The Bombesin Receptor Subtypes Have Distinct G Protein
Specificities*
Xiaoying
Jian
,
Eduardo
Sainz
,
William A.
Clark§,
Robert T.
Jensen¶,
James F.
Battey
, and
John K.
Northup§
From the
Laboratory of Molecular Biology, National
Institute on Deafness and Other Communication Disorders,
Rockville, Maryland 20850, ¶ Digestive Diseases Section, NIDDKD,
National Institutes of Health, Bethesda, Maryland 20892, and
§ Laboratory of Cellular Biology, National Institute on
Deafness and Other Communication Disorders,
Rockville, Maryland 20850
 |
ABSTRACT |
We used an in situ reconstitution
assay to examine the receptor coupling to purified G protein
subunits by the bombesin receptor family, including gastrin-releasing
peptide receptor (GRP-R), neuromedin B receptor (NMB-R), and bombesin
receptor subtype 3 (BRS-3). Cells expressing GRP-R or NMB-R catalyzed
the activation of squid retinal G
q and mouse
G
q but not bovine retinal G
t or bovine
brain G
i/o. The GRP-R- and NMB-R-catalyzed activations of G
q were dependent upon and enhanced by different

dimers in the same rank order as follows: bovine brain 
>
1
2
1
1. Despite these qualitative
similarities, GRP-R and NMB-R had distinct kinetic properties in
receptor-G protein coupling. GRP-R had higher affinities for bovine
brain 
,
1
1, and
1
2 and squid retinal G
q.
In addition, GRP-R showed higher catalytic activity on squid G
q. Like GRP-R and NMB-R, BRS-3 did not catalyze GTP
S
binding to G
i/o or G
t. However, BRS-3
showed little, if any, coupling with squid G
q but
clearly activated mouse G
q. GRP-R and NMB-R catalyzed
GTP
S binding to both squid and mouse G
q, with GRP-R activating squid G
q more effectively, and NMB-R also
showed slight preference for squid G
q. These studies
reveal that the structurally similar bombesin receptor subtypes, in
particular BRS-3, possess distinct coupling preferences among members
of the G
q family.
 |
INTRODUCTION |
Mammalian bombesin-like peptides, gastrin-releasing peptide
(GRP)1 and neuromedin B
(NMB), are widely distributed in the nervous system and the gut. They
regulate various physiological processes such as secretion, growth,
muscle contraction, and neuromodulation through high affinity receptors
(1, 2). Three pharmacologically and structurally distinct bombesin
receptor subtypes have been cloned and characterized in mammals as
follows: the GRP-preferring receptor (GRP-R), the neuromedin
B-preferring receptor (NMB-R), and bombesin receptor subtype 3 (BRS-3)
which has a structure related to GRP-R and NMB-R but for which no high
affinity, naturally occurring ligand has been identified as yet (2).
Comparison of the predicted amino acid sequences (2) of the bombesin
receptor subtypes shows all three to be structurally related members of the G protein-coupled receptor superfamily with pairwise sequence identity ranging from 48 to 54% (see Fig. 1). Upon agonist binding, G
protein-coupled receptors activate specific heterotrimeric G proteins,
which in turn regulate a variety of intracellular effectors such as
adenylyl cyclase, phospholipase C, ion channels, and
cGMP-phosphodiesterase (3).
Heterotrimeric G proteins are composed of three polypeptides as
follows: an
subunit and a 
dimer that acts as a functional monomer. Ligand-activated G protein-coupled receptors catalyze the
exchange of GTP for GDP bound to the G
subunit, resulting in
dissociation of the GTP-activated
subunit from both its cognate G
dimer and the receptor. The GTP-activated
subunit as well as dissociated G
dimer in turn regulate intracellular effectors. At least 20 different
subunits, 5
subunits, and 12
subunits have been identified to date. The G
subunits have been divided into
four groups based upon sequence homology and intracellular effector
regulation (4, 5). The G
q subfamily, which
includes G
q, G
11, G
14, and
G
15/16, stimulates phosphoinositide hydrolysis by
activating phospholipase C-
(6-10). In addition, G
subunits can also stimulate phospholipase C-
s in concert with
G
q (11, 12).
Given that the seven transmembrane domain receptor superfamily consists
of thousands of distinct receptors, and the family of heterotrimeric G
proteins involved in receptor coupling is also very diverse, a central
issue in receptor signaling is how these protein families contribute to
the diversity of receptor/G protein-mediated responses while conserving
the specificity of each response. One level of specificity is likely to
be determined by the thermodynamics of protein-protein interactions
between subunits of the heterotrimeric G protein and the receptor. An in situ reconstitution procedure has been used successfully
to study receptor-G protein interactions for baculovirus-infected Sf9 cell membranes expressing the 5-HT2c receptor
(13), and for mouse fibroblast cell membranes expressing stably
transfected GRP-R (14). This technique utilizes chaotrope-extracted
membrane fractions in which endogenous GTP-binding proteins as well as other extrinsic membrane proteins are removed or inactivated by urea,
while leaving uncoupled receptors fully functional when reconstituted
with agonist and purified G protein subunits.
Since mammalian bombesin receptors stimulate phosphoinositide
hydrolysis (15-17), it has been assumed that agonist stimulation of
bombesin receptors leads to activation of a G
q, which in
turn activates a phospholipase C-
isozyme. Antisense oligonucleotide injection of Xenopus oocytes (18) has identified
G
q as a mediator of the NMB-R response. However, in
Xenopus oocytes neither G
q nor
G
11 antisense injection had any effect on GRP-R signal
transduction, although the in situ reconstitution of GRP-R
with purified G protein subunits shows explicitly that GRP-R activates
a G
q but not G
i/o or G
t
(14). Such observations raise a possibility that the ambiguity in
the antisense oligonucleotide experiments could be due to a difference
in the relative affinity or activity of GRP-R for G
q as
compared with that of NMB-R for G
q. The assessment of
this possibility requires a quantitative comparison of GRP-R and NMB-R
coupling with purified G protein subunits in vitro.
In this report we compare the receptor-G protein interactions
within the structurally related bombesin receptor family using the
in situ reconstitution assay. We have quantitatively
examined the G protein activation by these related receptor structures using homogeneous preparations of defined G protein subunits. Our
studies revealed that whereas GRP-R and NMB-R selectively coupled with
squid and mouse G
q in an agonist- and
G
-dependent manner, their coupling properties were
distinct. GRP-R had higher affinities for G
q and G
dimers, higher catalytic activity for nucleotide exchange on
G
q, and a higher ratio of agonist stimulated to basal
activity than NMB-R. On the other hand, the structurally related BRS-3
was different from GRP-R and NMB-R in selectivity for
G
q. It strongly preferred mouse G
q over
squid G
q in the in situ reconstitution assay,
whereas GRP-R clearly preferred squid G
q, and NMB-R also
showed a slight preference for squid G
q.
 |
EXPERIMENTAL PROCEDURES |
Membrane Preparation--
Membranes were prepared from Balb 3T3
mouse fibroblast cells expressing mouse GRP-R (19), rat NMB-R (20),
human BRS-3 (17), or 4
BRS-3 (21). Sf9 cell membranes
expressing recombinant mouse GRP-R (22) or BRS-3 were also prepared.
Receptor-enriched membranes were obtained as a P2 fraction from these
cells as described previously (14).
Formation of Recombinant BRS-3 Baculovirus--
A cDNA
fragment encoding the open reading frame of the human BRS-3 (hBRS-3)
flanked by FLAG epitope tag at the 5' end was cloned into
EcoRI site of a transfer vector pBacPAK8
(CLONTECH). The sequence at the 5' end is
5'-AATTCGGCTTGCGCGCATGGACTACAGGACGACGATGACAAGGCTCAAAGGCAG-3'. The sequence at the 3' end is identical to that of the original hBRS-3 clone inserted into an EcoRI site (23). Insect cell
culture, transfection, plaque purification, and virus amplification of BRS-3 were carried out according to the manufacturer's protocol (CLONTECH).
Urea Extraction of Receptor-containing Membranes--
We
modified our previously published urea extraction procedure (13, 14).
The P2 membrane pellet was resuspended in ice-cold solution A (10 mM Hepes, pH 7.4, 1 mM EGTA, 100 µM 4-(2-aminoethyl)benzenesulfonyl fluoride HCl)
containing 7 M urea. After incubation in 7 M
urea for 30 min on ice, the membrane solution was diluted to less than 4 M urea with solution A and then sedimented at
142,000 × g for 30 min at 4 °C. Following a single
urea extraction and centrifugation, the membrane pellet was washed once
with solution A alone and recollected by sedimentation as before. The
final pellet was resuspended in solution A with 12% (w/v) sucrose, and
aliquots were frozen and stored at
80 °C.
Quantitation of Receptor Sites--
Receptor ligand-binding
sites were determined as described by Hellmich et al. (14).
Unextracted and 7 M urea-extracted GRP-R ligand-binding
sites were quantitated by analysis of binding to the radiolabeled GRP-R
antagonist 125I-labeled
[D-Tyr6]Bn-(6-13) methyl ester
(125I-Tyr-ME (24)). Unextracted GRP-R, NMB-R, BRS-3, and
4
BRS-3 were also assayed for ligand-binding sites with the
radiolabeled agonist 125I-labeled
[D-Tyr6,
-Ala11,Phe13,Nle14]Bn-(6-14)
(125I-Tyr-697 (25)). The receptor abundance of BRS-3
expressed in Sf9 cells was also estimated by Western blot using
FLAG-BAP as protein standard and anti-FLAG M2 monoclonal antibody
(Eastman Kodak Company) to detect FLAG fusion protein.
Purification of G Protein Subunits--
G proteins were isolated
from squid retina, bovine brain, bovine retina, and
baculovirus-infected Sf9 cells expressing recombinant mouse
q or
1
2. Squid retinal
G
q was purified as described by Hartman and Northup
(13). Bovine brain G
i/o and G
(26), bovine retina
G
t and
1
1 (27-29), and
recombinant mouse G
q (30) and
1
2 (31) expressed in Sf9 cells
were purified using previously published protocols. Bovine brain 
preparations were further purified by additional chromatography over
phenyl-Sepharose to remove GTP
S binding activity (13).
GDP/GTP
S Exchange Assay--
The receptor-catalyzed
GDP/GTP
S exchange on G
was determined essentially as described
previously (32) with the addition of 2 µM GDP to compete
for uncatalyzed GTP
S binding (14). Receptor-containing membranes
were mixed with G protein subunits and with or without agonist on ice
in a total volume of 30 µl. An addition of 20 µl of reaction
solution was used to initiate the reactions. The reactions contained a
final concentration of 50 mM MOPS, pH 7.5, 100 mM NaCl, 1 mM EDTA, 3 mM
MgSO4, 1 mM dithiothreitol, 3 mg/ml bovine serum albumin, 2 µM GDP, and [35S]GTP
S
(about 4-8 nM). Reactions were incubated at 30 °C for 10 or 15 min, terminated by adding 2 ml of ice-cold solution B (20 mM Tris/HCl, pH 8.0, 25 mM MgCl2,
100 mM NaCl), and filtered over nitrocellulose membranes on
a vacuum manifold. The filters were washed four times with 2 ml each of
ice-cold solution B and dried, and the bound radioactivity was counted
by liquid scintillation.
 |
RESULTS |
Urea-extracted cell membranes containing different heptahelical
receptors have been successfully reconstituted with purified G protein
subunits. These receptor membranes include rod outer segment disc
membranes of bovine retina (29), baculovirus-infected Sf9 cell
membranes containing the 5-HT2c receptor (13), and stably
transfected fibroblast cell membranes containing GRP-R (14). We applied
this in situ receptor reconstitution technique to the
bombesin receptor family to compare the G protein coupling properties
of bombesin receptor subtypes, which share 48 to 54% amino acid
homology (Fig. 1).

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Fig. 1.
Comparison of the primary structures of mouse
GRP-R, rat NMB-R, and human BRS-3. The predicted amino acid
sequences for mouse GRP-R (mGRPR), rat NMB-R (rNMBR), and human BRS-3
(hBRS3) are presented as the best sequence alignment. The seven
transmembrane (TM) helical domains predicted by hydropathy
plots are represented by TM-I to TM-VII with dark
underlines. The predicted four extracellular domains
(e1 to e4) and four intracellular domains
(i1 to i4) are designated. Bold amino acid
residues (indicated with *) are the positions mutated in 4 BRS-3 to
their counterparts in NMB-R and GRP-R.
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We modified the previously published urea extraction procedure to
obtain more consistent receptor recovery and G protein depletion (see
"Experimental Procedures"). Table I
summarizes the effects of the modified procedure on receptor-binding
sites and GTP
S binding activity of GRP-R- and NMB-R-containing
membranes. Compared with the 6 M urea extraction procedure
used previously, 7 M urea required only one instead of two
or three extractions, removed more endogenous GTP
S binding activity
(94-96% versus 92%), while consistently maintaining high
recovery of ligand-binding sites. The GRP-R-binding site abundance was
actually enriched more than 3-fold by 7 M urea extraction,
since 100% of the antagonist binding activity was recovered, whereas
71% of the membrane protein was removed. Furthermore, for both GRP-R
and NMB-R, agonist-stimulated GTP
S binding in the absence of
exogenous G proteins was also abolished more thoroughly by 7 M urea extraction than by 6 M urea extraction
used previously (data not shown), suggesting 7 M urea treatment resulted in a more homogeneous population of uncoupled receptors.
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Table I
Effects of 7 M urea extraction on ligand-binding sites and
endogenous GTP S binding activity of the GRP-R and NMB-R membranes
Membranes were prepared from Balb 3T3 mouse fibroblast cell lines
stably expressing GRP-R or NMB-R (see "Experimental Procedures").
After urea extraction, the membrane protein concentrations decreased
from 3.5 to 1 and from 6 to 1.6 µg/µl, for GRP-R and NMB-R,
respectively. For the receptor ligand binding assays, 3.5 µg of
membrane protein from unextracted GRP-R, 1 µg of membrane protein
from 7 M urea-extracted GRP-R, and 6 µg of membrane
protein from unextracted NMB-R membranes were used. GRP-R and NMB-R
concentrations were determined by Scatchard analysis of
125I-697 and 125I-ME binding as described under
"Experimental Procedures." GTP S binding assays of 0.75 nM GRP-R or 1.0 nM NMB-R, unextracted or 7 M urea-extracted, proceeded for 10 min at 30 °C, and
bound GTP S was determined as described under "Experimental
Procedures." All values presented are the means ± S.D. of data
obtained from three independent experiments.
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Table I also shows that 125I-697 (universal bombesin
receptor agonist) and 125I-ME (GRP-R-specific antagonist)
measured identical binding site abundance on membranes before urea
extraction, indicating that we could use 125I-697 to
determine the receptor concentration for NMB-R and BRS-3, for which a
radiolabeled high affinity antagonist is not available. Because of the
decreased affinities of uncoupled receptors for agonists,
125I-697-binding sites of 7 M urea-extracted
GRP-R, NMB-R, and BRS-3 could not be determined accurately.
Since 7 M urea extraction removed more endogenous GTP
S
binding from GRP-R-containing membranes, we tested whether these
uncoupled receptors could couple with purified squid retinal
G
q and bovine brain G
as was shown previously for
6 M urea-treated GRP-R (14). Fig.
2 shows the results for reconstitution of
membranes containing GRP-R and NMB-R either untreated or 7 M urea-extracted. To facilitate the comparison of the
efficiency of reconstitution, we have tested approximately equal
catalytic activities of the two receptor types at Km
concentrations of
q and near-saturating 
. Thus we
are directly comparing both the success of reconstitution and the
catalytic properties of the receptors. These experiments
demonstrated that (i) very little agonist-stimulated exchange of
GDP for GTP
S on exogenously added G
q was detected for
unextracted GRP-R or NMB-R (Fig. 2, A and C);
(ii) both 7 M urea-extracted GRP-R and 7 M
urea-extracted NMB-R coupled with squid G
q (Fig. 2,
B and D); (iii) like GRP-R, NMB-R-catalyzed
activation of G
q was also dependent on both agonist and

subunits (Fig. 2, B and D). Despite the
qualitative similarities between GRP-R and NMB-R in G protein coupling,
they were clearly different in the ratio of agonist-independent (basal)
to agonist-stimulated activity (Fig. 2, B and
D).

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Fig. 2.
Reconstitution of GRP-R and NMB-R with
G q and
G . P2 fractions of GRP-R
(A) and NMB-R (C) membranes and 7 M
urea-extracted GRP-R (B) and NMB-R (D) membranes
were assessed for agonist-stimulated GTP S binding with or without
reconstitution with G protein subunits as indicated. The
GRP-R-catalyzed GTP S binding reactions contained 0.5 nM
GRP-R, 70 nM squid G q, 270 nM
bovine brain G , and 1 µM GRP. The NMB-R reactions
contained 1.25 nM NMB-R, 120 nM squid
G q, 800 nM bovine brain G , and 1 µM NMB. G q concentration was determined by
GTP S binding, and  concentration was determined by Amido Black
staining. The GTP S binding assays proceeded for 15 min at 30 °C,
and bound GTP S was determined as described under "Experimental
Procedures." The values presented are the average and range of
duplicate determinations, and the results are representative of three
independent experiments. A and B, , no agonist
and , 1 µM GRP; C and D, , no
agonist and , 1 µM NMB.
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In order to obtain initial rate estimates for the
receptor-catalyzed GTP
S binding, we performed the progress analyses
for GRP-R- and NMB-R-catalyzed reactions shown in Fig.
3. For both 7 M
urea-extracted GRP-R and NMB-R and G protein alone, the binding of
GTP
S progressed at a very low rate; G protein-reconstituted receptors without agonist showed an increased rate of binding, whereas
the addition of agonist increased the reaction rate to the highest
values. Moreover, for all of these conditions the GTP
S binding was
approximately linear with time for the initial 10 min of the reaction.
Therefore we have used 10 min as a fixed time point in the GDP/GTP
S
exchange assay to measure the initial velocity of the
receptor-catalyzed activation in all subsequent experiments. The
greatly accelerated initial rates of GTP
S binding in the presence of
agonist represent our measure of receptor-catalyzed G protein
activation. The GRP-R, NMB-R, and BRS-3 were expressed at widely
varying abundance in the Balb 3T3 fibroblasts. Therefore, we have used
the modified GTP
S binding procedures (14) including trace
[35S]GTP
S and 2 µM GDP to suppress
residual nucleotide binding activity of the urea-extracted membranes
rather than our initial procedures that utilize 1 µM
GTP
S with no competing nucleotide (13). Our modified procedure also
accommodates the comparison of the family of G
proteins that
differ in spontaneous binding exchange rates. Because the
chemical concentration of GTP
S (4-8 nM) limits the binding reactions, the plateau values obtained in these experiments are
not stoichiometric binding of GTP
S to the G
q. Rather,
they represent consumption of the [35S]GTP
S trace in
the binding reactions. That these receptors are indeed catalytic was
demonstrated by additional experiments using 1 µM GTP
S
without competing GDP in which 1 nM GRP-R or NMB-R activated the entire 100 nM G
q in about 40 min (data not shown).

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Fig. 3.
Kinetics of GRP-R- and NMB-R-catalyzed
GTP S binding to
G q. Urea-extracted membranes
providing final concentrations of 0.5 nM GRP-R
(A) or 0.85 nM NMB-R (B) were assayed
alone ( ) or reconstituted with 840 nM bovine brain
 and 150 nM squid retinal G q.
Reconstituted membranes were assayed in the absence ( ) or presence
( ) of 1 µM GRP or NMB. The binding of GTP S to the G
protein subunits in the absence of membranes was also determined (×).
For all conditions, the reaction volumes were scaled up to 150 µl;
the binding reaction was conducted at 30 °C, and 10-µl aliquots
were removed at the indicated times for the determination of GTP S
binding as described under "Experimental Procedures." The
lines drawn for G protein-reconstituted samples are the
best-fit simple exponential curves using "Grafit."
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Our previous study has shown selective coupling of GRP-R with
G
q but not G
i/o or G
t
(14). In order to know whether the other members of the bombesin
receptor family share the same selectivity for G
q, we
tested the ability of urea-extracted membranes to catalyze exchange of
GDP for GTP
S on squid retinal G
q, bovine retinal
G
t, or bovine brain G
i/o in the presence
of bovine brain G
. Both GRP-R (Fig.
4A) and NMB-R (Fig.
4B) selectively catalyze the exchange reaction on squid
G
q in an agonist-dependent manner. However,
BRS-3 did not activate any of these G protein preparations using the
universal bombesin receptor agonist 697 (Fig. 4C). In order
to understand why BRS-3 failed to catalyze nucleotide exchange on all
tested G proteins, we have attempted to exclude the possibility that
peptide 697 is only a partial agonist of the BRS-3. We tested a mutated
BRS-3 receptor in which four amino acid residues critical for ligand
selectivity were replaced with their counterparts in NMB-R and GRP-R
(R127Q, S205P, H294R, and S315A, see Fig. 1). This mutant, 4
BRS-3,
displays 2 and 3 orders of magnitude increase in affinities for GRP
(21) and NMB (33), respectively. Fibroblast cells expressing 4
BRS-3
show NMB-stimulated inositol phosphate increases (33). As shown in Fig.
4D, 4
BRS-3 did not catalyze GTP
S binding on any of the
tested G
subunits in the presence of NMB. The enhanced GTP
S
binding on G
i/o in the presence of all of the bombesin
receptors seems to reflect a nonspecific interaction independent of
receptors, because the level of GTP
S binding was proportional to
total membrane protein concentration instead of receptor concentration
(data not shown).

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Fig. 4.
G coupling
selectivity of the GRP-R and NMB-R. Urea-extracted membranes
containing a final concentration of 0.5 nM GRP-R
(A), 0.5 nM NMB-R (B), 0.1 nM BRS-3 (C), or 0.16 nM 4 BRS-3
(D) were mixed with 250 nM bovine brain 
and 100 nM squid retinal G q, bovine retinal
G t, or bovine brain G i/o. GTP S binding
assays in the presence or absence of 1 µM agonist
(peptide 697 for GRP-R, NMB-R, and BRS-3; NMB for 4 BRS-3) proceeded
for 10 min at 30 °C, and bound GTP S was determined as described
under "Experimental Procedures." G subunit concentrations were
determined by GTP S binding. Membrane-independent background and
membrane-only GTP S binding were subtracted from the total binding to
give the values presented in the figure (membrane-independent binding
activities were as follows: 2.3 ± 0.1 fmol for G q
and  ; 2.1 ± 0.1 fmol for G t and  ; and
17.6 ± 0.6 fmol for G i/o and  ;
membrane-alone binding values were as follows: 0.46 ± 0.05 fmol
for GRP-R; 0.55 ± 0.01 fmol for NMB-R; 4.1 ± 0.1 fmol for
BRS-3; and 1.8 ± 0.2 fmol for 4 BRS-3). The values presented
are the means of triplicate determinations (bars, S.D.), and
the results are representative of three independent experiments.
A-C, , no agonist and , 1 µM 697;
D, , no agonist and , 1 µM NMB.
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The lack of coupling of BRS-3 and 4
BRS-3 with the G protein subunits
tested could be due to the low receptor abundance in Balb 3T3
fibroblast cells (0.26 and 0.33 pmol of receptor/mg of membrane protein
for BRS-3 and 4
BRS-3, respectively, versus 3.7 and 2.2 pmol/mg for GRP-R and NMB-R) or the absence of essential G protein
subunits. To achieve high receptor abundance, Sf9 cells were
used to express recombinant BRS-3 encoded by a baculovirus. To test the
possibility that BRS-3 can couple with a mammalian G
q
rather than squid G
q, recombinant mouse
G
q was purified from baculovirus-infected Sf9
cells and used in the reconstitution assays. GRP-R expressed in
Sf9 cells was also compared with that expressed in fibroblast
cells in order to establish that the receptors expressed in these
different cells have the same coupling properties. As shown in Fig.
5, GRP-R expressed in mouse fibroblast
cells and insect Sf9 cells behaved the same way. They activated
both mouse G
q and squid G
q but with
higher catalytic activity for the latter. Although BRS-3 expressed in
fibroblast cells failed to show agonist-stimulated activity with either
G
q (most likely due to the low receptor abundance),
Sf9 cell-expressed BRS-3 clearly showed coupling with mouse
G
q, but little if any coupling with squid
G
q. Another member of the bombesin receptor family,
NMB-R, falls in between GRP-R and BRS-3 in selectivity for mouse and squid G
q. NMB-R showed slightly more efficient coupling
with squid G
q than with mouse G
q.

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Fig. 5.
BRS-3 couples with mouse
G q. Balb 3T3 fibroblast
cell-expressed GRP-R (A, 1.25 nM), NMB-R
(E, 1.25 nM), and BRS-3 (C, 0.1 nM), as well as Sf9 cell-expressed GRP-R
(B, 20 nM) and BRS-3 (D, 20 nM) were assessed for agonist-stimulated GTP S binding
with or without G q subunit. All the receptor-containing
membranes were treated with 7 M urea prior to the in
situ reconstitution assay. The final concentrations of G
protein subunits in the reactions were 40 nM mouse or squid
G q and 200 nM bovine brain G . GTP S
binding reactions proceeded for 10 min at 30 °C, and binding was
determined as described under "Experimental Procedures." The values
presented are the average and range of duplicate determinations, and
the results are representative of two independent experiments.
A-E, , no agonist; , 1 µM 697.
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One of the unique advantages of this in situ reconstitution
technique is that it allows a quantitative assessment of receptor-G protein coupling. To determine how well a receptor couples with a G
protein as well as to compare coupling efficiency between different
receptors, we have performed saturation analysis of the
receptor-catalyzed GTP
S exchange with the G protein subunits. Fig.
6A shows the saturation of the
exchange reaction catalyzed by GRP-R and NMB-R with squid
G
q. The initial velocities conformed to a single-site
model with Km values of 58 nM for GRP-R and 112 nM for NMB-R. The catalytic activities of GRP-R and
NMB-R were also different, with
Vmax2
values of 8.5 × 10
3 MGTP
S bound
Mreceptor
1
s
1 for GRP-R and 4.1 × 10
3
MGTP
S bound
Mreceptor
1
s
1 for NMB-R. Fig. 6B shows the
saturation of the catalysis with bovine brain G
. These data also
fit well to a single-site model with a K1/2 of
115 nM for GRP-R and 238 nM for NMB-R. The
differences between GRP-R and NMB-R in affinity and catalytic activity
for G protein subunits were statistically significant, as summarized in
Table II.

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Fig. 6.
G protein subunit saturation of GRP-R- and
NMB-R-catalyzed GTP S binding.
A presents the G q saturation results. Varying
concentrations as indicated of squid G q were included in
reactions containing 0.5 nM GRP-R with 1 µM
GRP ( ) or 1.0 nM NMB-R with 1 µM NMB ( )
and 470 nM bovine brain  . B, presents the
G saturation results. Varying concentrations as indicated of
bovine brain  were included in reactions containing 0.5 nM GRP-R with 1 µM GRP ( ) or 1.0 nM NMB-R with 1 µM NMB ( ) and 150 nM G q. For all conditions the GTP S
binding reactions proceeded for 10 min at 30 °C, and bound GTP S
was determined as described under "Experimental Procedures." The
values presented are from single determinations. The lines drawn are
the best-fit curves for single site saturation using "Grafit." The
results are representative of three to six independent
experiments.
|
|
Given the diversity of G
dimers, the receptor-G protein coupling
selectivity is unlikely to be restricted to the G
subunit alone. To
address the question of whether the bombesin receptors also have
selectivity for 
dimers, we tested the ability of GRP-R (Fig.
7A) and NMB-R (Fig.
7B) to activate G
q with different 
dimers, including bovine brain 
, bovine retinal 
(
1
1), and
1
2. When tested at a concentration of
0.25 µM, bovine brain 
showed the greatest
enhancement of GRP-R- or NMB-R-catalyzed exchange reaction,
1
2 the second highest, whereas
1
1 hardly affected the binding of GTP
S
to G
q. At a concentration of 1 µM,
1
1 also enhanced the receptor-catalyzed
GTP
S binding but incompletely, whereas 0.74 µM of
1
2 produced the greatest enhancement.

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[in this window]
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|
Fig. 7.
G
coupling preference of the GRP-R and NMB-R. Urea-extracted
membranes providing a final concentration of 1.25 nM GRP-R
(A) or 2.5 nM NMB-R (B) were mixed
with 100 nM squid G q, 1 µM
agonist 697, and the indicated concentrations of  dimers.
BB is the phenyl-Sepharose isolated  fraction
from bovine cortex; 1 1 is the
bovine retinal  , and 1 2
is the recombinant dimer from baculovirus-infected Sf9 cells.
G dimer concentrations were determined by Amido Black staining.
GTP S binding reactions proceeded for 10 min at 30 °C, and binding
was determined as described under "Experimental Procedures." The
values presented are the means of triplicate determinations
(bars, S.D.), and the results are representative of three
independent experiments.
|
|
To compare the affinities of GRP-R and NMB-R for G
dimers, we
performed saturation analysis of the receptor-catalyzed GTP
S binding
with G
1
1 and
G
1
2. As shown in Fig.
8 and summarized in Table II, GRP-R
consistently showed higher affinity for the G
dimers that we
tested. For a given G
preparation, the ratio of
K1/2 of NMB-R and GRP-R ranged from 2.4- to
4.7-fold. Despite the quantitative differences between GRP-R and NMB-R,
they showed same rank order of preference among the three 
preparations: bovine brain 
>
1
2
1
1.

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[in this window]
[in a new window]
|
Fig. 8.
G 1 1
and
G 1 2
saturation of GRP-R- and NMB-R-catalyzed GTP S
binding. Varying concentrations as indicated of
G 1 1 and G 1 2
were included in reactions containing 100 nM squid retinal
G q and 1.0 nM GRP-R with 1 µM
GRP (A) or 1.4 nM NMB-R with 1 µM
NMB (B). The GTP S binding reactions proceeded for 10 min
at 30 °C, and bound GTP S was determined as described under
"Experimental Procedures." The values presented are from single
determinations. The lines drawn are the best-fit curves for
single site saturation. The results are representative of three to six
independent experiments.
|
|
 |
DISCUSSION |
In this study we adapted a published in situ receptor
reconstitution method utilizing membranes from cells expressing
recombinant GRP-R, NMB-R, BRS-3, or 4
BRS-3 which have been extracted
with 7 M urea to remove endogenous GTP-binding proteins.
The urea extraction procedure yielded a homogenous population of
uncoupled receptors with 100% recovery of receptor ligand-binding
sites. Such receptor preparations were functional when reconstituted
with heterotrimeric G protein subunits as shown by the assay measuring
the first biochemical event in G protein activation: receptor-catalyzed
exchange of GTP for GDP on a G
subunit. The in situ
receptor reconstitution technique has been used successfully in our
earlier studies using membranes from baculovirus-infected Sf9
cells expressing 5-HT2c receptor (13), membranes from
stably transfected fibroblast cells expressing GRP-R (14), and now
NMB-R and BRS-3. We believe that this method should be applicable to
study virtually any receptor-G protein coupling. The major limitation
we have observed is the receptor abundance in the membrane fraction.
We found all three bombesin receptors selectively coupled with a
G
q but not G
i/o or G
t.
However, three similar receptors were different in coupling selectivity
toward members of the G
q family. Although GRP-R and
NMB-R coupled to both squid and mouse G
q, GRP-R had a
much stronger preference for squid G
q, and NMB-R showed
only a slight preference for squid G
q. In contrast to GRP-R and NMB-R, the structurally related BRS-3 did not couple with
squid G
q, whereas it clearly coupled with mouse
G
q. Given that the differences between squid
G
q and mouse G
q structures are not much
greater than the ones among mouse G
q subtypes themselves (34-36), it will be interesting to investigate coupling of the bombesin receptors to various G
q family subtypes within
the same species in future studies.
The kinetic analysis of receptor-G protein interactions presented in
this report also revealed a quantitative difference between GRP-R and
NMB-R. The controlled, independent manipulation of receptor and G
protein subunit concentrations required for this analysis is not
possible using a whole cell system or prior reconstitution methods
using purified, detergent-solubilized receptors and G proteins in
phospholipid vesicles. GRP-R and NMB-R, although similar in their
selectivity for G
q and rank-order preference for G
in the receptor-G protein coupling, were different in the catalytic activity toward G
q and affinities for G proteins. GRP-R
showed higher catalytic activity on squid G
q and higher
affinities for both G
q and G
dimers than
NMB-R.
These results may partially explain an ambiguity noted in antisense
oligonucleotide experiments in which individual G
subunits were
depleted (18). In those experiments, Xenopus laevis oocytes expressing either GRP-R or NMB-R were microinjected with antisense phosphorothioate oligonucleotides complementary to specific regions of
either Xenopus G
q or G
11 to
deplete selectively G
q or G
11 protein.
Following application of agonist, the activity of the calcium-activated
chloride channel was measured under whole cell voltage clamp
conditions. These experiments showed that treatment with the
G
q antisense oligonucleotides could inhibit up to 74% of the response of the NMB-R but had no effect on the GRP-R response. G
11 antisense, on the other hand, had little effect on
either GRP-R- or NMB-R-mediated responses. The data reported here
showed GRP-R coupled more effectively with squid G
q than
with mouse G
q. Squid G
q is 74-78%
identical to mouse G
q, G
11, and
G
14. As the sequence identity between mouse
G
14 and G
q or G
11 is 80 or
81%, respectively, it is likely that GRP-R couples primarily with
G
14 instead of G
q or G
11.
It is also possible that due to the higher affinity as well as higher
catalytic activity for G
q, it would be easier to observe
the influence of G
q depletion on NMB-R-regulated
response than on GRP-R response. In those experiments, the antisense
depletion taking place might simply fail to reduce G
q to
a level that would impair GRP-R response.
GRP-R and NMB-R not only showed selective coupling with
G
q but also showed a clear discrimination between
different 
dimers. We provide two arguments that this result
suggests the different 
dimers have different affinity and/or
efficacy for bombesin receptors, rather than reflecting different
affinities of G
q for 
dimers. First, instead of a
uniform difference between G
dimers, GRP-R and NMB-R had
different K1/2 ratio for
1
1 and
1
2
(8-fold versus 4.6-fold). Second, rat 5-HT2c
receptor has also been shown to couple with squid retinal
G
q in the in situ reconstitution assay (13).
But unlike GRP-R or NMB-R, it has low affinity for both bovine brain

(estimated K1/2 is about 600 nM) and bovine retinal 
, i.e.
1
1 (13). Other receptor-G protein
coupling studies also supported the notion that receptors can have
different affinities for 
dimers. Studies of bovine rhodopsin
activation of
t have found differences in apparent affinity among tissue-derived 
dimers of defined compositions or
recombinant 
dimers, whereas
t shows essentially
no preference for 
dimers (31-32, 37). The fact that both GRP-R
and NMB-R preferred bovine brain 
over
1
2 and the diverse composition of bovine
brain 
dimers (37, 38) suggest there may be other 
dimer(s)
having higher affinity or/and efficacy than
1
2 in enhancing the catalytic activity of
GRP-R and NMB-R on G
q.
In situ receptor reconstitution has been proven to be a
useful methodology for detailed kinetic analysis of receptor-G protein coupling. It allows the identity and concentration of each coupling component to be defined and manipulated, while preserving the receptors
in their native phospholipid environment. By using this method, we
established a significant quantitative difference between GRP-R and
NMB-R for interaction with the same squid G
q and G
proteins and, in addition, a qualitative difference between those two
receptors and BRS-3 which did not interact with that same G
q. Combining the currently available high expression
systems (e.g. baculovirus infection of insect Sf9
cells, transfection of mouse fibroblast cells) with the in
situ receptor reconstitution technique, it should be feasible to
study functional coupling between any recombinant receptor and G
protein subunits, advancing our understanding of the molecular
mechanisms governing the signal transduction pathway for G
protein-coupled receptors.
 |
FOOTNOTES |
*
The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The amino acid sequences of these proteins can be accessed
through NCBI Protein Database under NCBI accession numbers 121639 (mouse GRP-R), 128394 (rat NMB-R), and 291877 (human BRS-3).
To whom correspondence should be addressed: Laboratory of
Cellular Biology, National Institute on Deafness and Other
Communication Disorders, 5 Research Ct., Rockville, MD 20850. Tel.:
301-496-9167; Fax: 301-480-8019; E-mail:
drjohn{at}codon.nih.gov.
2
These values almost certainly underestimate the
catalytic constants of GRP-R and NMB-R for G protein activation. Our
GTP
S binding reactions included 2 µM GDP to suppress
the receptor-independent binding to G
. Since the reactions included
carrier-free [35S]GTP
S at 4-8 nM, the
rates for GTP
S binding in the absence of competing GDP would be much higher.
 |
ABBREVIATIONS |
The abbreviations used are:
GRP, gastrin-releasing peptide;
NMB, neuromedin B;
GRP-R, GRP-preferring
receptor;
NMB-R, NMB-preferring receptor;
BRS-3, bombesin receptor
subtype 3;
G protein, guanine nucleotide-binding regulatory
protein;
GTP
S, guanosine 5'-o-(3-thiotriphosphate);
DTT, dithiothreitol;
5-HT, 5-hydroxytryptamine (serotonin);
125I-Tyr-ME, 125I-labeled
[D-Tyr6]Bn-(6-13) methyl ester;
125I-Tyr-697, [D-Tyr6,
-Ala11,Phe13,Nle14]Bn-(6-14);
MOPS, 4-morpholinepropanesulfonic acid.
 |
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