# A Segment of Five Amino Acids in the Second Extracellular Loop of the Cholecystokinin-B Receptor Is Essential for Selectivity of the Peptide Agonist Gastrin

Sandrine Silvente-Poirot, Stephen Wank

► To cite this version:

Sandrine Silvente-Poirot, Stephen Wank. A Segment of Five Amino Acids in the Second Extracellular Loop of the Cholecystokinin-B Receptor Is Essential for Selectivity of the Peptide Agonist Gastrin. Journal of Biological Chemistry, 1996, 271 (25), pp.14698-14706. 10.1074/jbc.271.25.14698. inserm-04974732

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# A Segment of Five Amino Acids in the Second Extracellular Loop of the Cholecystokinin-B Receptor Is Essential for Selectivity of the Peptide Agonist Gastrin*

(Received for publication, October 24, 1995, and in revised form, April 1, 1996)

Sandrine Silvente-Poirot‡ and Stephen A. Wank§

Digestive Diseases Branch, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-1804

The two known receptors mediating the actions of cholecystokinin (CCK) and gastrin, CCK type A (CCKAR) and CCK type B (CCKBR) receptors, are G protein-coupled receptors having approximately 50% amino acid homology. Both the CCKAR and CCKBR have high affinity for sulfated CCK peptides, while only the CCKBR has high affinity for gastrin peptides. To determine the structural basis for the selectivity of the CCKBR for gastrin, we first constructed a series of CCKB/AR chimeras in which restriction endonuclease-defined segments of the CCKBR were replaced with the corresponding segments of the CCKAR. Chimeras transiently expressed in COS-1 cells were screened for the selective loss of gastrin affinity according to the displacement of ¹²⁵I-labeled Bolton-Hunter-CCK-8 binding by gastrin-17-I and CCK-8. The sequence spanning from transmembrane domain III (TM III) to TM V was the only segment that resulted in the selective loss of gastrin affinity. This segment could account for 100% of the expected 300-fold lower affinity of gastrin-17-I observed for the control CCKAR compared to the control CCKBR. Using site-directed mutagenesis in this segment of the CCKBR, we identified a sequence of 5 amino acids in the second extracellular loop responsible for this 100-fold selective loss in gastrin affinity. ¹²⁵I-labeled Bolton-Hunter-CCK-8 binding displacement by L365,260 (a CCKBR selective antagonist) was unaffected by the changes in these 5 amino acids. These results present for the first time the identification of the amino acid sequence of the CCKBR conferring the majority of the selectivity for gastrin.

Cholecystokinin and gastrin peptides are two evolutionarily related families of regulatory peptides having high affinity for CCK<fnref n="1" /> receptors. Biological activity for CCK and gastrin resides in the amidated carboxyl terminus. Although both peptides have an identical carboxyl-terminal pentapeptide sequence, they differ in their selectivity for the two CCK receptor subtypes, CCKAR and CCKBR on the basis of a tyrosine sulfate at the seventh position (CCK) versus a variably sulfated tyrosine

in the sixth position (gastrin) from the carboxyl terminus (Fig. 1) (1-3).

CCKAR and CCKBR are seven transmembrane G protein-coupled receptors having approximately 50% amino acid homology (see Fig. 2) (4, 5). Although both CCKAR and CCKBR have nearly equally high affinity for sulfated CCK peptides, only the CCKBR has high affinity for the peptide agonist gastrin (6, 7). CCKAR and CCKBR can also be pharmacologically distinguished by the non-peptide-substituted benzodiazepine derivative antagonists, L-364,718 (CCKAR selective) and L-365,260 (CCKBR selective) (8, 9) (Fig. 1).

CCKAR are predominately found in the periphery where they regulate pancreatic secretion and gallbladder and gastrointestinal motility, but are also found in select areas of the central nervous system where they regulate satiety and analgesia. CCKBR are predominately present throughout the central nervous system where they regulate anxiety/panic attacks and dopamine release implicated in the pathogenesis of dopaminergic related movement and behavioral disorders in humans such as Parkinson's and Huntington's disease and schizophrenia, respectively. In the periphery, CCKBR (formerly referred to as gastrin receptors) regulate acid and histamine secretion as well as growth in the gastric mucosa and gastrointestinal motility (1-3).

While there is considerable data supporting agonist ligand binding to transmembrane areas for small amine ligands (e.g. epinephrine, histamine, 5-hydroxytryptamine, dopamine) less is known about peptide ligands, some of which may interact with both transmembrane and extracellular domains of G protein-coupled receptors (10, 11). Subtypes within a number of receptor families have been distinguished on the basis of their relative selectivity for agonists. The molecular basis for peptide ligand selectivity has been characterized for only a small group of G protein-coupled receptors including the second and third extracellular loops and the transmembrane domains VI and VII of the somatostatin types 1 and 2 receptor (12, 13), the fifth transmembrane segment of the neuromedin B receptor (14), the second extracellular loop of the κ-opiate receptor (15), the amino terminus, transmembrane, and extracellular loops of the neurokinin receptors (16-18), the transmembrane domain II, the second and third loops, and the flanking regions of the endothelin ETA receptor (19, 20), the first extracellular loop of the V1a vasopressin receptor (21) and the amino terminus of the interleukin 8 (22), vasoactive intestinal peptide and secretin (23, 24), and parathyroid hormone (25) receptors. These studies indicate that the epitopes for ligand binding selectivity varies within and between different receptor families and that any approach to discover the determinants responsible for peptide ligand selectivity must examine the entire receptor.

Little is known about the agonist and antagonist binding sites for cholecystokinin receptors. Studies on the binding site for the non-peptide antagonists, L-364,718 and L-365,260, as

<footnote>* 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.</footnote>

<footnote>‡ Supported by a research grant from the International Human Frontier Science Program. Present address: INSERM U 151, CHU Rangueil, L3 31054, Toulouse Cedex, France.</footnote>

<footnote>§ To whom correspondence and reprint requests should be addressed: Bldg. 10, Rm. 9C-103, National Institutes of Health, Bethesda, MD 20892-1804. Tel.: 301-496-4201; Fax: 301-402-0600.</footnote>

<footnote n="1">¹ The abbreviations used are: CCK, cholecystokinin; CCKAR, CCK type A receptor; CCKBR, CCK type B receptor; TM, transmembrane domain; BSA, bovine serum albumin; ET, endothelin.</footnote>
<figure><img src="image_2.png" /><figcaption>FIG. 1. Structure of CCK receptor peptide agonists and non-peptide antagonists. The primary sequence of CCK-8 and gastrin-17-1 are shown using single amino acid symbols. Full biological activity for CCK resides in the amidated, carboxyl-terminal heptapeptide containing a sulfated tyrosine at the seventh position from the carboxyl terminus (bold circle). Gastrin-17-1, the nonsulfated form of gastrin, has a tyrosine at the sixth position (bold circle) from the carboxyl terminus and shares the carboxyl-terminal pentapeptide with CCK, shown in black circles. Shown further below are the planar representations of the antagonists, L-365,260 (CCKBR preferring) and L-364,718 (CCKAR preferring).</figcaption></figure>

well as peptide-derived antagonist PD-135,158 identified several residues in the transmembrane domains of the CCKBR that are involved in subtype selectivity (26, 27). However, it is less clear that the transmembrane domains are involved in the selective binding of the agonist gastrin. Substitution of all divergent amino acids in the transmembrane domains of the human CCKBR with amino acids in equivalent positions of the CCKAR identified several amino acids that seemed weakly involved in the selective binding of gastrin. For example, the amino acid substitution that resulted in the largest decrease in gastrin affinity (17-fold) also caused an even greater decrease (29-fold) in the affinity of the nonselective agonist, CCK-8 (27).

In the present study, we used a different approach to determine the domains conferring subtype selectivity for gastrin in the rat CCKB receptor. First, we evaluated all areas of the CCKB receptor in a chimeric reductionist strategy. This initial approach followed by site-directed mutagenesis allowed us to localize a sequence of 5 amino acids within the second extracellular loop of the CCKB receptor essential for gastrin selectivity.

## EXPERIMENTAL PROCEDURES

*Construction of Chimeric Receptor cDNA*—Unique restriction endonuclease sites shared either naturally or introduced by site-specific mutagenesis (29) at five equivalent positions between TM I and TM VII in the CCKAR and CCKBR were chosen to create chimeric CCKB receptors (CCKB/AR). Segments of the CCKBR were replaced progressively or in segments by the equivalent CCKAR sequence. The *Bst*EII site at residue 87, *Msc*I site at residue 146, *Bsp*HI site at residue 187, *Avr*II site at residue 246, and *Asc*I site at residue 360 in the CCKBR were used for restriction endonuclease cleavage followed by ligation with T4 ligase of the corresponding CCKAR and CCKBR cDNA fragments (see Fig. 2). CCKB/AR chimeras containing smaller segments were constructed using overlapping mutagenic primers in a three-step polymerase chain reaction (30) using a mixture of *Pfu*I and *Taq* polymerases (ratio, 1/15) and oligonucleotide-directed mutagenesis (29) using the wild type CCKBR as a single strand template. To facilitate analysis of mutant constructs, mutated cDNA cassettes were generated as described above and were subsequently exchanged with the corresponding sequence in the CCKB cDNA using the shared restriction

endonuclease sites described above. All chimeric receptor cDNA sequences were confirmed using the dideoxy chain termination method (31) (ds Cycle Sequencing System, Life Technologies, Inc.).

*Transfection of Wild Type and Mutant Receptor cDNA into Mammalian Cells*—Two micrograms of pCDL-SRα containing either the wild type or mutant receptor cDNA were transfected into near confluent COS-1 cells (3 × 10⁶ cells/plate) using a DEAE/dextran method as described elsewhere (32). Approximately 48 h after transfection, cells were washed twice with cold phosphate-buffered saline, pH 7.4, containing 0.1% bovine serum albumin (BSA), scraped from the plate in cold Dulbecco's modified Eagle's medium containing 0.1% BSA, centrifuged 2 min (400 × g), and resuspended in Dulbecco's modified Eagle's medium with 0.1% BSA for radiolabeled binding assays.

*Binding of ¹²⁵I-Labeled Bolton-Hunter-CCK-8 to Transfected COS-1 Cells*—Approximately 48 h post-transfection, 2.5 × 10⁵ cells suspended in 0.5 ml of Dulbecco's modified Eagle's medium containing 0.1% BSA were incubated for 60 min at 37 °C with 50 pM of ¹²⁵I-labeled Bolton-Hunter-CCK-8 (2200 Ci/mmol) either with or without the indicated concentrations of unlabeled CCK-8 or gastrin-17-I. Cells associated with ¹²⁵I-labeled Bolton-Hunter-CCK-8 were separated from free radioligand by centrifugation (1 min, 13,000 × g) through a bed of 0.25 ml of phosphate-buffered saline containing 4% BSA at 4 °C in a 0.4-ml microcentrifuge tube and subsequently washed and centrifuged two times with 0.25 ml of phosphate-buffered saline with 4% BSA at 4 °C. Pelleted cells were assayed for gamma radioactivity (Packard, Auto-Gamma). Specific binding was equal to the total binding minus the nonspecific binding (amount of ¹²⁵I-labeled Bolton-Hunter-CCK-8 associated with the COS-1 cells in the presence of 1 µM CCK-8). Nonspecific binding was always less than 10% of total binding.

Ligand affinities were determined by radioligand dose inhibition studies with increasing concentrations of cold ligand performed in duplicate in at least three separate experiments. Curve fitting and $IC_{50}$ values were determined using the nonlinear curve fitting computer program, ALLFIT (33).

## RESULTS

To identify the domains in the rat CCKB receptor that confer high affinity for gastrin-17-I, the CCKAR and CCKBR were divided into six segments determined by five restriction endonuclease sites placed at equivalent positions in both receptors (Fig. 2). Segments of the CCKBR were subsequently replaced with the corresponding rat CCKAR sequence in single as well as multiple combinations of segments. The introduction of an *Avr*II site in the CCKAR and a *Bsp*HI site in the CCKBR resulted in amino acid substitutions CCKAR(Q238L) and CCKBR(V187T) (Fig. 2). ¹²⁵I-labeled BH-CCK-8, having equally high affinity for both CCKA and CCKB receptors, was used as the radioligand. Competition binding studies were performed to determine the affinities for both CCK-8 and gastrin-17-I for the restriction site modified recombinant rat CCKAR(Q238L) and CCKBR(V187T) receptors transiently expressed in COS-1 cells (Fig. 3). As expected, the nonselective ligand, CCK-8, displays a high and nearly equal affinity for both CCKAR and CCKBR with an $IC_{50}$ of 3.43 (± 0.91) nM and 3.61 (± 0.68) nM, respectively (Fig. 3, left panel, and Table I). Conversely, the selective ligand, gastrin-17-I, discriminates the two CCK receptors subtypes (Fig. 3, right panel). Gastrin-17-I has a low affinity for the CCKAR(Q238L), $IC_{50}$ = 1,140 (± 320) nM and a high affinity for the CCKBR(V187T), $IC_{50}$ = 4.32 (± 0.86) nM, similar to CCK-8 (Table I). The relative affinity of CCK-8 and gastrin-17-I for the CCKAR(Q238L) compared to CCKBR(V187T) (expressed as $IC_{50}$ CCKAR(Q238L)/$IC_{50}$ CCKBR(V187T)) is 1 and 263, respectively (Table I). These values agree closely with data for wild type rat CCKAR and CCKBR reported previously (3, 4) and indicate that the introduction of the restriction sites had no significant effect on their affinity for CCK-8 and gastrin-17-I. Therefore, they were subsequently used as controls for assessing changes in affinity resulting from receptor chimeric mutations.

The CCKB/AR chimera containing the smallest amount of CCKAR sequence without compromising the maximal selective loss in gastrin affinity was chimera Ch-1. Chimera Ch-1 is a
<figure><img src="image_3.png" /></figure>

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

FIG. 2. Schematic representation of the primary structure of the CCKA and CCKB receptors. Deduced amino acid sequence of the rat CCKAR and CCKBR illustrating putative transmembrane helices (boxed amino acids), asparagines for putative N-linked glycosylation sites (tridents), serine and threonine phosphorylation sites (-PO3), conserved cysteines in the first and second extracellular loops possibly forming a disulfide bridge (-S-S-), and a potential palmitoylated cysteine in the carboxyl terminus (jagged line extending into the membrane) conserved between the rat CCKAR and CCKBR. Naturally occurring or introduced restriction endonuclease sites shared between CCKA and CCKB receptors at equivalent positions are indicated along the sequence. Introduction of the AvrII site in the CCKAR and the BspHI site in the CCKBR results in a Q238L and a V187T mutation referred to as CCKAR(Q238L) and CCKBR(V187T), respectively. CCKAR and CCKBR homologous and nonhomologous amino acids are shown as open and closed circles, respectively. (-NH2, amino terminus; -COOH, carboxyl terminus).

CCKBR with the segment from the bottom of TM III to TM V (delimited by the restriction sites MscI at amino acid position 146 and AvrII at amino acid position 246, respectively; Fig. 4) replaced with the corresponding CCKAR sequence (Fig. 2). The Ch-1 chimera exhibits a high affinity for CCK-8 similar to that

of the CCKBR(V187T) IC50 = 6 (± 0.8) nM (Fig. 5, left panel, and Table I). In contrast to that observed for CCK-8, the chimera Ch-1 affinity for gastrin-17-1 is markedly decreased IC50 = 420 (± 3.0) nM (Fig. 5, right panel, and Table I). The decrease in binding affinity expressed as IC50 Ch-1/IC50 CCKBR(V187T)
is 97-fold (Table I). The selective loss of affinity of the chimera, Ch-1, for gastrin-17-I and not CCK-8 indicates the structural and functional integrity of the chimeric receptor. Other segments outside the region between the bottom of TM III and TM V alone or in combination with this region contributed less than a 3-fold loss of affinity for gastrin without affecting CCK-8 affinity (data not shown). These results indicated that the segment of the CCKBR between the bottom of TM III and TM V was the major area responsible for the selective affinity for gastrin and therefore further studies were confined to this area.

To determine the amino acids within the segment between the bottom of TM III and TM V of the CCKBR that confer subtype selectivity for gastrin-17-I, we constructed a series of CCKB/A receptor chimeras by replacing progressively fewer numbers of divergent amino acids from this segment of the CCKBR with corresponding amino acids from the CCKAR using site directed mutagenesis (Fig. 4). All subsequent chimeras were transiently expressed in COS-1 cells and assessed for their affinity for CCK-8 and gastrin-17-I by competition binding studies using ¹²⁵I-labeled Bolton-Hunter-CCK-8.

On the basis of studies of other peptide ligand receptors, we assumed that the interaction of CCK and gastrin would most likely occur within the extracellular loops and the outer part of

<figure><img src="image_5.png" /><figcaption>FIG. 3. Inhibition of ¹²⁵I-labeled Bolton-Hunter-CCK-8 binding to the CCKAR(Q238L) and CCKBR(V187T) receptors. Displacement of ¹²⁵I-labeled BH-CCK-8 by CCK-8 (left panel) and gastrin-17-I (right panel) in COS-1 cells expressing either the rat CCKA or CCKB wild type receptors that have been modified by introducing restriction endonuclease sites resulting in a Q238L or V187T mutation, respectively. Binding is expressed as the percent of specifically bound ¹²⁵I-labeled Bolton-Hunter-CCK-8. Each experiment was performed in duplicate, and the results given are the means ± S.E. from 12 experiments.</figcaption></figure>

the transmembrane spanning region. Therefore, chimera Ch-2 (Fig. 4) was constructed by replacing the entire second extracellular loop and the outer two-thirds of TM IV and TM V of the CCKBR(V187T) with the corresponding divergent amino acids from the CCKAR(Q238L). Similar to chimera Ch-1, the chimera Ch-2 has a high affinity for CCK-8, $IC_{50}$ = 5.02 (±0.75) nM, comparable to that of the control CCKBR(V187T) (Fig. 6a, left panel, and Table I) and a similar decrease in the affinity of gastrin-17-I, $IC_{50}$ = 370 (±7.0) nM (Fig. 6a, right panel, and Table I). The change in binding affinity expressed as $IC_{50}$ Ch-2/IC$_{50}$ CCKBR(V187T) is 85-fold (Table I). These data indicate that the second intracellular loop and the deeper third of the TM III, TM IV, and TM V do not contain amino acids necessary for gastrin-17-I selectivity.

To determine which amino acids of chimera Ch-2 were responsible for the decrease in gastrin-17-1 affinity, the sequence replaced by CCKAR amino acids was divided into four segments consisting of the outer two-thirds of TM IV, the amino and carboxyl-terminal halves of the second extracellular loop, and the outer two-thirds of TM V. A group of chimeras was constructed in which single as well as combinations of these segments replaced the corresponding segments of the CCKBR. Chimera Ch-3 (Fig. 4) in which the amino-terminal half of the second extracellular loop and the outer two-thirds of both TM IV and TM V were replaced with the corresponding CCKAR sequence was one of the most clearly informative chimeras from this group. Chimera Ch-3 displayed the same high affinity for both CCK-8 and gastrin-17-I as the control CCKBR(V187T) (Table I) and indicated that the area responsible for gastrin-17-1 selectivity resides in the carboxyl-terminal half of the second extracellular loop. This result was confirmed by a second group of chimeras constructed using oligonucleotide-directed mutagenesis of the wild type CCKBR to evaluate the carboxyl-terminal half of the second extracellular loop and the outer two-thirds of TM V. Chimera Ch-4 (Fig. 4) contained amino acid changes in both of these areas except for the 3 consecutive amino acids ARV (A212D, R213A, and V214M) in the second extracellular loop. The Ch-4 chimera exhibited a high affinity for CCK-8 similar to that of CCKBR(V187T), $IC_{50}$ = 5.88 (±1.3) nM (Fig. 6b, left panel, and Table I). In contrast to that observed for CCK-8, the Ch-4 chimera affinity for gastrin-17-I is markedly decreased, $IC_{50}$ = 684 (± 3.0) nM (Fig. 6b, right panel, and Table I). Similar to chimera Ch-2, the decrease in binding affinity for gastrin-17-1 expressed as $IC_{50}$ Ch-4/IC$_{50}$ CCKBR(V187T) is 158-fold (Table I). Chimera Ch-5 (Fig. 4) was constructed to determine the

<caption>TABLE I
Binding affinities of the agonists CCK-8 and gastrin-17-I and the nonpeptide antagonist L-365,260 for CCKA(Q238L) and CCKB(V187T) and chimeric receptors</caption>

Receptors were transiently expressed in COS-1 cells and ¹²⁵I-labeled Bolton-Hunter-CCK-8 displacement curves performed using increasing concentrations of CCK-8 and gastrin-17-I. Values of $IC_{50}$ are presented in nM as the mean ± S.E. for the number of experiments indicated within the parentheses. The factor $F_{mut}$ represents the effect of the mutations on the affinity of CCK-8 or gastrin-17-I and is calculated as the $IC_{50}$ of the experimental receptor (X) divided by the $IC_{50}$ of the CCKBR(V187T) (CCKBR).

<table><thead><tr><th rowspan="2"></th><th colspan="3">IC<sub>50</sub> (nM)</th><th colspan="2">F<sub>mut</sub> (IC<sub>50</sub>X/IC<sub>50</sub>CCKBR)</th></tr><tr><th>CCK-8</th><th>Gastrin-17-I</th><th>L-365,260</th><th>CCK-8</th><th>Gastrin-17-I</th></tr></thead><tbody><tr><td>CCKBR(V187T)</td><td>3.61 ± 0.68 (12)</td><td>4.32 ± 0.86 (12)</td><td>3.98 ± 2.1 (3)</td><td>1</td><td>1</td></tr><tr><td>CCKAR(Q238L)</td><td>3.43 ± 0.91 (9)</td><td>1140 ± 320 (9)</td><td>260 ± 12 (3)</td><td>0.95</td><td>263</td></tr><tr><td>Ch-1</td><td>6.00 ± 0.80 (4)</td><td>420 ± 3 (4)</td><td>ND<sup>a</sup></td><td>1.6</td><td>97</td></tr><tr><td>Ch-1rev</td><td>NB<sup>b</sup> (2)</td><td>NA<sup>c</sup></td><td>NA</td><td></td><td></td></tr><tr><td>CH-2</td><td>5.02 ± 0.75 (4)</td><td>370 ± 7 (4)</td><td>ND</td><td>1.38</td><td>85</td></tr><tr><td>Ch-3</td><td>3.40 ± 0.18 (2)</td><td>2.08 ± 0.49 (2)</td><td>ND</td><td>0.94</td><td>0.48</td></tr><tr><td>Ch-4</td><td>5.88 ± 1.30 (3)</td><td>684 ± 3 (3)</td><td>ND</td><td>1.6</td><td>158</td></tr><tr><td>Ch-5</td><td>1.43 ± 0.34 (2)</td><td>3.82 ± 0.77 (2)</td><td>ND</td><td>1.43</td><td>0.88</td></tr><tr><td>Ch-6</td><td>13.00 ± 5.80 (5)</td><td>718 ± 27 (5)</td><td>3.48 ± 2.7 (3)</td><td>3.6</td><td>166</td></tr><tr><td>Ch-6rev</td><td>NB (2)</td><td>NA</td><td>NA</td><td></td><td></td></tr></tbody></table>

<footnote><sup>a</sup> ND, not determined.</footnote>

<footnote><sup>b</sup> NB, no binding.</footnote>

<footnote><sup>c</sup> NA, not applicable.</footnote>
<figure><img src="image_6.png" /></figure>

<figure><img src="image_7.png" /></figure>

<figure><img src="image_8.png" /></figure>

<figure><img src="image_9.png" /></figure>

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<figure><img src="image_12.png" /></figure>

<figure><img src="image_13.png" /><figcaption>FIG. 4. Schematic representation of the chimeric receptors described in this study and summarized in Table I. The sequence between TM IV and TM V has been enlarged to indicate the CCKAR(Q238L) and CCKBR(V187T) nonhomologous amino acids that have been mutated (shown as closed circles).</figcaption></figure>
<figure><img src="image_14.png" /><figcaption>FIG. 5. Structure and ¹²⁵I-labeled Bolton-Hunter-CCK-8 displacement curves for the CCKBR chimera Ch-1. The structure of Ch-1 in which the segment of the CCKBR between MscI and AvrII was replaced with the equivalent segment from the CCKAR is shown on the far left. Filled circles represent amino acids mutated with the corresponding CCKAR amino acids. Displacement of ¹²⁵I-labeled Bolton-Hunter-CCK-8 by CCK-8 (left panel) and gastrin-17-I (right panel) in COS-1 cells expressing either the chimera Ch-1 or the CCKBR(V187T). Binding is expressed as the percent of specifically bound ¹²⁵I-labeled Bolton-Hunter-CCK-8. Each experiment was performed in duplicate, and the results given are the means ± S.E. from four separate experiments.</figcaption></figure>

contribution of TM V alone. As expected from previous results with chimera Ch-3, chimera Ch-5 maintains high affinity for CCK-8, $IC_{50} = 1.43 (± 0.34)$ nM and also does not affect gastrin binding, $IC_{50} = 3.82 (± 0.77)$ nM (Table I). Together these results suggest that only the substitution of the sequence QM-HRW (Q204M, M206R, H207F, R208L, and W209L, Fig. 2) in the amino-terminal half of the second extracellular loop of the CCKBR are necessary for gastrin selectivity. This is confirmed by chimera Ch-6 (Fig. 4) in which the substitution of the sequence QMHRW alone (constructed in the wild type receptor, i.e. without the V187T mutation) produces a similar shift in gastrin-17-I affinity as observed for the chimera Ch-1, $IC_{50} = 718 (±27.0)$ nM (Fig. 6c, right panel, and Table I). The decrease in binding affinity for gastrin-17-I expressed as $IC_{50}$ Ch-6/$IC_{50}$ CCKBR(V187T) is 166-fold compared to 97-fold for chimera Ch-1 (Table I). However, it should be noted that the affinity of chimera Ch-6 for CCK-8 is reduced by 3.6-fold ($IC_{50} = 13.0 (±5.8)$ nM) compared to CCKBR(V187T) (Fig. 6c, left panel, and Table I), while the affinity of chimera Ch-1 for CCK-8 is less affected, 1.6-fold (Table I). Thus, the substitution of the 5 amino acids QMHRW in the second extracellular loop and not the transmembrane domains in this region are responsible for the 100-fold decrease in gastrin-17-I affinity observed for the chimera Ch-1 and can account for the majority of the selectivity observed for gastrin-17-I by the CCKBR versus the CCKAR. In addition to the preservation of the high affinity for CCK-8, the replacement of these 5 amino acids did not interfere with the level of receptor expression as determined by the $B_{max}$ of 7.6 (± 5.3) pmol/10⁶ cells obtained for the chimeras Ch-6 compared to the $B_{max}$ of the CCKBR(V187T) of 7.2 (±5.1) pmol/10⁶ cells.

To further confirm the integrity of the chimera Ch-6 and to determine whether these same 5 amino acids, QMHRW, are involved in antagonist binding, we tested its affinity for the CCKB-selective, non-peptide antagonist, L-365,260. Compared to CCKBR(V187T), the affinity of the L-365,260 was unaffected, $IC_{50} = 3.45 (±2.7)$ nM (Table I), indicating that the 5 mutated amino acids are not directly involved in L-365,260 binding and that the overall structure of this chimera is well maintained.

To determine whether the same area spanning from the bottom of TM III to the end of TM V is also responsible for gastrin selectivity in the CCKAR, we constructed the reverse of chimera Ch-1, Ch-1rev (Fig. 4). The Ch-1rev chimera had undetectable binding at picomolar concentrations of ¹²⁵I-labeled Bolton-Hunter-CCK-8 and therefore precluded the evaluation of the relative affinity of gastrin-17-I. Micromolar concentra-

tions of CCK-8 and gastrin-17-I each failed to stimulate total inositol phosphates in COS-1 cells transiently transfected with the Ch-1rev chimera while cells transfected with either the CCKAR(Q238L) or the CCKBR(V187T) increased total inositol phosphates 7- and 3-fold, respectively. However, the Ch-1rev chimera bound the radiolabeled antagonist, ³H-labeled L-364,718, with a similar affinity (0.25 ± 0.03 nM) compared to the CCKAR(Q238L) (0.72 ± 0.16 nM), suggesting that the Ch-1rev chimera was not grossly misfolded as a result of the mutation.

Because the large area mutated in the Ch-1rev chimera affected both agonists, CCK-8 and gastrin-17-I, and because only the 5 amino acids, QMHRW, in the second extracellular loop of the CCKBR were necessary for most of the CCKBR selectivity for gastrin-17-I, the reverse of Ch-6, Ch-6rev, (Fig. 4) was constructed in the CCKAR by replacing the corresponding CCKAR sequence, MRFLL with the CCKBR sequence, QM-HRW, (M195Q, R197M, F198H, L199R, and L200W; Fig. 2). Similar to Ch-1rev, the Ch-6rev lost affinity for both agonists, CCK-8 and gastrin-17-I, as determined by ¹²⁵I-labeled Bolton-Hunter-CCK-8 binding and stimulation of total inositol phosphates, although it also maintained full affinity (0.05 ± 0.10 nM) for the antagonist, ³H-labeled L-364,718, compared to the CCKAR(Q238L) (0.72 ± 0.16 nM). Similar to Ch-1rev, these results indicate that Ch-6rev is also well expressed.

## DISCUSSION

Several studies examining peptide ligand binding domains of seven transmembrane G protein-coupled receptors indicate considerable diversity in both the number and location of ligand interacting determinants (10). Similarly, multiple ligand-receptor interaction sites have been found to be important for even the determination of ligand subtype specificity among some highly homologous members of the same family of G protein-coupled receptors. The two known members of the CCK family, CCKAR and CCKBR, are highly homologous and share a similar high affinity for the peptide agonist, CCK, yet they can be easily distinguished by the CCKBR subtype-selective, peptide agonist, gastrin (2). A recent study examining the structural basis for gastrin subtype selectivity using 58 chimeric receptors in which 1–4 divergent amino acids in the CCKBR were replaced with the corresponding amino acids from the CCKAR failed to identify any amino acids conferring subtype selectivity for gastrin (27). This report and the lack of consensus binding sites for ligand-receptor interaction among peptide ligand receptors led us to take a broad chimeric ap-
<figure><img src="image_15.png" /><figcaption>FIG. 6. Structure and ¹²⁵I-labeled Bolton-Hunter-CCK-8 displacement curves for the CCKBR chimeras. The structure of the CCKBR chimeras created by the replacement of amino acids in the region spanning from TM IV to TM V with the corresponding CCKAR amino acids are shown on the far left for chimeras Ch-2 (a), Ch-4 (b), and Ch-6 (c) (CCKBR amino acids that have been mutated with the corresponding CCKAR amino acids are shown as filled circles). Displacement of ¹²⁵I-labeled Bolton-Hunter-CCK-8 by CCK-8 (left panel) and gastrin-17-I (right panel) in COS-1 cells expressing either the chimera Ch-2 (a), Ch-4 (b), and Ch-6 (c) or the CCKBR(V187T). Binding is expressed as the percent of specifically bound ¹²⁵I-labeled Bolton-Hunter-CCK-8. Each experiment was performed in duplicate and the results given are the means ± S.E. from the number of separate experiments indicated in Table I.</figcaption></figure>

proach to determine the structural basis underlying gastrin selectivity. The CCKBR was chosen for mutagenesis in this study rather than the CCKAR as an initial approach to understanding the structural basis of gastrin affinity. This choice was made on the assumption that the complexity of ligand-receptor interaction may be simpler for the CCKBR, which, unlike the CCKAR, does not show selectivity of CCK/gastrin ligands on the basis of either the position or the presence of sulfation of the tyrosine (sixth *versus* seventh position from the carboxyl terminus for gastrin and CCK, respectively). Chimeras were examined for gastrin-17-I affinity in this initial study

so that the likelihood of finding a domain(s) directly responsible for gastrin selectivity would be increased because of both the altered position and lack of sulfation of the tyrosine in this form of gastrin.

Six segments of the CCKBR were replaced sequentially and in multiple combinations with the corresponding CCKAR segments. Ligand binding studies of these chimeras led to the identification of a large segment spanning from the bottom of TM III through TM V that could account for almost all of the 300-fold lower affinity for gastrin-17-I observed for a typical CCKAR (2). Construction of chimeras containing progressively
smaller contributions from the CCKAR to the CCKBR using site directed mutagenesis led to the discovery of a segment of five amino acids in the second extracellular loop of the CCKBR that constituted the major determinant for gastrin selectivity.

While other areas of the receptor may also be either directly or indirectly involved in ligand binding, their contribution must be small because the area in the second extracellular loop identified in this study can account for 100 of the 300-fold change in selectivity for gastrin. Our inability to detect other areas having a significant effect on gastrin affinity also supports the importance of this area. These data suggest that the transmembrane domains of the CCKBR are either not or only weakly involved in selective binding of gastrin. This is consistent with a recent publication that reported a minor role of the transmembrane domains of the CCKBR for gastrin selectivity while they appeared more involved in the non-selective binding of both gastrin and CCK peptides (27).

Studies using chimeric receptors to determine sites of ligand-receptor interaction cannot by themselves determine whether the mutated amino acids have a direct *versus* an indirect affect. As stated earlier, CCK and gastrin have a very similar biologically active carboxyl-terminal sequence differing only in the position of a variably sulfated tyrosine. Considering this similarity between CCK and gastrin and the present data demonstrating that a small replacement of 5 amino acids in the second extracellular loop of the CCKBR with the corresponding CCKAR sequence does not alter high affinity for CCK-8 and the CCKBR selective antagonist, L-365,260, suggests that the selective loss in gastrin affinity is not due to global changes in receptor conformation.

Although the replacement of the 5 amino acids, QMHRW, in the second extracellular loop of the CCKBR, Ch-6, results in the selective loss of gastrin-17-I, the reciprocal change in the corresponding sequence, MRFLL, of the CCKAR, Ch-6rev, results in the loss of CCK-8 and gastrin affinity as demonstrated by ¹²⁵I-labeled CCK-8 radioligand binding and total inositol phosphate stimulation by CCK-8 and gastrin-17-I. However, the affinity of the antagonist, L-364,718, remains unaffected and therefore suggests that the reciprocal chimera is expressed without gross conformational changes. The nonreciprocal effect of switching these 5 amino acids suggests that they do not by themselves form the basis for determining CCKAR and CCKBR subtype selectivity although they contribute to the CCKBR selectivity for gastrin, A related nonreciprocal situation has been reported for the endothelin (ET) type A and B receptors. The replacement of Tyr¹²⁹ in the ET-A receptor by the corresponding His¹⁵⁰ in the ET-B receptor results in increased affinity for the peptide agonists ET-3 and sarafotoxin and decreased affinity for the ET-A selective antagonist BQ123 similar to the ET-B receptor, while the reciprocal H150Y mutation of the ET-B receptor has no affect on the affinity for these ligands (28). However, unlike the ET-A and ET-B receptors, the reciprocal mutant, Ch-6rev, loses affinity for the agonists, CCK-8 and gastrin-17-I. Also, unlike the ET-A and ET-B receptors, the results of this study and another study (26) indicate that the binding sites for peptide agonists and antagonists within both the CCKAR and CCKBR do not overlap extensively.

Similar to the CCKBR, several studies of other peptide ligand receptors such as the somatostatin (types 1 and 2) (12), neurokinin (type 1) (17, 34), and opiate (μ and κ) receptors (15) also indicate that the second extracellular loop is important in determining peptide ligand selectivity. The importance of the second extracellular loop in ligand binding selectivity among these various receptors further supports that the 5 amino acids in the second extracellular loop of the CCKBR identified in this

study interact directly with the ligand.

Both extracellular and transmembrane domains have been reported to be involved in the binding pocket for other peptide receptors (10, 11), and the contribution of various extracellular domains to subtype selectivity of peptide ligands has been described (12–25). Our results do not rule out the possible contribution of the transmembrane domains in the formation of the gastrin binding pocket. The sequence of 5 amino acids identified here are in close proximity with a highly conserved cysteine (Cys²⁰⁵) that may form a disulfide bridge with another conserved cysteine (Cys¹²⁷) located at the top of TM III (Fig. 2). This disulfide bridge, shown to be important for proper folding of other G protein-coupled receptors (33), would bring the identified area in close proximity to the membrane as suggested by affinity cross-linking (34) and mutagenesis studies (17) of the interaction of substance P with the neurokinin 1 receptor. This would allow gastrin to interact with both the second extracellular loop, as demonstrated in this study, as well as with the transmembrane domains. Further studies altering sequences conserved among CCKAR and CCKBR will help to identify additional amino acids involved in the overall binding pocket for gastrin.

The present study provides an initial basis toward the future understanding of the molecular interactions underlying CCK receptor subtype selectivity for the peptide agonists CCK and gastrin. Clearly the extracellular domain plays a significant role in determining the selectivity for these as well as other peptide ligands. Future studies involving mutations of individual amino acids in the second extracellular loop of the CCKBR as well as in CCK and gastrin peptide ligands should help to define their direct points of interaction and ultimately the rational design of both agonist and antagonist subtype specific ligands.

## REFERENCES

1. Poirot, S. S., Dufresne, M., Vaysse, N., and Fourmy, D. (1993) *Eur. J. Biochem.* **215**, 513–529

2. Wank, S. A. (1995) *Am. J. Physiol.* **269**, G628–G646

3. Crawley, J. N., and R. L. Corwin. (1994) *Peptides* **15**, 731–755

4. Wank, S. A., Pisegna, J. R., and deWeerth, A. (1992) *Proc. Natl. Acad. Sci. U. S. A.* **89**, 8691–8695

5. Wank, S. A., Harkins, R. T., Jensen, R. T., Shapira, H., DeWeerth, A., and Slattery, T. (1992) *Proc. Natl. Acad. Sci. U. S. A.* **89**, 3125–3129

6. Saito, A., Sankaran, H., Goldfine, I. D., and Williams, J. A. (1981) *J. Neurochem.* **37**, 483–490

7. Soll, A. H., Amiran, D. A., Thosmas, L. P., Reedy, T. J., and Elashoff, J. D. (1984) *J. Clin. Invest.* **73**, 1434–1447

8. Chang, R. S., Lotti, V. J., Keegan, M. E., and Kunkel, K. A. (1986) *Biochem. Biophys. Res. Commun.* **134**, 895–899

9. Lotti, V. J., and Chang, R. S. L., (1989) *Eur. J. Pharmacol.* **163**, 273–280

10. Schwartz, T. W. (1994) *Curr. Opin. Biotechnol.* **5**, 434–444

11. Strader, C. D., Fong, T. M., Tota, M. R., Underwood D., and Dixon, R. A. F. (1994) *Annu. Rev. Biochem.* **63**, 101–132

12. Fitzpatrick, V. D., and Vandlen, R. L., (1994) *J. Biol. Chem.* **269**, 24621–24626

13. Kaupman, K., Bruns, C., Raulf, H., and Lubbert H. (1995) *EMBO J.* **14**, 727–735

14. Fathi, Z., Benya, R. V., Shapira, H., Jensen, R. T., and Battey J. F. (1993) *J. Biol. Chem.* **268**, 14622–14626

15. Wang, J. B., Johnson, P. S., Wu, J. M., Wang, W. F., and Uhl, G. R. (1994) *J. Biol. Chem.* **269**, 25966–25969

16. Gether, U., Johansen, T. E., and Schwartz T. W. (1993) *J. Biol. Chem.* **268**, 7893–7898

17. Fong, T. M., Huang, R. C., and Strader, C. D. (1992) *J. Biol. Chem.* **267**, 25664–25667

18. Fong, T. M., Yu, H., Huang, R. C., and Strader, C. D. (1992) *Biochemistry* **31**, 11806–11811

19. Adachi, M., Hashido, K., Trzeciak, A., Watanabe, T., Furuichi, Y., and Miyamoto C. (1993) *J. Cardiovasc. Pharmacol.* **22**, S121–S124

20. Krystek, S. R., Patel, P. S., Rose, P. M., Fisher, S. M., Kienzle, B. K., Lach, D. A., Liu, E. C.K., Lynch, J. S., Novotny, J., and Webb, M. L. (1994) *J. Biol. Chem.* **269**, 12383–12386

21. Chini, B., Mouillac, B., Ala, Y., Balestre, M., Kallmeyer, S., Hoflack, J., Elands, J., Hibert, M., Manning, M., Jard S., and C. Barberis. (1995) *EMBO J.* **14**, 2176–2182

22. LaRosa, G. J., Thomas, K. M., Kaufmann, M. E., Mark, R., White, M., Taylor, L., Gray, G., Witt D., and Navarro, J. (1992) *J. Biol. Chem.* **267**, 25402–25406

23. Holtmann, M. H., Hadac, E. M., and Miller, L. J. (1995) *J. Biol. Chem.* **270**, 14394–14398

24. Viladarga, J. P., De Neef, P., Di Paolo, E., Bollen, A., Waelbroeck, M., and
Robberecht, P. (1995) *Biochem. Biophys. Res. Commun.* **211**, 885-891

25. Juppner, H., Schipani, E., Bringhurst, F. R., McClure, I., Keutmann, H. T., Potts, J. T., Kronenberg, H. M., Abou-Samra, A. B., Segre, G. V., and Gardella, T. J. (1994) *Endocrinology* **134**, 879-884

26. Beinborn, M., Lee, Y.-M, McBride, E. W., Quinn, S. M., and Kopin, A. S. (1993) *Nature* **362**, 348-350

27. Kopin, A. S., E. W. McBride, S. M. Quinn, L. F. Kolakowski, Jr., and Beinborn, M. (1995) *J. Biol. Chem.* **270**, 5019-5023

28. Lee, J. A., Elliott, J. D., Sutiphong, J. A., Friesen, W. J., Ohlstein, E. H., Stadel, J. M., Gleason, J. G., and Peishoff, C. E. (1994) *Proc. Natl. Acad. Sci. U. S. A.* **91**, 7164-7168

29. Kunkel, T. A., Roberts J. D., and Zakour, R. A. (1987) *Methods Enzymol.* **154**, 367-382

30. Higuschi, R. (1989) in *PCR Technology* (Erlich, H. A., ed) pp. 61-70, Stockton Press, New York

31. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) *Proc. Natl. Acad. Sci. U. S. A.* **74**, 5463-5467

32. Cullen, B. R. (1987) *Methods Enzymol.* **152**, 684-704

33. DeLean, A., Munson, P. J., and Rodbard, D. (1978) *Am. J. Physiol.* **235**, E97-E102

34. Karnik, S. S., Sakmar, T. P., Chen H., and Khorona H. G. (1988) *Proc. Natl. Acad. Sci. U. S. A.* **85**, 8459-8463