©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Influence of Second and Third Cytoplasmic Loops on Binding, Internalization, and Coupling of Chimeric Bombesin/m3 Muscarinic Receptors (*)

(Received for publication, March 29, 1995; and in revised form, June 1, 1995)

Min-Jen Tseng Steve Coon Ed Stuenkel Valeria Struk Craig D. Logsdon (§)

From the Department of Physiology, University of Michigan, Ann Arbor, Michigan 48109-0622

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 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.


INTRODUCTION

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 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.

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)()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.

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 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.

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 -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.

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.


EXPERIMENTAL PROCEDURES

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 NaI in 20 µl of 0.5 M KPO buffer (pH 7.4). After incubation at 22 °C for 6 min, 300 µl of HO 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 NaPO, 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).

For analysis of the effects of GTPS 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) .

For measurement of arachidonic acid release, cells grown to near confluency in 6-well plates were incubated with 1 µCi of [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.

Analysis of intracellular [Ca] 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.


RESULTS

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.


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 [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.


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 [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.


Figure 3: A, time-course of specific binding and internalization of [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.



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 [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.

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 [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.


Figure 4: Binding characteristics of wild-type and mutant Bn receptors. Competitive inhibition of [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.





In order to more accurately estimate receptor binding parameters, competition experiments were performed using the bombesin antagonist [D-Tyr]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 Bvalues 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.


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-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.



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 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).


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] 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 GTPS on Binding to Wild-type and BM3L Receptors

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 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 GTPS 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 GTPS. Addition of GTPS 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.




DISCUSSION

The most well understood consequence of interaction between receptors and G proteins is the activation of G protein 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.

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 GTPS 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.

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: -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.

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-linked receptor to another does not necessarily lead to a well coupled receptor.

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. 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.

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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK41350 and by the Michigan Gastrointestinal Peptide Digestive Disease Center. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Box 0622 Dept. of Physiology, University of Michigan, 1150 W. Medical Center Dr., Ann Arbor, MI 48109. Tel.: 313-763-2539; Fax: 313-936-8813.

The abbreviations used are: Bn, bombesin; ACh, acetylcholine; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; SLO, streptolysin O; GTPS, guanosine 5`-3-O-(thio)triphosphate; PPI, polyphosphoinositides; CHO, Chinese hamster ovary.


ACKNOWLEDGEMENTS

We thank Drs. J. A. Williams and S. K. Fisher for helpful discussions throughout this work.


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