©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Carboxyl-terminal Domains Determine Internalization and Recycling Characteristics of Bombesin Receptor Chimeras (*)

(Received for publication, May 5, 1995; and in revised form, June 12, 1995)

Min-Jen Tseng Katharina Detjen Valeria Struk Craig D. Logsdon (§)

From theDepartment of Physiology, University of Michigan, Ann Arbor, Michigan 48109-0622

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

To investigate the role of the carboxyl terminus in the regulation of the bombesin (BN) receptor, we constructed two chimeric receptors with carboxyl termini transferred from either m3 muscarinic cholinergic (m3 ACh) (BMC) or cholecystokinin A (CCK(A)) (BCC) receptors and expressed them in Chinese hamster ovary cells. Previous studies showed that agonist treatment caused rapid internalization of CCK(A) but not m3 ACh receptors in these cells. In the current study we conducted separate analyses of ligand and receptor internalization and analyzed receptor recycling. Ligand internalization was assessed using acid washing. BN and CCK(A) receptors internalized ligand with 80 ± 3 and 85 ± 7% in an acid-resistant compartment at equilibrium. Ligand internalization of chimeric receptors generally assumed the properties of the donor receptors. Thus, BCC receptors internalized ligand to a similar extent as wild-type CCK(A) receptors (75 ± 3%), whereas, BMC receptors showed reduced ligand internalization (38 ± 1%). Receptor internalization was more directly assessed by determining agonist-induced loss of surface binding. BN and CCK(A) receptors were largely internalized (56 ± 8 and 50 ± 7%, respectively). BCC receptors were also extensively internalized (82 ± 3%). In contrast, BMC receptors were minimally internalized (22 ± 8%). Receptor recycling was assessed as recovery from agonist induced loss of binding. BN, CCK(A), and BMC receptors showed rapid recycling. In contrast, BCC receptors did not recycle. These data indicate that carboxyl-terminal structures determine both internalization of ligand-receptor complexes and subsequent receptor recycling.


INTRODUCTION

Receptor internalization is a ubiquitous process occurring for virtually all plasma membrane receptors. The endocytotic receptors, such as those for low density lipoprotein and transferrin, are constitutively internalized(1, 2) . In contrast receptors for hormones, neurotransmitters, and growth factors require agonist occupancy for internalization, suggesting the need for an agonist-dependent signal to initiate the internalization process. Tyrosine kinase activity is required for internalization of growth factor receptors that are tyrosine kinases, such as insulin (3) and epidermal growth factor (4) receptors. However, for the family of seven transmembrane G protein-coupled receptors the nature of the internalization signal is unknown. This signal is unlikely to be a common second messenger because receptors having diverse effects on intracellular second messengers are similarly internalized. However, whether the internalization signal involves activation of G proteins is less clear. On one hand, antagonists are not internalized and the internalization of partial agonists correlates with their abilities to activate G proteins(5) . Also, many uncoupled mutant receptors are not internalized, and the degree of uncoupling often correlates with the decrease in internalization(6, 7) . On the other hand, certain mutant receptors with reduced G protein coupling internalize normally(8, 9, 10, 11, 12) . One interpretation of these data is that both internalization and activation of heterotrimeric G proteins require agonist-induced conformational changes in the receptors and that the domains involved in internalization are near but separate from those involved in coupling to G proteins(6, 13) .

The search for internalization domains has occurred at a rapid pace because of the importance of internalization in receptor function and regulation. A large number of domains have been implicated in the internalization of specific G protein-linked receptors(14, 15, 16, 17, 18, 19) . However, these domains have localized to divergent regions of the various receptors and often involve sequences not conserved in other members of the receptor superfamily. Alterations in the carboxyl terminus have been found to influence internalization in the widest variety of receptors. Yet, the role of the carboxyl terminus remains uncertain as it has been found to be either necessary(11, 16, 20) , inhibitory(17, 21, 22, 23) , or not involved in internalization(24) , depending upon the receptor. One complication is that many of the studies have involved deletions, truncations, or multiple substitutions in the carboxyl terminus, such that it has often been difficult to discern whether the experimental alterations have been specific or may have resulted in nonspecific conformational effects.

An alternative to deletion and substitution experiments is the creation of receptor chimeras. This strategy offers substantial advantages for analysis of structure and function in that the expected outcome is the alteration or addition, rather than the loss of function(25) . We chose this approach to analyze the role of the carboxyl terminus in the internalization of the bombesin receptor. Bombesin receptors rapidly internalize ligand both in natively expressing cells, such as pancreatic acinar (26) and Swiss 3T3 (27) cells, as well as in transfected cell models, such as Balb C 3T3 cells(7, 11) . In addition, truncation or major alterations of the bombesin receptor carboxyl terminus were previously shown to block internalization in Balb C 3T3 cells(11) . As sources of donor carboxyl termini, we chose the m3 muscarinic cholinergic receptor (m3 ACh) (^1)and the cholecystokinin A (CCK(A)) receptor, as these receptors have different internalization characteristics. CHO cells show rapid agonist-induced internalization of CCK(A)(28) but not m3 ACh receptors(29) . Also, CCK(A) receptors are internalized via both clathrin-coated and -uncoated vesicles (28) whereas bombesin receptors are internalized exclusively via clathrin-coated vesicles(30) . As a working hypothesis we predicted that internalization of chimeric receptors would reflect the properties of the carboxyl-terminal donor receptors. Thus, a bombesin/m3 ACh chimeric receptor would show impaired internalization. In contrast, a bombesin/CCK(A) chimeric receptor would internalize to the same extent as wild-type BN or CCK(A) receptors. Ligand internalization, receptor internalization, and receptor recycling were separately analyzed. The data supported the working hypothesis and thus confirmed the previously proposed importance of carboxyl-terminal domains in receptor internalization. In addition, important differences were noted between internalization of ligand and receptor that reflected differences in the methodologies. Furthermore, we found that the carboxyl terminus influenced receptor recycling independently of internalization, thus indicating an additional role of the carboxyl terminus in receptor regulation.


EXPERIMENTAL PROCEDURES

[I-Tyr^4]Bombesin (81.4 TBq/mmol) and [I-BH]CCK(8) (81.4 TBq/mmol) were obtained from DuPont NEN. myo-[^3H]Inositol (19.1 Ci/mmol) was obtained from Amersham Corp. 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 (Madison, WI). Oligonucleotides were synthesized by an Applied Biosystems 380B DNA synthesizer. Bombesin and CCK8 were obtained from Bachem (Torrance, CA). Trichloroacetic acid was obtained from J. T. Baker. Soybean trypsin inhibitor type I-S, bovine serum albumin, fraction V, and all non-specified reagents were obtained from Sigma. Dulbecco's modified Eagle's medium, fetal bovine serum, penicillin, streptomycin, G418, Lipofectin reagent, and amphotericin B were obtained from Life Technologies, Inc. Tissue culture plastic-ware (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, CCK(A), and m3 Muscarinic Receptors

The plasmid containing the 1.4-kilobase mouse bombesin receptor cDNA (31) was kindly provided by Dr. Battey. The 1.4-kilobase EcoRI DNA fragment was subcloned into pBluescript SK- plasmid to obtain the pBRR plasmid. The plasmid containing the full-length of rat CCK(A) receptor cDNA (32) was kindly provided by Dr. S. Wank and was subcloned into pGEM-2 (pGCCK) and the expression vector pTEJ-8 in a previous study(33) . The full-length clone of human m3 muscarinic receptor cDNA (34) was a kind gift of Dr. E. Peralta and was subcloned as a 2.1-kilobase EcoRI to BamHI DNA fragment into pBluescript SK-plasmid (pHm3).

Construction of Bombesin Carboxyl-terminal Chimeric Receptors

Polymerase chain reaction (PCR) methodology was used to construct chimeric receptor cDNAs in which the carboxyl terminus of the mouse BN receptor was replaced by the analogous portion of the human m3 muscarinic receptor (BMC) or rat CCK(A) receptor (BCC).

BMC

Steps in the construction of this chimeric receptor were as follows: (a) primers with sequences 5`-CCTTTG CTCTTTATCTGCTGAACAAAACATTCAGAACCAC-3` (BSMC) and 5`-ATTAACCCTCACTA AAG-3` (T3) were used in a 30-cycle PCR reaction containing the ScaI-linearized pHm3 plasmid. This reaction amplified a DNA fragment coding the carboxyl terminus of the m3 ACh receptor fused at one end to sequences coding for the seventh transmembrane segment of the BN receptor. (b) The amplified DNA fragment from (a) was used to prime a 10-cycle PCR reaction containing XbaI-linearized pBRR plasmid followed by addition of the flanking primers, SK (5`-TCTAGAACTAGTGGATC-3`) and KS (5`-CGAGGTCGACGGTATCG-3`), and 30 additional cycles. The final reaction product was cut with EcoRI and BamHI, the amplified chimeric bombesin/m3 muscarinic receptor DNA was cloned into pBluescript, and DNA sequences were verified by sequencing.

BCC

A similar PCR protocol was used to obtain the BCC receptor chimera in which the carboxyl terminus of the rat CCK(A) receptor was substituted for the corresponding portion of the BN receptor. (a) The two primers with sequences 5`-TTGCTCTTTATCTGCTGAGCAAACGCTTTCGCCTGGGCTT-3` (BCC) and 5`-ATTTAGGTG ACACTATA-3` (SP6) were used in a PCR reaction containing ScaI-linearized pGCCK. This reaction amplified a DNA fragment coding for the carboxyl terminus of the CCK(A) receptor fused at one end to sequences coding for the seventh transmembrane segment of the BN receptor. (b) The amplified DNA fragment from (a) was used to prime a 10-cycle PCR reaction containing XbaI-linearized pBRR plasmid followed by addition of the flanking primers, KS and SP6, and 30 additional cycles. The final reaction product was cut with HindIII and BamHI, the amplified chimeric bombesin/CCK(A) receptor DNA was cloned into pBluescript, and DNA sequences were verified by sequencing.

All receptors were subcloned into pTEJ-8 for expression in cell lines.

Transfection of Cell Lines

The CHO-K1 cells stably expressing CCK(A) receptors have been described previously(33) . The same Lipofectin-mediated transfection method was used to construct stable CHO-K1 cell lines expressing BN, BMC, or BCC chimeric receptors. Cells were grown to 30-40% confluence in 60-mm dishes and transfected with 1 µg of plasmid DNA using Lipofectin reagent for 6-8 h in serum-free medium. Cells were then returned to 5% fetal bovine serum, cultured 36 h, then 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^4]bombesin. Two or more clones were tested in all assays to control for clonal variation. CHO-K1 cells were routinely cultured in DMEM media supplemented with 5% fetal bovine serum in a humidified atmosphere of 5% CO(2).

Binding Assays

Binding was conducted to cells plated in 24-well dishes at 2-3 10^5 cells/ml the day before the binding assay. For cell binding assays [I-Tyr^4]bombesin or [I-BH]CCK(8) (10 pM) was added to HR buffer (5 mM NaCl, 4.7 mM KCl, 1 mM Na(2)PO(4), 1.28 mM CaCl(2), 10 mM HEPES, pH 7.4, with 0.5% bovine serum ablumin, and 0.1 mg/ml soybean trypsin inhibitor). Cells were incubated to equilibrium (overnight at 4 °C) then washed twice with ice-cold phosphate-buffered saline (PBS). The cells were then solubilized with 1 ml of 0.1 N NaOH and radioactivity quantified in a -counter. Nonspecific binding was determined in the presence of 100 nM bombesin or CCK(8). Protein contents were determined on samples after counting. Binding affinity and capacity were calculated using the Ligand analysis program(35) .

Internalization of Ligand and Receptor

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 determination of receptor internalization, we measured the loss of surface binding after agonist treatment. The analysis of agonist-induced loss of binding was conducted on cells plated in 24-well dishes at 2-3 10^5 cells/ml the day before the assay. Incubations were performed at 37 °C with 100 nM of bombesin (for BN and BN chimeric receptor) or CCK(8) (for CCK(A) receptor). After specified periods of time, the cells were washed with 1 ml of ice-cold PBS and incubated in 1 ml of a low pH ligand removal solution (90 mM NaCl, 50 mM sodium citrate, 0.2 mM Na(2)HPO(4), 0.1% bovine serum albumin, pH 5) (36) for 10 min at 4 °C. Cells were then washed with PBS at 4 °C. This procedure removed >90% of receptor-associated ligand and rebinding was not impaired by this treatment (data not shown). The acid wash solution utilized for analysis of ligand internalization was not suitable for this assay as the cells did not rebind labeled agonist after the acid wash treatment (data not shown). Cells were then incubated in binding buffer (HR buffer) with I-bombesin or I-CCK(8) (10 pM) overnight at 4 °C. Nonspecific binding was determined in the presence of 100 nM bombesin or CCK(8). Incubation was terminated by washing the cells with 1 ml of ice-cold PBS twice. The cells were then removed from the plate with 0.1 N NaOH, and cell-associated radioactivity was counted in a -counter.

Recovery from Agonist-induced Loss of Binding

For determination of receptor recycling, we measured the recovery from agonist-induced loss of surface binding. After the cells were incubated with 100 nM of bombesin or CCK(8) (for CCK(A) receptor cells) for 30 min at 37 °C, the receptor-associated ligand was removed by PBS and low pH ligand removal solution washes. Cells were then incubated with 1 ml of HR binding buffer at 37 °C. After an indicated time, the cells were cooled to 4 °C and incubated with I-bombesin or I-CCK(8) (10 pM) overnight. The cells were then washed twice with ice-cold PBS, solubilized with 1 ml of 0.1 N NaOH and radioactivity was detected in a -counter.

Measurement of Polyphosphoinositide Generation

For measurement of total inositol phosphate release, cells were cultured in 24-well multi-well dishes at 3 10^5 cells/ml for 24 h in the presence of 1.5 µCi of myo-[^3H]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 30 min. The 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 KHCO(3), and diluted with 2.5 ml of water. Analysis of total [^3H]inositol phosphates was carried out by the method described by Berridge et al.(37) .


RESULTS

Construction and Expression of Bombesin Carboxyl-terminal Chimeric Receptors

In order to determine the role of the carboxyl terminus in the internalization of bombesin receptors, two chimeric bombesin receptors bearing carboxyl termini from either the m3 muscarinic receptor or the CCK(A) receptor were constructed by PCR methodology. Schematic representations of the carboxyl termini of the bombesin receptor (BN), the chimeric bombesin/m3 muscarinic receptor (BMC), and the chimeric bombesin/CCK(A) receptor (BCC) are shown in Fig.1.


Figure 1: Schematic representations of the amino acid composition of BN, BCC, and BMC receptor carboxyl termini. A, diagram showing the location of potentially phosphorylated residues. B, diagram indicating conserved sequences (boxes), potential phosphorylation sites (bold), and consensus protein kinase C sequences (underlined) between BN, CCK(A), and m3 ACh receptors. Chimeric receptors were produced by a PCR based strategy. All constructs were confirmed by sequencing.



To obtain CHO cell lines with stable expression of chimeric bombesin receptors (BMC and BCC), 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-bombesin binding. Clones with approximately equal levels of binding were chosen and further characterized. Competition binding experiments with I-bombesin and increasing concentrations of unlabeled bombesin were performed at 4 °C in order to determine if replacing the carboxyl terminus of the BN receptor with the analogous portion of either m3 ACh or CCK(A) receptors altered the affinity of the receptor for agonist. Computer aided non-linear least-squares analysis of bombesin competition binding curves fit the behavior of a single class of binding sites for each cell line. The two BN chimeric receptors had affinities (K) for bombesin similar to the wild-type BN receptor (Table1). The receptor number (B(max)) of the selected clonal cell lines bearing chimeric receptors was the same (BMC) or somewhat lower (BCC) than that of the clone bearing the wild-type BN receptor (Table1).



The ability of the BN chimeric receptors to mediate bombesin-induced stimulation of polyphosphoinositide (PPI) metabolism was examined in the stable cell lines. Both BMC and BCC receptors induced pronounced PPI hydrolysis responses indicating that the substitution of foreign carboxyl termini did not greatly affect the ability of the receptors to activate G proteins. The response of BMC bearing cells was comparable in magnitude to that of the wild-type BN receptor bearing cells, whereas the BCC bearing cells had a somewhat lesser response (Fig.2). Activation of mutant receptors also elicited increases in intracellular Ca mobilization when analyzed using fura-2 as an indicator (data not shown).


Figure 2: Effects of bombesin on total PPI hydrolysis in CHO cells bearing bombesin receptors. Confluent cells were incubated with 1.5 µCi/ml myo-[2-^3H]inositol for 24 h after which they were exposed to the specified concentrations of bombesin for 30 min. Isolation of [^3H]inositol phosphates was performed as detailed under ``Experimental Procedures.'' Data are expressed as the fold increase over basal PPI release and represents the mean ± S.E. of three separate experiments, with each value measured in triplicate in each experiment.



Internalization of Ligand

Ligand internalization was assessed using an acid stripping method that has been previously described(38) . In this assay, cells were incubated with a tracer amount of labeled ligand for various amounts of time at 37 °C before being placed on ice to inhibit further internalization. Cells were then washed with cold buffer to remove unbound ligand and treated with low pH buffer to remove surface-bound ligand. The radioactivity in the low pH wash and in the cells were counted separately to determine the distribution of ligand in acid removable (surface) and acid-resistant (internalized) compartments. BN and CCK(A) receptor bearing cells rapidly internalized ligand (Fig.3), as expected. At equilibrium BN and CCK(A) receptor bearing cells had 80 ± 3 and 85 ± 7% of total specifically bound ligand distributed in an acid resistant compartment (Fig.4). BCC receptor bearing cells also rapidly internalized ligand (Fig.3). The extent of internalization in these cells (75 ± 3%, n = 3) was not statistically different than in the parental wild-type receptor bearing cells (Fig.4). In contrast, BMC receptor bearing cells internalized only half as much ligand (38 ± 1%, n = 3) (Fig.4). Reduced internalization was predicted for the BMC receptor based upon the lack of rapid internalization previously reported for the m3 ACh receptor in CHO cells(29) . Thus, these observations generally confirmed the working hypothesis.


Figure 3: Time course of specific binding and internalization of [I-Tyr^4]bombesin in CHO cells bearing wild-type BN and chimeric BCC and BMC receptors. Cells were incubated with 10 pM [I-Tyr^4]bombesin at 37 °C for the indicated times, then the cells were washed at 4 °C. Surface-bound (acid removable, open symbols) and intracellular (acid-resistant, closed symbols) 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 is given as percent of total tracer specifically bound and is from a representative of three to five separate experiments.




Figure 4: Ligand and receptor internalization by wild-type and chimeric receptors. For analysis of ligand internalization CHO cells bearing BN, BCC, BMC, and CCK(A) receptors were incubated with 10 pM [I-Tyr^4]bombesin or I-CCK(8) at 37 °C for 90 min then the cells were washed at 4 °C. Internalized (acid-resistant) radioactivity was determined. Nonspecific binding, defined as binding in the presence of 100 nM bombesin or CCK(8), was determined similarly, and values have been subtracted. Data shown are expressed as the percentage of total tracer specifically bound and represent the mean ± S.E. of three separate experiments. For analysis of receptor internalization, cells were treated with 100 nM agonist (bombesin or CCK(8)) for 30 min at 37 °C then placed on ice, washed with PBS and low pH buffer, and binding of I-bombesin or I-CCK(8) was conducted overnight at 4 °C. Receptor internalization is calculated as the reduction in the percentage of the surface binding in cells not treated with agonist and data represent means ± S.E. for three to seven experiments.



Internalization of Receptors

In order to directly assess the effects of agonist treatment on receptor internalization, we analyzed the ability of agonist to induce a loss of surface binding. Cells were treated with agonist (100 nM bombesin or CCK(8)) for different times at 37 °C, then receptor-associated agonist was removed with cold PBS and low pH washes. The extent of loss of surface radioligand binding was subsequently determined by comparing the level of I-ligand specifically bound after incubation overnight at 4 °C in treated and untreated cells. The loss of surface binding was rapid for all receptors with maximal effects observed after 30 min and no further decrease noted out to 9 h (Fig.5). However, the extent varied with 56 ± 8% (n = 4), 50 ± 7% (n = 5), 22 ± 8% (n = 4), and 82 ± 3% (n = 5) of surface binding lost in cells bearing BN, CCK(A), BMC, and BCC receptors, respectively (Fig.4). Thus, the loss of surface binding qualitatively paralleled the extent of ligand internalization. BMC receptors showed reduced levels of both parameters. However, internalization of ligand quantitatively exceeded the loss of surface receptors for all except the BCC receptors.


Figure 5: Agonist-induced receptor internalization in cells bearing BN, BCC, BMC, or CCK(A) receptors. Cells were treated with 100 nM agonist (bombesin or CCK(8)) for various times at 37 °C, then placed on ice, washed with PBS and low pH buffer, and binding of I-bombesin or I-CCK(8) was conducted overnight at 4°. The reduction in surface binding after agonist treatment is presented as a percentage of the binding in cells not treated with agonist, and data represent means ± S.E. for three to seven experiments.



Because a reduction in surface binding might involve alterations in receptor affinity, we conducted agonist binding competition studies before and after agonist treatment (Table1). No significant changes were noted in receptor affinities. Reductions of receptor numbers entirely accounted for the observed changes in total specific binding.

To investigate whether the loss of binding reflected internalization via clathrin-coated vesicles, the effects of high sucrose concentrations known to block this pathway (39) were examined. Sucrose treatment completely blocked internalization of wild-type BN receptors (92 ± 2% inhibition, n = 3). In contrast, BCC receptor internalization was only partially inhibited (72 ± 4% inhibition, n = 3).

Receptor Recycling

Wild-type BN (30) and CCK(A)(28) receptors have been reported to recycle after internalization. To determine if receptor recycling might account for the observed differences in the extent of ligand versus receptor internalization, we assessed receptor recycling by analyzing the recovery of surface binding after agonist-induced receptor internalization. Cells were incubated with 100 nM of bombesin or CCK(8) (for CCK(A) receptors) for 30 min at 37 °C. After removal of surface binding by cold PBS and low pH washes, the cells were allowed to recover at 37 °C for various times. Surface receptor levels were then determined by measuring the binding of radioligand overnight at 4 °C. Surface binding in BN, CCK(A), and BMC receptor-bearing cells rapidly recovered after agonist treatment and returned to nearly control levels within 90 min (Fig.6). This recovery was not prevented by cycloheximide indicating the lack of requirement for protein synthesis (data not shown). In contrast, BCC receptor-bearing cells did not recover indicating the inability of these receptors to recycle. Competition binding assays conducted on cultures of cells after the recovery period indicated that changes in receptor numbers accounted for the observed recovery of surface binding (Table1).


Figure 6: Recovery of surface binding to cells bearing BN, BCC, BMC, or CCK(A) receptors after agonist-induced receptor internalization. Cells treated with 100 nM agonist (bombesin or CCK(8)) for 30 min at 37 °C were washed with PBS and low pH buffer, then allowed to recover for various times at 37 °C before being placed on ice, and binding of I-bombesin or I-CCK(8) was conducted overnight at 4 °C. Binding to surface receptors is presented as a percentage of the binding to cells not treated with agonist, and data represent means ± S.E. for four to nine experiments.




DISCUSSION

The majority of G protein-coupled receptors internalize, and therefore internalization might be expected to involve a common mechanism. However, thus far, generalized domains and mechanisms for receptor internalization have been difficult to ascertain. Several complications have hindered the search for common structural features related to receptor internalization. These include: 1) receptor conformation can be indirectly influenced by structural manipulations; 2) internalization occurs via multiple mechanisms; 3) methodologies for investigating receptor internalization vary between receptor classes and are not strictly comparable; and 4) receptor internalization is cell type-dependent. The current study has investigated the role of the carboxyl terminus in bombesin receptor internalization and has attempted to address these issues.

In the current study, we utilized a chimeric approach. Previous studies have demonstrated that mutation or truncation of sequences in the third cytoplasmic loop(6, 24, 40, 41, 42) , the second cytoplasmic loop(6) , and the carboxyl terminus(15, 16, 17, 18, 20, 21, 29, 43) reduced internalization of specific receptors. However, interpretation of mutation studies must take into account the possibility of secondary conformational effects since receptors exist as three-dimensional entities. Thus, mutations of specific domains that are not themselves directly required for internalization may interfere with conformational changes in domains that are required for internalization. Secondary conformational effects are also possible in chimeric receptors. However, the advantage of the chimeric approach is that the predicted outcome is the retention or acquisition of function, rather than its loss, and it is unlikely that a function would be nonspecifically acquired. In the current study, substitution of the carboxyl terminus from the CCK(A) receptor produced a chimeric receptor with a fully intact ability to internalize. It has previously been shown that truncation of the bombesin receptor carboxyl terminus blocked ligand internalization (11) . Thus, the restoration of the ability to internalize achieved by addition of the CCK(A) carboxyl terminus strongly supports the hypothesis that this domain is an important determinant of this function. Furthermore, substitution with the carboxyl terminus from the m3 ACh receptor, which itself is not rapidly internalized in these cells(29) , produced a receptor with reduced internalization properties. Thus, the internalization characteristics of the chimeric receptors were specific, generally reflected the characteristics of the donor receptors, and strongly indicated a role of the carboxyl terminus in receptor internalization.

In the current study, internalization of wild-type bombesin receptors into CHO cells was completely blocked by hypertonic sucrose treatment, suggesting that internalization occurred exclusively via clathrin-coated vesicles, as has been previously reported in other cells(30) . However, internalization of the bombesin/CCK(A) chimera was not completely blocked by sucrose treatment, suggesting the participation of other non-clathrin mediated internalization pathways. Previously it was shown that the CCK(A) receptor is internalized into CHO cells via both coated and uncoated vesicles and that the latter pathway is not inhibited by hypertonic media(28) . Therefore, the carboxyl terminus of the CCK(A) receptor may have transferred the ability to interact with a clathrin-independent pathway to the chimeric receptor. Morphological investigations will be needed to verify this hypothesis. It is unclear how these morphologically distinct pathways correspond to known regulatory mechanisms such as sequestration and down-regulation. Both sequestration and down-regulation involve receptor internalization but appear to involve separate mechanims as they can be independently affected by structural manipulations(8, 9, 15, 18) . Moreover, it is unknown whether the mechanisms used for the internalization of peptide ligands are the same as those used for receptor sequestration or down-regulation, or whether they represent yet an additional internalization pathway. The existence of multiple internalization pathways interacting with separate structural domains may provide a partial explanation for the difficulty in generalizing the results obtained from studies on internalization of specific receptors.

In the current study both ligand and receptor internalization were independently measured. In the case of the bombesin/m3 ACh receptor, alterations in receptor internalization fit the predictions based on the characteristics of the carboxyl-terminal donor. Previously we found that m3 ACh receptor sequestration was not increased by agonist in CHO cells(29) . Accordingly, internalization of the bombesin/m3 ACh receptor chimera was nearly blocked. Ligand internalization characteristics are not known for m3 ACh receptors. Unfortunately, the methods and terminology used to describe internalization for peptide binding receptors are different compared to adrenergic and muscarinic receptors. ``Sequestration'' originally referred to the loss of binding of hydrophilic ligands while the binding of hydrophobic ligands remained unchanged in studies of adrenergic and muscarinic receptors (44, 45) . For peptide binding receptors what is sometimes referred to as sequestration is derived from measurements of ligand internalization using acid washing because hydrophobic ligands are generally not available. However, several important differences exist between ligand internalization and receptor sequestration. First, sequestration is concentration dependent and requires concentrations of agonist that activate second messenger pathways. In contrast, ligand internalization occurs with tracer concentrations of labeled ligand that are below the threshold of activating second messengers. Second, because tracer concentrations of ligand only occupy a small fraction of receptors, the internalization of a large proportion of tracer is accomplished by the internalization of only a small proportion of receptors. Third, the accumulation of intracellular ligand is influenced by a large number of factors other than the rate of receptor internalization, including the rates of ligand degradation and release, and receptor recycling. Thus, ligand internalization measured by acid washing does not accurately reflect the process of receptor sequestration. In contrast, agonist-induced loss of binding closely matches the classic sequestration assays as both follow receptors rather than ligand, occur in response to activating concentrations of agonist, and are not affected by ligand degradation. Therefore, comparison between agonist-induced loss of binding and sequestration is more appropriate than between acid washing and sequestration.

In the current study, receptor recycling was estimated from recovery of surface binding after agonist-induced loss. Recovery measured in this assay reflected recycling rather than de novo receptor synthesis since the time course was rapid and recovery was not blocked by protein synthesis inhibitors. Rapid recycling of wild-type bombesin and CCK(A) receptors was observed, as was previously reported (28, 30) . In contrast, the bombesin/CCK(A) chimeric receptor did not recycle. Interestingly, the proportion of internalized ligand exceeded the proportion of internalized receptors for bombesin and CCK(A) receptors while these parameters were equal in the non-recycling chimeric receptor. One likely explanation is that receptor recycling occurred more rapidly than the efflux of internalized ligand, such that ligand normally accumulated relative to receptors. Thus, for the recycling-deficient bombesin/CCK(A) chimeric receptor, ligand and receptor internalization occurred to the same extent. Also, for the internalization-deficient bombesin/m3 ACh chimeric receptor, receptor recycling provides an explanation for the unexpectedly high extent of ligand accumulation.

The lack of recycling of the chimeric bombesin/CCK(A) receptor observed in the current study was not predicted from the behavior of the wild-type CCK(A) receptor, which has been previously reported to recycle in CHO cells(28) . Thus, the lack of recycling of the chimeric receptor may represent a nonspecific interference with normal receptor trafficking mechanisms. Nevertheless, this observation indicates that receptor internalization and recycling mechanisms are separate and both are influenced by the receptor carboxyl terminus. Recent studies on the epidermal growth factor receptor have indicated that internalization and recycling are separately regulated and involve separate signals(46) . For the epidermal growth factor receptor it appears that the default pathway is recycling and that lysosomal targeting of receptors is due to specific and saturable endosomal retention. Also, epidermal growth factor receptors require occupancy for endosomal retention such that the nature of the ligand, especially its rate of dissociation, influence recycling(47) . Whether these characteristics are also important for G protein-linked receptors is unknown. The current study provides the first indication of a role for specific receptor domains in coupling to this important regulatory phenomenon. Further experiments will be necessary to define more precisely the structures and mechanisms involved in endosomal retention of bombesin and other G protein-linked receptors.

Receptor internalization is cell type-specific. For example, muscarinic cholinergic (mACh) receptors are down-regulated but not sequestered in CHO cells (29) and JEG-3 cells (43) and are sequestered but not down-regulated in HEK293 cells(42) . Cell-specific differences in internalization have also been reported for luteinizing hormone/chorionic gonadotropin receptors(17) . Thus, the fact that previous studies on receptor internalization have been conducted in a variety of cell models increases the probability that different pathways of internalization may have been investigated. In the current study a comparison was made between the internalization of chimeric receptors and wild-type receptors in the same cell model, in order to avoid these complications.

Many previous studies of receptor internalization have focused on the role of potential sites of phosphorylation. Recently, evidence has been obtained for G protein-coupled receptor kinase (GRK2) involvement in internalization of mACh receptors(48) . In the current study the number of potential carboxyl-terminal phosphorylation sites varied from 16 for Bn (8 Ser, 7 Thr, 1 Tyr) to 10 for CCK(A) (5 Ser, 3 Thr, 2 Tyr), and five for m3 ACh (1 Ser, 3 Thy, 1 Tyr). While the m3 ACh carboxyl terminus possessed the least potential phosphorylation sites and was the least internalized, no definitive conclusions can be drawn from these observations. Because phosphorylation of tyrosine residues is important in the internalization of growth factor receptors, tyrosines have been targeted as potentially important in signaling internalization(12, 15, 43) . In the current study all receptor carboxyl termini possessed at least 1 tyrosine and no obvious pattern or relationship between tyrosines and internalization or recycling could be discerned. Other investigations have scrutinized the potential role of protein kinase C in agonist-induced receptor internalization. Specifically, an involvement of protein kinase C was suggested in bombesin receptor internalization as elimination of a carboxyl-terminal consensus site reduced (11) and preincubation of cells with activators of protein kinase C increased bombesin internalization(7) . In the current study all receptor carboxyl termini possessed potential protein kinase C phosphorylation sites and no obvious relationship between numbers or location of potential sites with receptor trafficking was apparent.

In summary, we have shown that substitution of the carboxyl terminus of the bombesin receptor with termini from either CCK(A) or m3 ACh receptors had profound effects on receptor internalization and recycling. In general the chimeric receptors acquired the internalization characteristics of the carboxyl-terminal donor receptors. The CCK(A) receptor carboxyl terminus allowed rapid ligand internalization, and it may have conveyed to the bombesin receptor chimera the ability to internalize via a non-clathrin-mediated pathway. The bombesin/m3 ACh chimeric receptor internalized poorly. Additionally the bombesin/CCK(A) receptor was deficient in its ability to recycle, demonstrating that internalization and recycling pathways are separable. In conclusion, these results indicate that separate carboxyl-terminal domains determine receptor interactions with distinct cellular trafficking mechanisms.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK41225 and by Michigan Gastrointestinal Peptide Digestive Disease Center Grant DK34933. 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: Dept. of Physiology, Box 0622, The University of Michigan, 1150 W. Medical Center Dr., Ann Arbor, MI 48109. Tel.: 313-763-2539; Fax: 313-936-8813.

^1
The abbreviations used are: m3 ACh, m3 muscarinic cholinergic receptor; CCK cholecystokinin; CHO, Chinese hamster ovary; PCR, polymerase chain reaction; PBS, phosphate-buffered saline; PPI, polyphosphoinositide; BN, bombesin.


ACKNOWLEDGEMENTS

We thank Dr. J. A. Williams for helpful discussions throughout this work.


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