium. Cells were
then returned to 10% fetal bovine serum, cultured 36 h, and then
removed from the dishes by brief trypsin EDTA treatment and replated at
reduced density in 150-mm plates in the presence of 1 mg/ml (active)
G418. G418-resistant colonies were selected and screened for bombesin
and mutant receptors by binding of
[
I-Tyr
]bombesin. Two or more clones
were tested in all assays to control for clonal variation.
Binding Assays
Binding was conducted to cells plated in 24-well dishes at 1
10
cells/ml the day before the binding assay. For
agonist cell binding assays
[
I-Tyr
]bombesin (10 pM)
was added to HR buffer (5 mM NaCl, 4.7 mM KCl, 1
mM Na
PO
, 1.28 mM CaCl
, 10 mM Hepes (pH 7.4) with 0.5% bovine
serum albumin and 0.1 mg/ml soybean trypsin inhibitor). For antagonist
binding assays
[
I-D-Tyr
]bombesin(6, 7, 8, 9, 10, 11, 12, 13) methyl
ester (10 pM) was utilized. Cells were incubated to
equilibrium (2 h at 37 °C) and then washed twice with ice-cold
phosphate-buffered saline (PBS). The cells were then scraped into 1 ml
of 0.1 N NaOH and were counted in a
-counter. Nonspecific
binding was determined in the presence of 100 nM bombesin.
Protein contents were determined on samples after counting. Binding
affinity and capacity were calculated using the Ligand analysis program (20) or with Prism (Graphpad Software Inc., San Diego, CA). For
determination of ligand internalization, we utilized an acid washing
procedure. For acid washing experiments, after the PBS wash, the cells
were given an additional wash with 1 ml of acid wash buffer (0.2 M acetic acid, 0.5 M NaCl, pH 2) at 4 °C for 5 min,
which was collected and counted separately from the cells. This wash
was found to remove >90% of either agonist or antagonist tracer from
the surface of cells after 4 h of binding at 4 °C (data not shown).
S on binding properties of
bombesin receptors, cells were permeabilized with SLO (0.4 IU/ml) in
permeabilization buffer (120 mM KCl, 5 mM EGTA, 1
mM MgCl, 1 mM MgATP, 1.078 mM
CaCl
, glucose 5.6 mM, 30 mM Hepes (pH 7)
with 0.5% bovine serum albumin and 0.1 mg/ml soybean trypsin inhibitor)
for 5 min at 37 °C and then washed with the same buffer lacking
SLO. Binding with [
I-Tyr
]bombesin
was conducted in permeabilization buffer at 37 °C for the indicated
times.
Cross-linking Experiments
The cells were seeded at a concentration of 10 cells/well in a 6-well dish and grown to confluence. The cells
were then washed on ice 3 times with ice-cold binding buffer without
bovine serum albumin.
[
I-Tyr
]Bombesin (0.5 nM)
was added to the cells in 0.8 ml of binding buffer and incubated for 4
h on ice. The cells were then washed 3 times with ice-cold binding
buffer and once with PBS. The cross-linking reagent, EGS, was dissolved
in dimethyl sulfoxide and then diluted to 1 mM in PBS. One ml
of PBS containing 1 mM ethylene glycol-bis(succinic acid N-hydroxysuccinimide) was added to each well and incubated at
room temperature for 20 min. The cross-linking reaction was terminated
by the addition of 10 µl of 2 M Tris-HCl (pH 8.0), and the
cells were washed once with PBS. Sample buffer (2% sodium dodecyl
sulfate, 10% glycerol, 100 mM dithiothreital, 60 mM
Tris (pH 6.8), 0.001% bromphenol blue) (80 µl) preheated to 95
°C was added to the cells, and the cells were scraped into an
Eppendorff tube. The samples were boiled and separated on a 8%
SDS-PAGE. The gel was dried and exposed on Kodak XAR-5 film for 2 weeks
at -70 °C.
Second Messenger Generation Assays
For measurement of total inositol phosphate release, cells
were cultured in 6-well multiwell dishes at 1 10
cells/ml for 24 h in the presence of 1.5 µCi of myo-[
H]inositol. The cells were then
washed twice with HR buffer containing 10 mM lithium.
Incubation with the indicated concentrations of bombesin was conducted
in the same buffer for the times indicated. Incubation was terminated
by addition of an equal volume of 20% ice-cold trichloroacetic acid.
After centrifugation at 2000
g for 20 min, 0.9 ml of
each supernatant was washed twice with water-saturated diethyl ether,
neutralized with 100 µl of 1 M KHCO
, and
diluted with 2.5 ml of water. Analysis of total
[
H]inositol phosphates was carried out by the
method described by Berridge et al.(21) .
H]arachidonic acid for 24 h. The cells were then
washed with PBS and incubated in HR buffer plus or minus bombesin (10
nM) for 30 min. The incubation medium was then removed and
counted in a scintillation counter.
]
was conducted on cells
grown to 30-50% confluence on glass coverslips. Cells were
incubated with 5 µM fura-2 at 37 °C for 30 min in an
incubator and then washed and resuspended in HR buffer. Coverslips
containing cells were transferred to a closed chamber, mounted on the
stage of a Zeiss Axiovert inverted microscope, and continuously
superfused at 1 ml/min with HR buffer at 37 °C. Measurement of
emitted fluorescence and calibration of these signals to yield a
measurement of intracellular Ca
was performed using
an Attofluor digital imaging system (Rockville, MD) exactly as
described previously(22) . Cells were scored as to whether or
not they showed a response to the addition of 10 nM bombesin
to the superfusate.
Data Analysis
All values were represented as the mean ± S.E.
Student's two-tailed t test, unpaired, or when
appropriate, paired, was used for statistical analysis of the data.
Construction and Expression of the Bombesin/m3 ACh
Receptor Chimeric Receptors
In order to investigate the
structural basis for differences between Bn and m3 ACh receptors, PCR
technology was utilized to produce chimeric bombesin receptors bearing
the second, third, or both cytoplasmic loops from the m3 ACh receptor.
A schematic representation of the putative seven membrane-spanning
domain topography of the bombesin receptor, the m3 muscarinic receptor
(m3 ACh) and the chimeric bombesin/m3 muscarinic receptors with the
second (BM2L), third (BM3L), or both (BM23L) cytoplasmic loops
transfered from the m3 ACh into the Bn receptor are shown in Fig. 1. Nucleotide sequences were confirmed by sequencing.
I-Tyr
]bombesin binding assays.
Northern blot hybridization of mRNA isolated from transfected cells and
hybridized with a radiolabeled bombesin receptor cDNA probe or a probes
for the cytoplasmic loops of the m3 muscarinic receptor confirmed the
transcription of the receptors' genes in the transfected CHO
cells (data not show). To confirm the expression of the BM3L mutant
phenotype, the Bn and BM3L cell lines were labeled with
[
I-Tyr
]bombesin and cross-linked
with EGS. Cellular proteins were subjected to SDS-PAGE and analyzed
autoradiographically. The wild-type Bn receptor ran as a broad band
with an apparent molecular mass of
95 kDa (Fig. 2). The
BM3L receptor chimera had an apparent molecular mass of
120 kDa
supporting the transfer of the larger third cytoplasmic loop from the
m3 ACh receptor into the Bn receptor.
I-Tyr
]bombesin and the
cross-linking reagent EGS in the presence and absence of excess
unlabeled bombesin. Cells were solubilized, samples were run on an SDS
page gel, and the gel was exposed to autoradiography. The cross-linked
wild-type receptor ran as a broad band, likely indicating extensive
glycosylation. Its estimated median molecular mass was
95 kDa. The
cross-linked BM3L receptor also ran as a broad band; however, in this
case the estimated median molecular mass was
120 kDa. This likely
reflects the increased mass of the chimeric
receptor.
Receptor Binding and Ligand Internalization
Characteristics of Wild-type and Mutant Bn Receptors
Next we
examined the binding of
[I-Tyr
]bombesin to the wild-type
and mutant receptor-bearing CHO cells. When cultured cells were
incubated with 10 pM
[
I-Tyr
]bombesin in the presence and
absence of excess nonradioactive bombesin, saturable binding was
observed. A steady state of binding was reached after 90-120 min
at 37 °C, at which time binding of the radiolabeled tracer averaged
approximately 30%/well in all cell lines examined (Fig. 3A). Nonspecific binding averaged 9 ± 2%
of specific binding n = 6 and did not differ
significantly between the cell lines.
I-Tyr
]bombesin in CHO cells
bearing wild-type Bn and chimeric BM3L receptors. Cells were incubated
with 10 pM [
I-Tyr
]bombesin
at 37 °C for the indicated times, and then the cells were washed at
4 °C. Surface-bound (acid-removable, triangles) and
intracellular (acid-resistant, circles) radioactivity were
determined as described under ``Experimental Procedures.''
Nonspecific binding, defined as binding in the presence of 100
nM bombesin, was determine similarly, and values have been
subtracted from each point. Each point represents the mean ±
S.E. of three separate experiments. B, extent of ligand
internalization in CHO cells bearing Bn, BM2L, BM3L, or BM23L
receptors. Cells were incubated with 10 pM
[
I-Tyr
]bombesin at 37 °C for 2
h, and then the cells were washed at 4 °C. Intracellular
(acid-resistant) radioactivity was determined as described under
``Experimental Procedures.'' Nonspecific binding, defined as
binding in the presence of 100 nM bombesin, was determined
similarly, and values have been subtracted from each point. Data
presented are the acid-resistant binding as a percentage of the total
specific binding and represent the mean ± S.E. of three separate
experiments.
I-Tyr
]bombesin specifically
associated with the cells was acid-resistant (presumably internalized)
at equilibrium (Fig. 3B). Similarly, in BM2L-bearing
cells 75 ± 1%, n = 3, of
[
I-Tyr
]bombesin specifically
associated with the cells after 120 min was acid-resistant. In
contrast, in BM3L-bearing CHO cells, only 20 ± 2%, n = 3, and in BM23L only 16 ± 1%, n =
4, of the [
I-Tyr
]bombesin
specifically associated with the cells was acid-resistant (Fig. 3B). The relative lack of internalization of the
BM3L mutant was also observed when the receptors were expressed in
COS-1 and NIH3T3 cells (data not shown). Thus, chimeric Bn receptors
containing the third cytoplasmic loop from the m3 ACh receptor were
severely limited in their ability to internalize ligand.
I-Tyr
]bombesin was carried out at
37 °C in the presence of increasing amounts of unlabeled bombesin,
the percent of [
I-Tyr
]bombesin
bound decreased, with the decrease being half-maximal at 5-10
nM and maximal at 30-100 nM bombesin for Bn,
BM2L, BM3L, and BM23L receptor-bearing cells (Fig. 4). Computer
analysis of the binding data from all cells fit the behavior of a
single class of binding sites for each cell line with a K
of
5-10 nM (Table 1). Rapid ligand internalization complicated the
analysis of agonist binding competition experiments conducted at 37
°C and therefore yielded only estimates of binding parameters.
However, no evidence for the existence of dual binding affinities was
observed in mutant receptors.
I-Tyr
]bombesin binding by bombesin
at 37 °C is shown. CHO cells bearing wild-type Bn, BM2L, BM3L, and
BM23L receptors were incubated for 2 h with 10 pM
[
I-Tyr
]bombesin and indicated
concentration of nonradioactive bombesin. Each point is mean ±
S.E. of three to eight experiments. Insert, Scatchard plot
from experiments with Bn, BM2L, BM3L, and BM23L receptor-bearing cells.
Primary data from the competition curves were analyzed by nonlinear
curve fitting, and the line shown in the Scatchard plot was drawn from
the best-fitting parameters for the K and B
of a single class of sites.
, Bn;
, BM2L;
,
BM3L;
, BM23L.
]bombesin
6-13 methyl ester(18) . Control experiments showed that
the antagonist was internalized only to a small and equal extent in all
cells (20 ± 3% acid-resistant, n = 8).
Competition binding experiments using the antagonist showed that the
affinity and B
values did not differ
greatly among the Bn receptors (Table 1).
Signal Transduction in Cells Bearing Wild-type and Mutant
Bn Receptors
In order to investigate the coupling of the Bn
receptors to signal transduction pathways, we investigated the effects
of bombesin on phospholipid metabolism. It has previously been shown
that bombesin receptors are able to couple to the activation of a
phospholipase C, causing the rapid hydrolysis of polyphosphoinositides
(PPI) and leading to the release of inositol phosphates(24) .
We found that activation of the wild-type Bn receptor increased the
hydrolysis of PPI in a time-dependent manner, which was linear for at
least 30 min (data not shown). The ability of Bn receptors to induce
PPI hydrolysis was also dose-dependent with maximal effects observed at
10 nM bombesin (Fig. 5A). In contrast, BM3L
receptor-bearing cells showed no increase of PPI hydrolysis when
treated with concentrations of bombesin that should occupy all
receptors (100 nM). We found that cells bearing BM2L
receptors, similar to those bearing wild-type Bn receptors, showed a
greater than 10-fold increase of total inositol phosphates released
after 30 min of bombesin (10 nM) stimulation (Fig. 5B). In BM23L receptor-bearing cells, similar to
those bearing BM3L receptors, bombesin did not induce a measurable
increase in PPI hydrolysis. Activation of bombesin receptors has also
been shown to activate a phospholipase A2 leading to the release of
arachidonic acid(25) . Bombesin (10 nM) treatment of
Bn cells stimulated the release of 479 ± 32% (n = 3) of basally released
[H]arachidonate. In contrast, BM3L cells treated
with bombesin (10 nM) only released 103 ± 5% (n = 3) of basally released arachidonate.
H]inositol for 24 h after which
they were exposed to bombesin for 30 min. Isolation of
[
H]inositol phosphates was performed as detailed
under ``Experimental Procedures.'' A, concentration
dependence of bombesin stimulation of total inositol phosphate release
in CHO cells bearing Bn or BM3L receptors. Each data point is expressed
as the percentage of basal PPI hydrolysis and represents the mean
± S.E. of three separate experiments, with each value measured
in triplicate in each experiment. B, effects of bombesin
treatment on release of total inositol phosphates in CHO cells bearing
Bn, BM2L, BM3L, or BM23L receptors. Cells were exposed to 10 nM bombesin for 30 min. Each data point is expressed as the
percentage of basal PPI release and represents the mean ± S.E.
of three to five separate experiments, with each value measured in
triplicate in each experiment.
levels using fura-2 measurements in
an Attofluor cell imager (Fig. 6A). CHO cells bearing
the wild-type Bn receptor responded to high doses of bombesin (10
nM) with an increase in intracellular Ca
,
which averaged 360 ± 32 nM, n = 3
separate experiments (43 cells). The majority of the wild-type
receptor-bearing cells responded even at relatively low doses of
bombesin (0.1 nM) (78 ± 13%, n = 4
experiments, 77 cells), and virtually all cells responded to 1 nM bombesin (91 ± 7%, n = 4 experiments, 77
cells). In contrast, the BM3L-bearing CHO cells did not respond to low
doses of bombesin, but a significant fraction of these cells responded
to 10 nM bombesin (38 ± 5%, n = 12
experiments, 322 cells). The responding BM3L-bearing cells also showed
a lesser increase in
[Ca
]
, which averaged
142 ± 17 nM, n = 4 separate experiments
(96 cells). BM2L-bearing cells responded similarly to Bn-bearing cells
with 100% of the cells (3 runs, 108 cells) responding at 0.1 nM bombesin. BM23L receptor-bearing cells, like BM3L-bearing cells,
responded weakly with only 17 ± 3%, n = 3 runs
(118 cells) of the cells, indicating any rise in
[Ca
]
when activated by
10 nM bombesin. Control experiments showed no significant
response of untransfected CHO cells to bombesin (data not shown).
] was counted. Results are expressed as
the number of cells that responded as a percentage of total cells and
are means ± S.E. for three to five
experiments.
Effects of GTP
Chimeric Bn receptors expressing the m3 ACh third
cytoplasmic loop-bound ligand with high affinity but did not couple
normally to second messenger generation. Therefore, it was of interest
to further examine the interaction of these receptors with G proteins.
Thus, we tested the effects of the nonhydrolyzable GTP analog GTPS on Binding to Wild-type and BM3L
Receptors
S
on binding wild-type and BM3L receptors. Initially attempts were made
to conduct these experiments using membrane preparations. Membranes
from Bn receptor-bearing cells showed high levels of specific binding.
However, membranes prepared from BM3L receptor-bearing cells possessed
extremely low levels of specific binding that were insufficient for
further studies (data not shown). As an alternative approach, we
utilized SLO to permeabilize CHO cells and allow access of GTP
analogues to intracellular sites. Using this method, reasonable levels
of specific binding were observed, and GTP
S inhibited agonist
binding in both wild-type Bn (63 ± 3% reduction, n = 3, p < 0.05) and mutant BM3L (27 ± 6% n = 3, p < 0.05) receptor-bearing cells. To
confirm that this inhibition of binding was due to affects on receptor
affinity, competition binding experiments were then conducted with
permeabilized cells in the presence and absence of GTP
S. Addition
of GTP
S to permeabilized cells decreased the binding affinity of
both wild-type Bn and BM3L receptors with little effect on receptor
number (Table 2). Interestingly, these experiments also revealed
that SLO permeabilization itself significantly decreased the binding
affinity of mutant BM3L receptors while it had little effect on the
binding affinity of wild-type Bn receptors (Table 2). Taken together
these data suggest that BM3L receptors interact with G proteins but
that the interaction is highly labile.
subunits
leading to the release of GDP and the subsequent binding of GTP, which
causes the release of the
and
subunits from the
receptor and allows their interaction with effectors (for review, see (1) ). However, interactions between receptors and G proteins
are responsible for a number of other phenomena. The interactions
between G proteins and receptors occur within the cytoplasmic domains
of the receptors, and particular importance has been ascribed to the
second and primarily the third cytoplasmic loops. We found that
substitution of the second cytoplasmic loop from the m3 ACh had little
influence on the characteristics or functioning of the Bn receptor. In
contrast, substitution of the third cytoplasmic loop from the m3 ACh
receptor into the bombesin receptor had a number of consequences on
receptor function. The most prominent effects were a severe reduction
in ligand internalization and receptor-effector coupling. However,
receptor binding affinity was not greatly affected. These results have
several implications for our understanding of the functioning of these
receptors.
S to decrease binding affinity, and their ability
to couple, albeit weakly, to increases in
[Ca
]
. However, these
chimeric receptors were not fully able to activate G proteins, as was
clear from their inability to activate full second messenger responses,
particularly hydrolysis of polyphosphoinositides and release of
arachidonic acid. Thus, these chimeric receptors substantially
separated the effects of receptor G protein interaction from those of
receptor G protein activation. Interestingly, the interaction of the
chimeric receptors bearing the third cytoplasmic loop from the m3 ACh
receptor with G proteins was very labile, as indicated by the
observations that permeabilization alone caused a significant reduction
in binding affinity and preparation of isolated membranes nearly
abolished specific binding.
-adrenergic receptors that are functionally
uncoupled(35) ; yeast G protein-coupled pheromone receptors
expressed in yeast lacking functional G proteins(36) ; and
uncoupled neurotensin receptor mutants(37) . Taken together,
these data suggest that the heterotrimeric G protein itself is not
involved in receptor sequestration.
-linked
receptor to another does not necessarily lead to a well coupled
receptor.
. One
possible explanation is that the Ca
response observed
was not dependent upon generation of inositol phosphates, as has been
suggested in other systems(41) . Another possibility is that
the sensitivity of the biochemical assay for phosphatidylinositol
hydrolysis was not sufficient to detect the small level of increase in
inositol phosphate hydrolysis necessary for the Ca
response.
S, guanosine
5`-3-O-(thio)triphosphate; PPI, polyphosphoinositides; CHO,
Chinese hamster ovary.
We thank Drs. J. A. Williams and S. K. Fisher for
helpful discussions throughout this work.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.