# Ligand-induced Internalization of Cholecystokinin Receptors

Markus Pohl, Sandrine Silvente-Poirot, Joseph Pisegna, Nadya Tarasova, Stephen Wank

► To cite this version:

Markus Pohl, Sandrine Silvente-Poirot, Joseph Pisegna, Nadya Tarasova, Stephen Wank. Ligand-induced Internalization of Cholecystokinin Receptors. Journal of Biological Chemistry, 1997, 272 (29), pp.18179-18184. 10.1074/jbc.272.29.18179. inserm-04974735

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# Ligand-induced Internalization of Cholecystokinin Receptors

DEMONSTRATION OF THE IMPORTANCE OF THE CARBOXYL TERMINUS FOR LIGAND-INDUCED INTERNALIZATION OF THE RAT CHOLECYSTOKININ TYPE B RECEPTOR BUT NOT THE TYPE A RECEPTOR*

(Received for publication, January 22, 1997, and in revised form, May 13, 1997)

Markus Pohl‡, Sandrine Silvente-Poirot‡§, Joseph R. Pisegna‡¶, Nadya I. Tarasova||**, and Stephen A. Wank‡‡

From the ‡Digestive Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892 and ¶Molecular Aspects of Drug Design Section, ABL-Basic Research Program, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702

Internalization of a variety of different heptahelical G protein-coupled receptors has been shown to be influenced by a number of different structural determinants of the receptors, including the carboxyl terminus. To investigate the role of the carboxyl terminus of cholecystokinin (CCK) receptors in receptor internalization, the rat wild type (WT) CCK-A receptor (WT CCKAR) and the rat WT CCK-B receptor (WT CCKBR) were truncated after amino acid residue 399 (CCKAR Tr399) and 408 (CCKBR Tr408), thereby deleting the carboxyl-terminal 45 and 44 residues, respectively. All WT and mutant CCK receptors were stably expressed in NIH/3T3 cells. Internalization of the CCKAR Tr399 was not significantly different from the WT CCKAR. In contrast, internalization of the CCKBR Tr408 was decreased to 26% compared with the WT CCKBR internalization of 92%. The mutation of all 10 serine and threonine residues (as potential phosphorylation sites) in the carboxyl terminus of the CCKBR to alanines (mutant CCKBR ΔS/T) could account for the majority of this effect (39% internalization). All mutant receptors displayed similar ligand binding characteristics, G protein coupling, and signal transduction as their respective WT receptors, indicating that the carboxyl termini are not necessary for these processes. Thus, internalization of the CCKBR, unlike that of the CCKAR, depends on the carboxyl terminus of the receptor. These results suggest that, despite the high degree of homology between CCKAR and CCKBR, the structural determinants that mediate the interaction with the endocytic pathway reside in different regions of the receptors.

Many G protein-coupled receptors (GPCRs)¹ undergo internalization (or sequestration) to intracellular sites within minutes after agonist exposure. Internalized receptors are either degraded in lysosomes (1) or undergo resensitization and reinsertion into the plasma membrane (2–4). The relationship between internalization and desensitization is unsettled for most GPCRs and may vary between different receptors and different cell systems. For some receptors, receptor internalization seems to be a major factor in the acute desensitization process that dampens the receptor-mediated cellular response to agonist stimulation (5, 6). For other receptors, receptor internalization makes very little (7) or no (8, 9) contribution to receptor desensitization, arguing that these two processes are unrelated.

The search for the structural basis of GPCRs that couples to the endocytic machinery has revealed a variety of different structural determinants for different receptors that influence receptor internalization. Although for many receptors the carboxyl terminus has been shown to influence receptor internalization (5, 8, 10–18), a variety of other structural determinants including areas within the second (19) and third (20) intracellular loop and a conserved NPXnY motif near the seventh transmembrane domain (21) have been found to be critical for internalization for different GPCRs. However, a universal structural element that mediates internalization has not been identified and may not exist. The signaling mechanism that allows the agonist-occupied receptor to couple to the endocytic machinery and to internalize is unknown for the majority of the GPCRs. For some receptors, however, receptor phosphorylation has been suggested to be implicated in receptor internalization because the mutation of potential serine and threonine phosphorylation sites caused the loss of receptor internalization (5, 10, 15, 17). Only recently, functional studies on the β₂-adrenergic receptor have demonstrated a mechanism in which phosphorylation of the carboxyl terminus of the receptor leads to receptor internalization by enhancing the affinity for β-arrestin, an adaptor molecule between the receptor and clathrin-coated vesicles (22, 23).

Cholecystokinin (CCK) receptors belong to the superfamily of GPCRs (24). There are two types of CCK receptors, which have nearly 50% amino acid homology and can be distinguished on the basis of their affinities to the agonists cholecystokinin octapeptide (CCK-8) and gastrin. The cholecystokinin type A receptor (CCKAR) binds CCK-8 with much greater affinity than gastrin, whereas the cholecystokinin type B receptor

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Present address: INSERM U 151, CHU Rangueil, L3, 31054 Toulouse Cedex, France.

¶ Present address: Center for Ulcer Research and Education, VA/UCLA, Digestive Diseases Center, West Los Angeles Veterans Administration Medical Center, Los Angeles, CA 90073.

** Sponsored by the NCI, DHHS, National Institutes of Health.

‡‡ To whom correspondence should be addressed: Bldg. 10, Room 9C-103, National Institutes of Health, Bethesda, MD 20892-1804. Tel.: 301-496-4201; Fax: 301-402-0600.

¹ The abbreviations used are: GPCR, G protein-coupled receptor; CCK, cholecystokinin; CCK-8, CCK octapeptide; CCKAR, cholecystokinin type A receptor; CCKBR, cholecystokinin type B receptor; DMEM, Dulbecco's modified Eagle's medium; ¹²⁵I-BH-CCK-8, ¹²⁵I-Bolton-Huntler-labeled CCK-8; BSA, bovine serum albumin; WT, wild type; CCKAR

Tr399, CCKAR truncated after amino acid residue 399; CCKBR Tr408, CCKBR truncated after amino acid residue 408; PBS, phosphate-buffered saline; RG-CCK-8, rhodamine green-conjugated CCK-8; RB-conA, rhodamine B-conjugated concanavalin A.
(CCKBR) binds CCK-8 and gastrin nearly equally well. The signal transduction mechanisms are similar for both the CCKAR and the CCKBR. Both receptors couple to a pertussis toxin-insensitive G protein and elicit the production of inositol phosphates and diacylglycerol (24).

Stimulation of the CCKAR promotes enzyme secretion in the pancreas, smooth muscle contraction in the gallbladder, and regulation of satiety in selective areas of the central and peripheral nervous systems. The CCKBR is present in the stomach where it mediates acid secretion from parietal cells and is also found throughout the central nervous system where it regulates dopamine release and anxiety (24).

Both CCKARs and CCKBRs have been shown to undergo ligand-induced internalization in pancreatic acini (25) or transfected NIH 3T3 cells (26), respectively. They have also been shown to undergo desensitization in their native cells (27–29), but the impact of receptor internalization in this process is unclear. The CCKAR is phosphorylated predominantly (>95%) in the third intracellular loop within seconds after agonist stimulation (30), in part by protein kinase C and in part by another heparin-inhibitable enzyme, probably a G protein receptor kinase (31). The pattern of ligand-induced phosphorylation of the CCKBR has not been previously demonstrated.

In this study, we investigated the structural determinants that mediate internalization of CCK receptors. Deletion of the 44 carboxyl-terminal amino acid residues profoundly decreased internalization of the CCKBR. Interestingly, a deletion of the corresponding amino acids in the CCKAR did not affect internalization. The mutation of all serine and threonine residues in the carboxyl terminus to alanines decreased internalization of the CCKBR to almost the same degree as truncation of the carboxyl terminus.

## EXPERIMENTAL PROCEDURES

**Materials**—NIH/3T3 cells were obtained from the American Type Culture Collection (Rockville, MD). Dulbecco's modified Eagle's medium (DMEM), calf serum, trypsin-EDTA, and aminoglycoside G418 were from Life Technologies, Inc. (Gaithersburg, MD). Sulfated cholecystokinin octapeptide (CCK-8) was purchased from Research Plus, Inc. (Bayonne, NJ). $^{125}$I-Bolton-Hunter-labeled CCK-8 ($^{125}$I-BH-CCK-8) (2200 Ci/mmol) was obtained from NEN Life Science Products. $^{45}$Ca$^{2+}$ (10–40 mCi/mg Ca$^{2+}$) and [γ-³²P]ATP, triethylammonium salt (6000 Ci/mmol) were from Amersham Life Science, Inc. Potassium thiocyanate, sodium fluoride, aluminum chloride, and EGTA were purchased from Sigma. Bovine serum albumin (BSA) was from ICN Biomedicals Inc. (Aurora, OH). Hydrofluor was from National Diagnostics, Inc. (Atlanta, GA).

**Mutant CCK Receptor Construction**—The wild type (WT) rat CCKAR and CCKBR were subcloned into the vector pCDL/SRα containing a neomycin resistance gene (32). Truncated receptor mutants were created by introducing a stop codon using the polymerase chain reaction. The WT CCKAR was truncated after amino acid residue 399 (CCKAR Tr399), and the WT CCKBR was truncated after amino acid residue 408 (CCKBR Tr408) (see Fig. 1). All serine and threonine residues in the carboxyl terminus were mutated to alanines by site-directed mutagenesis (Muta-Gene® phagemid in vitro mutagenesis kit, Bio-Rad) for both the CCKAR and the CCKBR and are referred to as CCKAR ΔS/T and CCKBR ΔS/T (see Fig. 1). All mutations were confirmed by DNA sequence analysis using the dsDNA cycle sequencing system (Life Technologies, Inc.).

**Stable Expression of CCK Receptors in NIH/3T3 Cells**—All WT and mutant receptors were stably transfected into NIH/3T3 cells by electroporation (500 microfarads, 0.25 kV, Gene Pulser®, Bio-Rad) of $2 \times 10^7$ cells in a volume of 0.25 ml with 20 µg of the linearized recombinant vector containing the respective WT or mutant receptor cDNA in the presence of 500 µg/ml salmon sperm DNA as a carrier. Cell clones stably expressing the receptors were then selected for G418 resistance (250 µg of G418/ml) and $^{125}$I-BH-CCK-8 binding. Cells were maintained in DMEM, 10% calf serum/G418 (250 µg/ml) at 37 °C in a 6% CO$_2$ atmosphere.

**Radioligand Binding Displacement Studies**—Transfected NIH/3T3 cells were plated in 24-well tissue culture plates and assayed the

following day for radioligand binding. For binding displacement studies, cells were incubated for 90 min with 50 pM $^{125}$I-BH-CCK-8 in the absence or presence of increasing concentrations of unlabeled CCK-8 in DMEM, 0.1% BSA at 37 °C (0.5 ml final volume). Nonspecific binding was defined as total binding in the presence of 1 µM unlabeled CCK-8 and was always <15% that of total binding. After termination of the binding reaction by washing the cells two times with phosphate-buffered saline (PBS; 8.1 mM NaH$_2$PO$_4$, 1.5 mM KH$_2$PO$_4$, 138 mM NaCl, 2.7 mM KCl, pH 7.4) containing 4% BSA at room temperature, cells were solubilized with 1 ml of 1% SDS, and radioactivity was detected in a Packard Autogamma counter (Packard Instrument Co). The half-maximal inhibition of binding (IC$_{50}$) was determined with the nonlinear curve fitting computer program ALLFIT (33).

**Radioligand Stripping Studies**—Cells were prepared as mentioned above and then incubated with 50 pM $^{125}$I-BH-CCK-8 at 37 °C for various time intervals between 5 and 120 min. At the times indicated, cells were rapidly washed two times with PBS, 4% BSA and subjected to 0.5 M KSCN for 10 min at room temperature. Cell-associated radioactivity was measured after solubilizing the cells with 1 ml of 1% SDS. In all cases, parallel incubations were performed in the presence of 1 µM unlabeled CCK-8 to determine nonsaturation binding at each time point. Internalized receptor was defined as cell-associated radioactivity after stripping and expressed as the percent of the total binding.

**Laser Scanning Confocal Microscopy**—NIH/3T3 cells expressing WT or mutant CCK receptors were plated in Nunc cover glass chamber slides and assayed the following day. For internalization studies, cells were treated with Rhodamine Green-conjugated CCK-8 (RG-CCK-8) for 15 min at 37 °C, labeled with the cell surface marker Rhodamine B-conjugated concanavalin A (RB-conA) for 2 min at 4 °C, and observed under a laser scanning confocal microscope (Zeiss inverted LSM 410). RG-CCK-8 was excited using a 488-nm argon/krypton laser, and emitted fluorescence was detected through a 515–540-nm band-pass filter. Fluorescence of RB-conA was excited with a 568-nm helium/neon laser, and fluorescence was detected with a 590-nm band-pass filter. All observations were performed using a pinhole of 40, a × 63 oil immersion lens, and an electronic zoom of 3.2 to yield a 2016-fold magnification.

**Binding Studies in the Presence of Aluminum Fluoride**—NIH/3T3 cells expressing WT or mutant CCK receptors were scraped out of the tissue culture flasks and resuspended at a concentration of $10^6$ cells/ml in DMEM, 0.1% BSA. Following a 30-min preincubation of the cells with 30 mM NaF and 10 µM AlCl$_3$ at 37 °C, $^{125}$I-BH-CCK-8 was added to a concentration of 50 pM in a final volume of 0.5 ml and allowed to bind for 90 min at 37 °C. Samples of 0.2 ml of the cell suspension were transferred into 1 ml of PBS, 4% BSA. Cell-associated $^{125}$I-BH-CCK-8 was separated from free radioligand by centrifugation (1 min, 13,000 × g) and subsequently washed and re centrifuged two times with 1 ml of PBS, 4% BSA. Gamma radioactivity associated with the pelleted cells was determined in a Packard Autogamma counter. Control experiments were performed as described above either using 30 mM NaCl in place of NaF or in the absence of NaF and AlCl$_3$. For all experiments, nonsatutable binding was determined in the presence of 1 µM CCK-8. Results are expressed as percent of total binding of transfected cells in the absence of NaF and AlCl$_3$.

**Measurement of $^{45}$Ca$^{2+}$ Efflux**—Similar to a previously described method (34, 35), cells were plated in 24-well tissue culture plates and loaded with $^{45}$Ca$^{2+}$ for 12–16 h with a concentration of 8 µCi/ml. After washing the cells seven times with DMEM containing 3 mM EGTA at room temperature, cells were stimulated with increasing concentrations of unlabeled CCK-8 at 37 °C in a final volume of 0.5 ml. After a 2-min stimulation, 0.1 ml of the supernatant was sampled for $^{45}$Ca$^{2+}$ efflux into Hydrofluor, and radioactivity was measured using a Packard 2500 TR liquid scintillation analyzer. The half-maximal stimulation (EC$_{50}$) was calculated with the ALLFIT program (33).

## RESULTS

To investigate the role of the carboxyl terminus in the internalization of cholecystokinin receptors, we initially truncated the CCKAR after amino acid residue 399 (mutant CCKAR Tr399) and the CCKBR after residue 408 (mutant CCKBR Tr408), thereby deleting the last 45 and 44 residues of the carboxyl terminus, respectively (Fig. 1). These truncation sites were chosen to eliminate all 8 potential phosphorylation sites in the carboxyl terminus of the CCKAR and all 10 potential phosphorylation sites in the carboxyl terminus of the CCKBR. To examine a possible specific involvement of potential phosphorylation sites, all serine and threonine residues in the car-
<figure><img src="image_2.png" /></figure>

FIG. 1. Amino acid models of the rat CCKAR and the rat CCKBR illustrating the receptor mutations. The models show the predicted amino acid sequence of the WT CCKAR (upper panel) and the WT CCKBR (lower panel). The mutant CCKAR Tr399 was constructed by truncation of the carboxyl terminus after amino acid residue 399, as indicated by the *black bar*. The mutant CCKAR ΔS/T was constructed by mutation of all eight serine and threonine residues in the carboxyl terminus (indicated by the *enlarged and embolded circles*) to alanines. In parallel, the mutant CCKBR Tr408 was constructed by truncation of the carboxyl terminus after amino acid residue 408 as indicated by the *black bar*. The mutant CCKBR ΔS/T was constructed by mutation of all 10 serine and threonine residues in the carboxyl terminus (indicated by the *enlarged and embolded circles*) to alanines.

boxyl terminus were mutated to alanines, producing the mu-
tants CCKAR ΔS/T and CCKBR ΔS/T (Fig. 1).

NIH/3T3 cells were stably transfected with either WT or
mutant CCK receptor subcloned into the pCDL/SRα vector
containing the neomycin resistance gene. At least five G418-
resistant clones were assayed for ¹²⁵I-BH-CCK-8 radioligand
binding for each WT and mutant transfection. Cell clones with
similar receptor densities (receptors/cell = 87 × 10³ for the WT
CCKAR, 291 × 10³ for the CCKAR ΔS/T, 69 × 10³ for the
CCKAR Tr399, 66 × 10³ for the WT CCKBR, 86 × 10³ for the
CCKBR ΔS/T, and 26 × 10³ for the CCKBR Tr408) were chosen
for further studies. ¹²⁵I-BH-CCK-8 binding competition curves
demonstrated specific high affinity binding of CCK-8 for all WT
and mutant CCKARs and CCKBRs (Fig. 2). All receptors had a
similar affinity for CCK-8 with IC₅₀s between 0.37 ± 0.11 and
0.7 ± 0.02 nM for CCKAR constructs (Fig. 2, *left panel*) and
0.42 ± 0.05 and 0.53 ± 0.05 nM for CCKBR constructs (Fig. 2,
*right panel*).

To investigate the effect of the deletion of the carboxyl ter-
minus or the mutation of the serine and threonine residues to
alanines on receptor-mediated ligand internalization, radioli-
gand stripping experiments were performed. NIH/3T3 cells
expressing the WT CCKAR or WT CCKBR internalized radio-
labeled CCK-8 rapidly and to a high degree (Fig. 3). For both
WT receptors, >50% of the radioligand was internalized before
15 min. The internalization process reached a plateau after 60
min, with a maximal internalization of 89 ± 2.7% (WT CCKAR)
and 92 ± 3.3% (WT CCKBR) of the radioligand at 120 min (Fig.

<figure><img src="image_3.png" /></figure>

FIG. 2. Displacement of ¹²⁵I-BH-CCK-8 binding to NIH/3T3 cells stably expressing WT or mutant CCK receptors. NIH/3T3 cells stably expressing the indicated WT or mutant CCKAR (left panel) or CCKBR (right panel) were incubated with ¹²⁵I-BH-CCK-8 (50 pM) either alone or with the indicated concentrations of unlabeled CCK-8. Data are presented as percent of saturable binding in the absence of unla- beled ligand. Each value represents the mean ± S.E. of at least three experiments performed in duplicate.

<figure><img src="image_4.png" /></figure>

FIG. 3. Internalization of ¹²⁵I-BH-CCK-8 by NIH/3T3 cells sta-
bly expressing WT or mutant CCK receptors. NIH/3T3 cells ex-
pressing the indicated WT or mutant CCKAR (left panel) or CCKBR
(right panel) were incubated with ¹²⁵I-BH-CCK-8 (50 pM). At the indi-
cated time points, cells were subjected to a 0.5 M KSCN solution.
Internalized radioligand is defined as the ¹²⁵I-BH-CCK-8 that could not
be stripped by KSCN and is expressed as percent of the total saturably
bound ¹²⁵I-BH-CCK-8 in control cells processed in parallel without
KSCN exposure. Each value represents the mean ± S.E. of at least
three experiments performed in duplicate.

3). Cells expressing the CCKAR Tr399 as well as cells express-
ing the CCKAR ΔS/T displayed nearly the same degree and
kinetics of internalization as the WT CCKAR (Fig. 3, *left
panel*). In contrast, the internalization of ¹²⁵I-BH-CCK-8 was
profoundly attenuated in cells transfected with the mutant
CCKBRs. At 15 min, 89% of the radioligand was stripped with
KSCN for both the truncated and the ΔS/T CCKBR mutants,
equivalent to 11% internalization (Fig. 3, *right panel*). After a
120-min incubation, only 26 ± 0.8 and 39 ± 5.3% of the radio-
ligand was internalized in cells transfected with the CCKBR
Tr408 and CCKBR ΔS/T, respectively (Fig. 3, *right panel*).
There was a small, however significant, difference in internal-
ization between CCKBR Tr408 and CCKBR ΔS/T for the later
time points (90 and 120 min). To exclude that differences in
internalization were due to clonal variation, KSCN-stripping
experiments were also performed on a population of transiently
transfected COS-7 cells with similar results. To further dem-
onstrate that the inability to strip radioligand at 37 °C was
indeed due to its internalization, we performed KSCN-strip-
ping studies after 4 °C binding, a temperature at which inter-
<figure><img src="image_5.png" /><figcaption>FIG. 4. Internalization of fluorescent Rhodamine Green-conjugated CCK-8 by NIH/3T3 cells stably expressing WT or mutant CCK receptors assessed by confocal laser scanning microscopy. Shown are representative confocal images of NIH/3T3 cells stably expressing the WT CCKAR (A), the CCKAR $\Delta$S/T (B), the WT CCKBR (C), or the CCKBR $\Delta$S/T (D) after a 15-min incubation with Rhodamine Green-conjugated CCK-8. Cell surfaces labeled with Rhodamine B-conjugated concanavalin A are shown in red. Internalized Rhodamine Green-conjugated CCK-8 is shown in green. Colocalization of concanavalin A and Rhodamine Green-conjugated CCK-8 on the cell surface appears as yellow.</figcaption></figure>

nalization does not occur. As expected, even after 180 min of binding with cells stably expressing either of the six receptor constructs with 50 pM $^{125}$I-BH-CCK-8 at 4 °C, 85–94% of the radioligand could still be stripped by KSCN (data not shown).

To confirm the effects of the mutations on internalization and to assure that KSCN-resistant $^{125}$I-BH-CCK-8 represented internalized receptor, laser scanning confocal microscopy studies were performed. Cells expressing the WT CCKAR or WT CCKBR internalized the majority of the ligand RG-CCK-8 within 15 min away from the cell surface (marked in red with RB-conA) to the interior of the cell, as evidenced by the green intracellular fluorescence (Fig. 4, A and C). Consistent with the radioligand stripping studies, cells transfected with CCKAR Tr399 and CCKAR $\Delta$S/T (Fig. 4B) showed a pattern similar to the WT CCKAR. However, cells expressing CCKBR Tr408 or CCKBR $\Delta$S/T (Fig. 4D) showed colocalization of RB-conA and RG-CCK-8 on the cell surface, which appears as a persistent yellow surface pattern on the overlay of green and red fluorescent images, suggesting that few of the receptors were internalized. Confocal microscopy studies on a population of transiently transfected COS-7 cells with either receptor showed similar results (data not shown), again excluding that differences between WT and mutant receptors were due to clonal variation.

For some receptors, intact G protein coupling may be necessary for the internalization process. To determine whether the decreased internalization of the CCKBR Tr408 and the CCKBR $\Delta$S/T was due to an inability to couple to G proteins, we measured the effect of aluminum fluoride on ligand binding. The presence of 30 mM NaF plus 10 $\mu$M $AlCl_3$, concentrations previously shown to effectively activate G proteins in intact cells (36, 37) resulting in a decreased ligand affinity of the subse-

<figure><img src="image_6.png" /><figcaption>FIG. 5. Effect of aluminum fluoride on $^{125}$I-BH-CCK-8 binding to NIH/3T3 cells stably expressing WT or mutant CCK receptors. NIH/3T3 cells stably expressing WT or mutant CCKARs (left panel) or CCKBRs (right panel) were incubated with $^{125}$I-BH-CCK-8 (50 pM) in the presence of either 30 mM NaCl plus 10 $\mu$M $AlCl_3$ (shaded bars) or 30 mM NaF plus 10 $\mu$M $AlCl_3$ (open bars). Data are presented as percent of total saturable binding in medium without additions. Each value represents the mean ± S.E. of at least three experiments performed in duplicate.</figcaption></figure>

quently uncoupled receptor, decreased the total binding of $^{125}$I-BH-CCK-8 for all cells transfected with either of the six receptor constructs to <8% compared with the control with no NaF or $AlCl_3$ in the incubation medium (Fig. 5). Control experiments using 30 mM NaCl plus 10 $\mu$M $AlCl_3$ showed a minor effect on total binding (81–99% compared with the control with no additions) (Fig. 5), confirming that the effect of aluminum fluoride was specific and not due to a change in ionic strength or osmolarity.

To exclude that differences in internalization between mutant and WT receptors were due to an altered signal transduction, we determined whether the mutations of the receptors had an effect on the signal transduction cascade by examining $^{45}$Ca$^{2+}$ efflux as a biologic response to CCK-8 stimulation. CCK-8-stimulated $^{45}$Ca$^{2+}$ efflux was maximal at 2 min, in agreement with a previous study (34), and resulted in a 3- to 5-fold increase over basal for all WT and mutant receptors. CCK-8 stimulated $^{45}$Ca$^{2+}$ efflux through WT CCKAR, CCKAR Tr399, and CCKAR $\Delta$S/T in a dose-dependant manner, with nearly identical EC$_{50}$s (between 1.14 ± 0.06 and 1.5 ± 0.33 nM) (Fig. 6, left panel). Similarly, no significant difference was observed between WT CCKBR, CCKBR Tr408, and CCKBR $\Delta$S/T (EC$_{50}$s between 0.85 ± 0.1 and 1.65 ± 0.2 nM) (Fig. 6, right panel).

DISCUSSION

The present study examines the role of the carboxyl terminus of cholecystokinin receptors in regulating ligand-stimulated internalization. CCKAR and CCKBR were mutated by either truncation of the carboxyl terminus or by replacement of serine and threonine residues within the carboxyl terminus with ala- nines. WT and mutant receptors stably expressed in NIH/3T3 cells displayed similar ligand binding, G protein coupling, and signal transduction, suggesting that the carboxyl termini are not necessary for these processes. Receptor internalization as- sessed by KSCN stripping of the radioligand and confocal mi- croscopy, was reduced to 26% in the CCKBR truncated in the carboxyl terminus after amino acid residue 408 compared with the WT CCKBR internalization of 92%. The mutation of all 10 serine and threonine residues in the carboxyl terminus of the CCKBR could account for the majority of this effect. In con- trast, similar mutations of the CCKAR involving either trun- cation of the carboxyl terminus after amino acid residue 399 or
<figure><img src="image_7.png" /><figcaption>FIG. 6. Ability of CCK-8 to stimulate <sup>45</sup>Ca<sup>2+</sup> efflux in NIH/3T3 cells stably expressing WT or mutant CCK receptors. NIH/3T3 cells stably expressing WT or mutant CCKARs (left panel) or CCKBRs (right panel) were stimulated with the indicated concentrations of CCK-8 following a 12-16-h preincubation with <sup>45</sup>Ca<sup>2+</sup> (8 µCi/ml). Data are presented as percent of maximal stimulation with 1 µM CCK-8. Each value represents the mean ± S.E. of at least three experiments performed in duplicate.</figcaption></figure>

mutation of all eight serine and threonine residues in the carboxyl terminus to alanines failed to affect internalization.

The carboxyl terminus has been shown to influence internal-
ization in a number of G protein-coupled receptors (5, 8, 10–
18). However, these truncations have been shown to have un-
predictable effects on internalization. To investigate the role of
the carboxyl terminus of CCK receptors for internalization, we
truncated the carboxyl terminus including all potential phos-
phorylation sites for both receptors. Truncation of the CCKAR
after amino acid residue 399 failed to affect its internalization,
whereas truncation of the CCKBR after amino acid residue 408
profoundly attenuated agonist-induced receptor internaliza-
tion. This suggests that for the CCKBR, unlike for the CCKAR,
structural determinants within the carboxyl terminus are re-
quired for internalization. However, a conformational change
induced by the truncation of the receptor that alters the acces-
sibility or affinity of the endocytic machinery to another un-
known internalization motif cannot be ruled out. A permissive
effect of the carboxyl terminus for internalization, like for the
CCKBR, has been shown for a number of other GPCRs, such as
the angiotensin II (8), the yeast α-pheromone (14), the thyro-
tropin-releasing hormone (13), and the gastrin-releasing pep-
tide receptors (15). All of these truncations involved the re-
moval of the carboxyl terminus and caused an attenuation of
receptor internalization without affecting G protein coupling.
On the other hand, an inhibiting influence of the carboxyl
terminus on internalization was discovered for the luteinizing
hormone receptor and the avian β-adrenergic receptor. Re-
moval of the last 43 amino acid residues of the carboxyl termi-
nus of the luteinizing hormone receptor causes the receptor to
internalize faster compared with the full-length receptor (12).
Carboxyl-terminal truncation enables the avian β-adrenergic
receptor to internalize, whereas the WT receptor is not able to
internalize (11). Similar to the CCKAR, truncation of the car-
boxyl terminus of the Hm1 muscarinic cholinergic receptor,
which eliminated all potential phosphorylation sites, did not
change receptor internalization. Instead, a serine- and threo-
nine-rich region in the third cytoplasmic loop was found to be
critical (20).

A variety of structural determinants within the carboxyl
terminus of various receptors have been found to influence
internalization. To determine whether potential phosphoryla-
tion sites within the carboxyl terminus of the CCKBR are
involved in receptor internalization, we mutated all serine and
threonine residues to alanines. This resulted in a reduced

internalization to nearly the same degree as the deletion of the
carboxyl terminus, raising the possibility of a specific role of
these residues, perhaps as phosphorylation sites, in the signal-
ing for internalization. The mutation of serine and threonine
residues in the carboxyl terminus as potential phosphorylation
sites has been shown to attenuate receptor internalization for a
number of other GPCRs, such as the human β₂-adrenergic (10),
the gastrin-releasing peptide (15), and the m₃-muscarinic ace-
tylcholine receptors (5). For the human β₂-adrenergic receptor,
the best characterized receptor for internalization, β-arrestin,
acting as an adaptor molecule between the receptor and clath-
rin-coated pits (23), binds to the receptor with much higher
affinity when it is phosphorylated in the carboxyl terminus
(22). However, an additional domain, a NPXnY motif conserved
in many GPCRs, has been found to be critical for the internal-
ization of the human β₂-adrenergic receptor (21), suggesting
that the signaling involved in internalization may be more
complicated. Furthermore, it is possible that the serine and
threonine residues in the carboxyl terminus of the CCKBR do
not serve as phosphorylation sites but as a part of a larger
internalization consensus motif in the carboxyl terminus as has
been found for other GPCRs, such as the yeast α-pheromone
(14) and the thyrotropin-releasing hormone receptors (13). The
slightly but significantly greater extent of internalization of the
CCKBR ΔS/T compared with the CCKBR Tr408 (39% versus
26%) may therefore reflect an incomplete disruption of such a
motif or that another structural determinant within the car-
boxyl terminus influences internalization. This could be the
putative palmitoylation site that is present in the CCKBR ΔS/T
but absent in the CCKBR Tr408 and was found to be important
for internalization of the thyrotropin-releasing hormone recep-
tor (13).

The confocal microscopy studies visualized the internaliza-
tion of the fluorescent agonist in cells expressing WT or mutant
CCK receptors. Cells transfected with the WT CCKAR or WT
CCKBR showed accumulation of the fluorescent ligand in in-
tracellular compartments. For the CCKAR, these results are
consistent with the localization of CCKARs expressed in trans-
fected CHO cells by an antiserum directed against the amino
terminus of the receptor (38). For the CCKBR, a study on
NIH/3T3 cells transfected with the WT CCKBR showed that
gastrin was internalized through a clathrin-dependent mecha-
nism accumulating in endosomes and lysosomes (26), a known
pathway of internalization for other GPCRs (1). Altogether,
these studies indicate that the internalization of the fluores-
cent ligand in this study reflected the internalization of the
receptor. The intracellular pattern of fluorescence for the WT
and mutant CCKARs and the WT CCKBR was consistent with
the resistance to KSCN stripping of the radioligand. Unlike the
WT CCKBR, the confocal pictures of the CCKBR mutants
showed that the fluorescent ligand was not translocated to the
cell interior, but stayed on the cell surface, suggesting an
interruption of the coupling to the endocytic pathway by the
truncation or mutation of serine and threonine residues, re-
spectively. This surface pattern was consistent with suscepti-
bility of the radioligand to KSCN stripping.

The relationship between G protein coupling and receptor
internalization is still unsettled. Earlier studies on the β-adre-
nergic and muscarinic receptors suggested a functional rela-
tionship between G protein coupling and receptor internaliza-
tion (39–41). On the other hand, the existence of mutant
receptors of the human β₂-adrenergic, the α-pheromone, and
the angiotensin II receptors that are defective in G protein
coupling but internalize to the same extent as their respective
WT receptors argues that these two processes are unrelated
(42–44). A third type of relationship between G protein cou-
pling and internalization is suggested by a recent study on the neurotensin receptor in which a G protein that was not involved in the signal transduction cascade of the receptor was found to affect internalization (18). Regardless, the aluminum fluoride experiments and $ ^{45}Ca^{2+} $ efflux studies showed full G protein coupling and intact signal transduction of all mutant and WT CCKARs and CCKBRs. This excludes the possibility that the internalization defect of the mutants CCKBR Tr408 and CCKBR $\Delta S/T$ results from a perturbation of G protein coupling. Finally, these results also indicate that the carboxyl-terminal 45 and 44 amino acids of the CCKAR and CCKBR, respectively, are unnecessary for G protein coupling and signal transduction.

We have shown that the internalization of the CCKBR, unlike that of the CCKAR, depends on structural determinants in the carboxyl-terminal part of the receptor. Therefore, despite a 48% homology in amino acid sequence between the CCKAR and the CCKBR and similar signal transduction pathways, the structural motifs that allow the interaction with the internalization pathway seem to reside in different parts of the receptors. The demonstration that the internalization of CCKBR $\Delta S/T$ is impaired almost to the same extent as the internalization of the CCKBR Tr408 would suggest that the serine and threonine residues in the carboxyl terminus are specifically involved in the internalization process. The results of this study will now allow the investigation of the potential relationship between ligand-induced internalization and phosphorylation of the serine and threonine residues in the carboxyl terminus of the CCKBR and the degree to which these processes influence receptor desensitization.

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