The Bombesin Receptor Subtypes Have Distinct G Protein Specificities*

Xiaoying JianDagger , Eduardo SainzDagger , William A. Clark§, Robert T. Jensen, James F. BatteyDagger , and John K. Northup§parallel

From the Dagger  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

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We used an in situ reconstitution assay to examine the receptor coupling to purified G protein alpha  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 Galpha q and mouse Galpha q but not bovine retinal Galpha t or bovine brain Galpha i/o. The GRP-R- and NMB-R-catalyzed activations of Galpha q were dependent upon and enhanced by different beta gamma dimers in the same rank order as follows: bovine brain beta gamma  > beta 1gamma 2 >> beta 1gamma 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 beta gamma , beta 1gamma 1, and beta 1gamma 2 and squid retinal Galpha q. In addition, GRP-R showed higher catalytic activity on squid Galpha q. Like GRP-R and NMB-R, BRS-3 did not catalyze GTPgamma S binding to Galpha i/o or Galpha t. However, BRS-3 showed little, if any, coupling with squid Galpha q but clearly activated mouse Galpha q. GRP-R and NMB-R catalyzed GTPgamma S binding to both squid and mouse Galpha q, with GRP-R activating squid Galpha q more effectively, and NMB-R also showed slight preference for squid Galpha q. These studies reveal that the structurally similar bombesin receptor subtypes, in particular BRS-3, possess distinct coupling preferences among members of the Galpha q family.

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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 alpha  subunit and a beta gamma dimer that acts as a functional monomer. Ligand-activated G protein-coupled receptors catalyze the exchange of GTP for GDP bound to the Galpha subunit, resulting in dissociation of the GTP-activated alpha  subunit from both its cognate Gbeta gamma dimer and the receptor. The GTP-activated alpha  subunit as well as dissociated Gbeta gamma dimer in turn regulate intracellular effectors. At least 20 different alpha  subunits, 5 beta  subunits, and 12 gamma  subunits have been identified to date. The Galpha subunits have been divided into four groups based upon sequence homology and intracellular effector regulation (4, 5). The Galpha q subfamily, which includes Galpha q, Galpha 11, Galpha 14, and Galpha 15/16, stimulates phosphoinositide hydrolysis by activating phospholipase C-beta (6-10). In addition, Gbeta gamma subunits can also stimulate phospholipase C-beta s in concert with Galpha 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 Galpha q, which in turn activates a phospholipase C-beta isozyme. Antisense oligonucleotide injection of Xenopus oocytes (18) has identified Galpha q as a mediator of the NMB-R response. However, in Xenopus oocytes neither Galpha q nor Galpha 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 Galpha q but not Galpha i/o or Galpha 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 Galpha q as compared with that of NMB-R for Galpha 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 Galpha q in an agonist- and Gbeta gamma -dependent manner, their coupling properties were distinct. GRP-R had higher affinities for Galpha q and Gbeta gamma dimers, higher catalytic activity for nucleotide exchange on Galpha 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 Galpha q. It strongly preferred mouse Galpha q over squid Galpha q in the in situ reconstitution assay, whereas GRP-R clearly preferred squid Galpha q, and NMB-R also showed a slight preference for squid Galpha q.

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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 4Delta 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 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 4Delta BRS-3 were also assayed for ligand-binding sites with the radiolabeled agonist 125I-labeled [D-Tyr6,beta -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 alpha q or beta 1gamma 2. Squid retinal Galpha q was purified as described by Hartman and Northup (13). Bovine brain Galpha i/o and Gbeta gamma (26), bovine retina Galpha t and beta 1gamma 1 (27-29), and recombinant mouse Galpha q (30) and beta 1gamma 2 (31) expressed in Sf9 cells were purified using previously published protocols. Bovine brain beta gamma preparations were further purified by additional chromatography over phenyl-Sepharose to remove GTPgamma S binding activity (13).

GDP/GTPgamma S Exchange Assay-- The receptor-catalyzed GDP/GTPgamma S exchange on Galpha was determined essentially as described previously (32) with the addition of 2 µM GDP to compete for uncatalyzed GTPgamma 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]GTPgamma 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.

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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 4Delta BRS-3 to their counterparts in NMB-R and GRP-R.

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 GTPgamma 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 GTPgamma 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 GTPgamma 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 GTPgamma 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." GTPgamma 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 GTPgamma S was determined as described under "Experimental Procedures." All values presented are the means ± S.D. of data obtained from three independent experiments.

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 GTPgamma S binding from GRP-R-containing membranes, we tested whether these uncoupled receptors could couple with purified squid retinal Galpha q and bovine brain Gbeta gamma 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 alpha q and near-saturating beta gamma . 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 GTPgamma S on exogenously added Galpha q was detected for unextracted GRP-R or NMB-R (Fig. 2, A and C); (ii) both 7 M urea-extracted GRP-R and M urea-extracted NMB-R coupled with squid Galpha q (Fig. 2, B and D); (iii) like GRP-R, NMB-R-catalyzed activation of Galpha q was also dependent on both agonist and beta gamma 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 Galpha q and Gbeta gamma . 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 GTPgamma S binding with or without reconstitution with G protein subunits as indicated. The GRP-R-catalyzed GTPgamma S binding reactions contained 0.5 nM GRP-R, 70 nM squid Galpha q, 270 nM bovine brain Gbeta gamma , and 1 µM GRP. The NMB-R reactions contained 1.25 nM NMB-R, 120 nM squid Galpha q, 800 nM bovine brain Gbeta gamma , and 1 µM NMB. Galpha q concentration was determined by GTPgamma S binding, and beta gamma concentration was determined by Amido Black staining. The GTPgamma S binding assays proceeded for 15 min at 30 °C, and bound GTPgamma 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.

In order to obtain initial rate estimates for the receptor-catalyzed GTPgamma S binding, we performed the progress analyses for GRP-R- and NMB-R-catalyzed reactions shown in Fig. 3. For both M urea-extracted GRP-R and NMB-R and G protein alone, the binding of GTPgamma 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 GTPgamma 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/GTPgamma S exchange assay to measure the initial velocity of the receptor-catalyzed activation in all subsequent experiments. The greatly accelerated initial rates of GTPgamma 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 GTPgamma S binding procedures (14) including trace [35S]GTPgamma 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 GTPgamma S with no competing nucleotide (13). Our modified procedure also accommodates the comparison of the family of Galpha proteins that differ in spontaneous binding exchange rates. Because the chemical concentration of GTPgamma S (4-8 nM) limits the binding reactions, the plateau values obtained in these experiments are not stoichiometric binding of GTPgamma S to the Galpha q. Rather, they represent consumption of the [35S]GTPgamma S trace in the binding reactions. That these receptors are indeed catalytic was demonstrated by additional experiments using 1 µM GTPgamma S without competing GDP in which 1 nM GRP-R or NMB-R activated the entire 100 nM Galpha q in about 40 min (data not shown).


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Fig. 3.   Kinetics of GRP-R- and NMB-R-catalyzed GTPgamma S binding to Galpha q. Urea-extracted membranes providing final concentrations of 0.5 nM GRP-R (A) or 0.85 nM NMB-R (B) were assayed alone (triangle ) or reconstituted with 840 nM bovine brain beta gamma and 150 nM squid retinal Galpha q. Reconstituted membranes were assayed in the absence (open circle ) or presence () of 1 µM GRP or NMB. The binding of GTPgamma 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 GTPgamma S binding as described under "Experimental Procedures." The lines drawn for G protein-reconstituted samples are the best-fit simple exponential curves using "Grafit."

Our previous study has shown selective coupling of GRP-R with Galpha q but not Galpha i/o or Galpha t (14). In order to know whether the other members of the bombesin receptor family share the same selectivity for Galpha q, we tested the ability of urea-extracted membranes to catalyze exchange of GDP for GTPgamma S on squid retinal Galpha q, bovine retinal Galpha t, or bovine brain Galpha i/o in the presence of bovine brain Gbeta gamma . Both GRP-R (Fig. 4A) and NMB-R (Fig. 4B) selectively catalyze the exchange reaction on squid Galpha 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, 4Delta BRS-3, displays 2 and 3 orders of magnitude increase in affinities for GRP (21) and NMB (33), respectively. Fibroblast cells expressing 4Delta BRS-3 show NMB-stimulated inositol phosphate increases (33). As shown in Fig. 4D, 4Delta BRS-3 did not catalyze GTPgamma S binding on any of the tested Galpha subunits in the presence of NMB. The enhanced GTPgamma S binding on Galpha i/o in the presence of all of the bombesin receptors seems to reflect a nonspecific interaction independent of receptors, because the level of GTPgamma S binding was proportional to total membrane protein concentration instead of receptor concentration (data not shown).


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Fig. 4.   Galpha 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 4Delta BRS-3 (D) were mixed with 250 nM bovine brain beta gamma and 100 nM squid retinal Galpha q, bovine retinal Galpha t, or bovine brain Galpha i/o. GTPgamma S binding assays in the presence or absence of 1 µM agonist (peptide 697 for GRP-R, NMB-R, and BRS-3; NMB for 4Delta BRS-3) proceeded for 10 min at 30 °C, and bound GTPgamma S was determined as described under "Experimental Procedures." Galpha subunit concentrations were determined by GTPgamma S binding. Membrane-independent background and membrane-only GTPgamma 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 Galpha q and beta gamma ; 2.1 ± 0.1 fmol for Galpha t and beta gamma ; and 17.6 ± 0.6 fmol for Galpha i/o and beta gamma ; 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 4Delta 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.

The lack of coupling of BRS-3 and 4Delta 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 4Delta 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 Galpha q rather than squid Galpha q, recombinant mouse Galpha 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 Galpha q and squid Galpha q but with higher catalytic activity for the latter. Although BRS-3 expressed in fibroblast cells failed to show agonist-stimulated activity with either Galpha q (most likely due to the low receptor abundance), Sf9 cell-expressed BRS-3 clearly showed coupling with mouse Galpha q, but little if any coupling with squid Galpha q. Another member of the bombesin receptor family, NMB-R, falls in between GRP-R and BRS-3 in selectivity for mouse and squid Galpha q. NMB-R showed slightly more efficient coupling with squid Galpha q than with mouse Galpha q.


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Fig. 5.   BRS-3 couples with mouse Galpha 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 GTPgamma S binding with or without Galpha 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 Galpha q and 200 nM bovine brain Gbeta gamma . GTPgamma 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.

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 GTPgamma 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 Galpha 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 MGTPgamma S bound Mreceptor-1 s-1 for GRP-R and 4.1 × 10-3 MGTPgamma S bound Mreceptor-1 s-1 for NMB-R. Fig. 6B shows the saturation of the catalysis with bovine brain Gbeta gamma . 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 GTPgamma S binding. A presents the Galpha q saturation results. Varying concentrations as indicated of squid Galpha 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 (open circle ) and 470 nM bovine brain beta gamma . B, presents the Gbeta gamma saturation results. Varying concentrations as indicated of bovine brain beta gamma were included in reactions containing 0.5 nM GRP-R with 1 µM GRP () or 1.0 nM NMB-R with 1 µM NMB (open circle ) and 150 nM Galpha q. For all conditions the GTPgamma S binding reactions proceeded for 10 min at 30 °C, and bound GTPgamma 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.

                              
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Table II
Summary of GRP-R and NMB-R affinity and catalytic activity for squid retinal Galpha q and different Gbeta gamma dimers

Given the diversity of Gbeta gamma dimers, the receptor-G protein coupling selectivity is unlikely to be restricted to the Galpha subunit alone. To address the question of whether the bombesin receptors also have selectivity for beta gamma dimers, we tested the ability of GRP-R (Fig. 7A) and NMB-R (Fig. 7B) to activate Galpha q with different beta gamma dimers, including bovine brain beta gamma , bovine retinal beta gamma (beta 1gamma 1), and beta 1gamma 2. When tested at a concentration of 0.25 µM, bovine brain beta gamma showed the greatest enhancement of GRP-R- or NMB-R-catalyzed exchange reaction, beta 1gamma 2 the second highest, whereas beta 1gamma 1 hardly affected the binding of GTPgamma S to Galpha q. At a concentration of 1 µM, beta 1gamma 1 also enhanced the receptor-catalyzed GTPgamma S binding but incompletely, whereas 0.74 µM of beta 1gamma 2 produced the greatest enhancement.


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Fig. 7.   Gbeta gamma 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 Galpha q, 1 µM agonist 697, and the indicated concentrations of beta gamma dimers. BBbeta gamma is the phenyl-Sepharose isolated beta gamma fraction from bovine cortex; beta 1gamma 1 is the bovine retinal beta gamma , and beta 1gamma 2 is the recombinant dimer from baculovirus-infected Sf9 cells. Gbeta gamma dimer concentrations were determined by Amido Black staining. GTPgamma 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 Gbeta gamma dimers, we performed saturation analysis of the receptor-catalyzed GTPgamma S binding with Gbeta 1gamma 1 and Gbeta 1gamma 2. As shown in Fig. 8 and summarized in Table II, GRP-R consistently showed higher affinity for the Gbeta gamma dimers that we tested. For a given Gbeta gamma 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 beta gamma preparations: bovine brain beta gamma  > beta 1gamma 2 >> beta 1gamma 1.


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Fig. 8.   Gbeta 1gamma 1 and Gbeta 1gamma 2 saturation of GRP-R- and NMB-R-catalyzed GTPgamma S binding. Varying concentrations as indicated of Gbeta 1gamma 1 and Gbeta 1gamma 2 were included in reactions containing 100 nM squid retinal Galpha q and 1.0 nM GRP-R with 1 µM GRP (A) or 1.4 nM NMB-R with 1 µM NMB (B). The GTPgamma S binding reactions proceeded for 10 min at 30 °C, and bound GTPgamma 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
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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 4Delta BRS-3 which have been extracted with 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 Galpha 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 Galpha q but not Galpha i/o or Galpha t. However, three similar receptors were different in coupling selectivity toward members of the Galpha q family. Although GRP-R and NMB-R coupled to both squid and mouse Galpha q, GRP-R had a much stronger preference for squid Galpha q, and NMB-R showed only a slight preference for squid Galpha q. In contrast to GRP-R and NMB-R, the structurally related BRS-3 did not couple with squid Galpha q, whereas it clearly coupled with mouse Galpha q. Given that the differences between squid Galpha q and mouse Galpha q structures are not much greater than the ones among mouse Galpha q subtypes themselves (34-36), it will be interesting to investigate coupling of the bombesin receptors to various Galpha 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 Galpha q and rank-order preference for Gbeta gamma in the receptor-G protein coupling, were different in the catalytic activity toward Galpha q and affinities for G proteins. GRP-R showed higher catalytic activity on squid Galpha q and higher affinities for both Galpha q and Gbeta gamma dimers than NMB-R.

These results may partially explain an ambiguity noted in antisense oligonucleotide experiments in which individual Galpha 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 Galpha q or Galpha 11 to deplete selectively Galpha q or Galpha 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 Galpha q antisense oligonucleotides could inhibit up to 74% of the response of the NMB-R but had no effect on the GRP-R response. Galpha 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 Galpha q than with mouse Galpha q. Squid Galpha q is 74-78% identical to mouse Galpha q, Galpha 11, and Galpha 14. As the sequence identity between mouse Galpha 14 and Galpha q or Galpha 11 is 80 or 81%, respectively, it is likely that GRP-R couples primarily with Galpha 14 instead of Galpha q or Galpha 11. It is also possible that due to the higher affinity as well as higher catalytic activity for Galpha q, it would be easier to observe the influence of Galpha 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 Galpha q to a level that would impair GRP-R response.

GRP-R and NMB-R not only showed selective coupling with Galpha q but also showed a clear discrimination between different beta gamma dimers. We provide two arguments that this result suggests the different beta gamma dimers have different affinity and/or efficacy for bombesin receptors, rather than reflecting different affinities of Galpha q for beta gamma dimers. First, instead of a uniform difference between Gbeta gamma dimers, GRP-R and NMB-R had different K1/2 ratio for beta 1gamma 1 and beta 1gamma 2 (8-fold versus 4.6-fold). Second, rat 5-HT2c receptor has also been shown to couple with squid retinal Galpha q in the in situ reconstitution assay (13). But unlike GRP-R or NMB-R, it has low affinity for both bovine brain beta gamma (estimated K1/2 is about 600 nM) and bovine retinal beta gamma , i.e. beta 1gamma 1 (13). Other receptor-G protein coupling studies also supported the notion that receptors can have different affinities for beta gamma dimers. Studies of bovine rhodopsin activation of alpha t have found differences in apparent affinity among tissue-derived beta gamma dimers of defined compositions or recombinant beta gamma dimers, whereas alpha t shows essentially no preference for beta gamma dimers (31-32, 37). The fact that both GRP-R and NMB-R preferred bovine brain beta gamma over beta 1gamma 2 and the diverse composition of bovine brain beta gamma dimers (37, 38) suggest there may be other beta gamma dimer(s) having higher affinity or/and efficacy than beta 1gamma 2 in enhancing the catalytic activity of GRP-R and NMB-R on Galpha 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 Galpha q and Gbeta gamma proteins and, in addition, a qualitative difference between those two receptors and BRS-3 which did not interact with that same Galpha 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).

parallel 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 GTPgamma S binding reactions included 2 µM GDP to suppress the receptor-independent binding to Galpha . Since the reactions included carrier-free [35S]GTPgamma S at 4-8 nM, the rates for GTPgamma 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; GTPgamma 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,beta -Ala11,Phe13,Nle14]Bn-(6-14); MOPS, 4-morpholinepropanesulfonic acid.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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