Introduction

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Vasoactive intestinal peptide (VIP) is a widely distributed neurotransmitter that may play a role in a number of physiological and pathophysiological processes (Dockray, 1994; Gozes and Brenneman, 2000). Important possible pathophysiological effects include its growth effects on cancer cells (Moody, 1996; Jiang et al., 1997; Zia et al., 2000); a potential treatment role for VIP has been proposed in asthma (Groneberg et al., 2001), impotence (Sandhu et al., 1999), various central nervous system disorders through a neuroprotective effect (Gozes and Brenneman, 2000), and various inflammatory disorders, such as rheumatoid arthritis, because of its potent anti-inflammatory effects (Gomariz et al., 2001; Delgado et al., 2002). Furthermore, use of VIP in localizing various tumors such as breast and gastrointestinal cancers by imaging methods has been proposed because these tumors have a high density of VIP receptors (Virgolini, 1997).

Two receptor subtypes designated VPAC₁ and VPAC₂ are identified (Harmar et al., 1998; Ulrich et al., 1998). These receptors have distinct pharmacologies (Harmar et al., 1998; Ulrich et al., 1998; Nicole et al., 2000) and differ in their distribution in the central nervous system and peripheral tissues (Dockray, 1994; Usdin et al., 1994; Waschek et al., 1995; Ito et al., 2000; Reubi et al., 2000; Delgado et al., 2002). Unlike the pharmacology (Harmar et al., 1998) and the physiology of VPAC₁ (Dockray, 1994; Gaudin et al., 1996; Ito et al., 2000; Nicole et al., 2000), VPAC₂ has been poorly described.

Simplified agonists or antagonists of VIP that retain high affinity, with high selectivity for VPAC₂, and that are metabolically stable would be of value for investigating its physiological or pathological role in vivo or to use as a therapeutic agent. To eventually develop such analogs, it is necessary to understand the VIP pharmacophore for VPAC₂ and how it is similar to or different from the closely related receptor, VPAC₁. However, minimal information exists on the pharmacophore of VIP for VPAC₂. There is only one study (Nicole et al., 2000) that has examined the VIP pharmacophore for human VPAC₂ (hVPAC₂) using alanine scanning, and no study has examined the importance of the orientation of the amino acid substitutions using D-amino acid scanning of VIP. Furthermore, a recent study (Igarashi et al., 2002) demonstrated with VPAC₁ that significant differences in the VIP pharmacophore can exist between the human receptor and that in animals commonly used in many experimental studies, such as rats (Igarashi et al., 2002). Also, a recent study (Igarashi et al., 2002) demonstrates that the type of cell in which VPAC₁ is stably expressed can have a significant effect on the VIP pharmacophore. Therefore, to be certain that the VIP pharmacophore obtained in VPAC₂-transfected cells is reflective of the native receptor, it is essential the results be compared with that of a cell expressing native receptors. This has not been done in detail in any study. This can be difficult because many cells possess both VPAC₁ and VPAC₂, and normally VPAC₁ is expressed in greater numbers (Waschek et al., 1995; Busto et al., 1999; Ito et al., 2000; Reubi et al., 2000). Whether any of these points also applies to VPAC₂ and are important in establishing the true VIP pharmacophore is at present unknown.

Therefore, the goal of the present study was to determine the VIP pharmacophore for VPAC₂. To accomplish this, we performed systematic alanine scanning and D-amino acid scanning of VIP and determined its effect on affinity and potency, as well as efficacy, for VPAC₂ receptor activation in both human and rat VPAC₂ stably transfected CHO cells and PANC1 cells, and compared the results with Sup T₁ lymphoblastoma cells, which natively possess only hVPAC₂ (Igarashi et al., 2002).

	Materials and Methods

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Chinese hamster ovary (CHO) cells, PANC1 human pancreatic cancer cells, and Sup T₁ human lymphoblastoma cells were obtained from the American Type Culture Collection, Manassas, VA. Porcine vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating polypeptide (PACAP) (1-27) were obtained from Bachem Biosciences (King of Prussia, PA). Basal medium Eagle amino acid mixture, basal medium Eagle vitamin solution, fetal bovine serum, pcDNA 3.1(+), pcDNA 3.1(), and LipofectAMINE transfection reagent were obtained from Invitrogen (Carlsbad, CA); geneticin (G418 sulfate) was from Mediatech (Herndon, VA). Bacitracin, soybean trypsin inhibitor, 3-isobutyl-1-methylxanthine (IBMX), and alumina were obtained from Sigma-Aldrich (St. Louis, MO). The Dowex AG 50W-X4 resin was from Bio-Rad (Hercules, CA). Bovine serum albumin (BSA) fraction V was from ICN Pharmaceuticals Biochemicals Division (Aurora, OH). [¹²⁵I]VIP (2200 Ci/mmol) and [¹²⁵I]PACAP(1-27) (2200 Ci/mmol) were obtained from PerkinElmer Life Sciences (Boston, MA); Na¹²⁵I (2200 Ci/mmol) and [2-³H]adenine (22 Ci/mmol) were from Amersham Biosciences Inc. (Piscataway, NJ); 1,3,4,6-Tetrachloro 3,6-diphenylglycouril (IODO-GEN) was obtained from Pierce Chemical Co. (Rockford, IL); HEPES was obtained from Roche Diagnostics (Indianapolis, IN). Dulbecco's modified Eagle's medium (DMEM), RPMI 1640 medium, and Ham's F-12K medium were obtained from Biofluids (Rockville, MD). The standard incubation solution contained 24.5 mM HEPES (pH 7.45), 98 mM NaCl, 6 mM KCl, 2 mM KH₂PO₄, 5 mM sodium pyruvate, 5 mM sodium fumarate, 5 mM sodium glutamate, 2 mM glutamine, 11.5 mM glucose, 0.5 mM CaCl₂, 1 mM MgCl₂, 1% (w/v) BSA, 0.2% (w/v) soybean trypsin inhibitor, 1% (v/v) amino acid mixture, and 1% (v/v) essential vitamin mixture.

Preparation of Peptides. Native VIP has an alanine in positions 4 and 18, so that the remaining 26 amino acids were replaced with alanine sequentially. Therefore, 26 VIP analogs with a single alanine substitution in VIP numbered from the amino terminal amino acid position 1 to position 28 in the carboxyl terminus of VIP were synthesized. In addition, 28 VIP analogs with a single D-amino acid substitution in VIP numbered from the amino-terminal amino acid to position 28 in the carboxyl terminus of VIP were synthesized. Synthesis was performed using standard solid phase methods as described previously (Sasaki and Coy, 1987; Igarashi et al., 2002). [Lys¹⁵,Arg¹⁶,Leu²⁷]VIP(1-7)GRF(8-27) (a VIP analog selective for VPAC₁) (Gourlet et al., 1997a; Harmar et al., 1998; Ito et al., 2000) and Ro 25-1553 (a cyclic VIP analog selective for VPAC₂) (O'Donnell et al., 1994; Gourlet et al., 1997b) were also synthesized using a similar procedure. Homogeneity of the peptides was assessed by thin-layer chromatography and analytical reverse phase HPLC, and purity was at least 97% for each peptide.

Transfection of CHO or PANC1 Cells with hVPAC₂ and rVPAC₂ and Selection of Stable Transfectants. Construction of the rat VPAC₂ receptor (rVPAC₂), and hVPAC₂ receptor expression vector was described previously (Ito et al., 2000, 2001; Igarashi et al., 2002). Construction of hVPAC₂ or rVPAC₂ stably transfected CHO or PANC1 cells (hVPAC₂ or rVPAC₂/CHO or PANC1 cells) was described previously (Ito et al., 2001). Briefly, to select stably transfected CHO and PANC1 cells containing hVPAC₂ or rVPAC₂, after transfection with LipofectAMINE, individual colonies were isolated and expanded, and cloned cells were screened for VPAC receptor expression by receptor binding of [¹²⁵I]VIP or [¹²⁵I]PACAP(1-27). For rat and human VPAC receptors in each cell type, at least four clones were isolated, and binding of [¹²⁵I]VIP or [¹²⁵I]PACAP(1-27) was assessed in more detail by dose-inhibition curves for VIP and related peptides. For each cell type and species the affinities of the different clones were similar for VIP and the other peptides tested. The clone showing the highest binding was selected for additional studies.

Cell Culture. The hVPAC₂/CHO and rVPAC₂/CHO cells were grown in Ham's F-12K medium supplemented with 2 mM L-glutamine, 10% (v/v) fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 300 µg/ml G418. The rVPAC₂/PANC1 cells and hVPAC₂/PANC1 cells were grown in DMEM supplemented with 10% (v/v) fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, and 300 µg/ml G418. Sup T₁ human lymphoblastoma cells were grown in RPMI 1640 medium supplemented with 2 mM L-glutamine, 10% (v/v) fetal bovine serum, and antibiotics (penicillin/streptomycin), adjusted to contain 1.5 g/l sodium bicarbonate, 4.5 g/l glucose, 10 mM HEPES, and 1.0 mM sodium pyruvate as recommended by the American Type Culture Collection. They were maintained in incubators at 37°C in an atmosphere of 5% CO₂ and 95% air.

Preparations of [¹²⁵I]Ro 25-1553. [¹²⁵I]Ro 25-1553 at a specific activity of 2200 Ci/mmol was prepared by a modification of the methods described previously (Zhou et al., 1989). Briefly, 0.8 µg of IODO-GEN in chloroform was transferred to a vial, dried under a stream of nitrogen, and washed with 100 µl of 0.5 M KH₂PO₄ (pH 8.0). To this vial, 20 µl of 0.5 M KH₂PO₄ (pH 8.0), 8 µg of peptide in 4 µl of water, and 2 mCi (20 µl) of Na¹²⁵I were added, mixed gently, and incubated at room temperature for 6 min. The incubation was stopped by the addition of 100 µl of distilled water. The iodination mixture was applied to a Sep-Pak (Waters Associates, Milford, MA), and free ¹²⁵I was eluted with 5 ml of water followed by 5 ml of 0.1% (v/v) trifluoroacetic acid. The radiolabeled peptides were eluted with 200 µl of sequential elutions (10 times) with 60% acetonitrile in 0.1% trifluoroacetic acid. The two or three fractions with the highest radioactivity were combined and purified on reverse-phase, high performance liquid chromatography with a Vydac C18 column (0.46 × 25 cm; Vydac/The Separations Group, Hesperia, CA). The column was eluted with a linear gradient of acetonitrile in 0.1% trifluoroacetic acid (v/v) from 16 to 60% acetonitrile in 60 min, and 1-ml fractions were collected and assayed for radioactivity and receptor binding. The pH of the pooled fractions was adjusted to 7 using 0.2 M Tris (pH 9.5), and radioligands were stored in aliquots with 0.5% bovine serum albumin (w/v) at -20°C.

Binding Studies. Both the binding of [¹²⁵I]VIP to hVPAC₂/CHO and rVPAC₂/CHO cells, and the binding of [¹²⁵I]PACAP(1-27) to hVPAC₂/PANC1 and rVPAC₂/PANC1 cells were performed by incubation in standard incubation solution containing 0.05% (w/v) bacitracin for 60 min at room temperature. To assess VPAC₂ affinities in Sup T₁ human lymphoblastoma cells, binding was performed using [¹²⁵I]Ro 25-1553 in standard incubation solution containing 0.05% (w/v) bacitracin for 60 min at 37°C, because [¹²⁵I]VIP and [¹²⁵I]PACAP(1-27) were rapidly degraded in these cells even with protease inhibitors present. The separation of bound from free radioactivity was obtained by centrifugation of cells through 2% (w/v) BSA in standard incubation solution. The tubes were washed twice with 2% (w/v) BSA in standard incubation solution, and radioactivity was counted. Nonsaturable binding for [¹²⁵I]VIP, [¹²⁵I]PACAP(1-27), or [¹²⁵I]Ro 25-1553 was less than 5% of total binding.

cAMP Assay. hVPAC₂/PANC1 (0.05 × 10⁶ cells) and hVPAC₂/CHO (0.05 × 10⁶ cells) were plated on 24-well plates and incubated for 48 h at 37°C with medium containing 10% FBS (v/v). The medium was then replaced with medium supplemented with 2% FBS (v/v) and 2 µCi/ml [2-³H]adenine. Cells were incubated for an additional 48 h at 37°C. The medium was removed and cells were incubated in 500 µl of DMEM containing 1% (w/v) BSA and 0.5 mM IBMX, with or without peptides at various concentrations for 1 h at 37°C. Reactions were terminated by the addition of 120 µl of cAMP stopping solution [2% SDS (w/v), 25 mM cAMP] followed by 1 ml of Tris (50 mM, pH 7.4). Samples were stored at 20°C until analyzed. The amount of cAMP formation was determined using a modification of a method reported previously using the Dowex AG 50W-X4 anion exchange resin column and alumina column (Benya et al., 1994). For studying cAMP generation in Sup T₁ cells, 20 ml of medium containing 2.0 to 4.0 × 10⁶ cells/ml, supplemented with 2% FBS (v/v) and 2 µCi/ml [2-³H]adenine, were incubated for 48 h at 37°C. Then the solution containing the cells was centrifuged and the cell pellet was washed with DMEM twice, after which it was suspended with 50 ml of DMEM containing 1% (w/v) BSA and 0.5 mM IBMX. Next, 500 µl of this cell solution was added to each tube with or without peptides at various concentrations and incubated for 1 h at 37°C. The procedure of termination of the reaction and column processing was the same as above. For all peptides, the EC₅₀ was calculated, which was the concentration of the peptide that gave half-maximal stimulation of a maximally effective concentration of VIP (1 µM) or the peptide tested. The EC₅₀ was calculated using the curve-fitting program KaleidaGraph.

Statistical Analysis. The results are mean ± S.E.M. of three or more experiments. IC₅₀ and EC₅₀ were calculated using the curve-fitting program KaleidaGraph. Statistical comparisons were made using Student's t test.

	Results

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Preparation of Cells Containing Human or Rat VPAC₂ Receptors. To access the pharmacology of VIP at the VPAC₂ receptor, we prepared cell lines with VPAC₂ stably transfected, because many native cells contain more than one subtype of VIP/PACAP receptor (Waschek et al., 1995; Wei and Mojsov, 1996; Fruhwald et al., 1999). VIP can also interact with both VPAC₁ and, at high concentrations, with the PACAP receptor (Alexander and Peters, 1997). hVPAC₂-transfected PANC1 cells (hVPAC₂/PANC1 cells) were used because in our previous study (Igarashi et al., 2002), when hVPAC₁ was stably transfected in the PANC1 cells, the pharmacophore correlated closely with that of the native hVPAC₁ in T47D human breast cancer cells. hVPAC₂-transfected CHO cells (hVPAC₂/CHO cells) were also made because this cell line has been extensively used to characterize the cell biology and pharmacology of VIP receptors in previous studies by others (Gaudin et al., 1996; Gourlet et al., 1997a,b). Furthermore, to confirm that the pharmacology in the transfected cell lines was similar to that of the native receptor, we screened various cell lines reported to contain hVPAC₂ (Robberecht et al., 1996; Vertongen et al., 1996). We found, using reverse transcription-polymerase chain reaction with Southern blotting, that Sup T₁ human lymphoblastoma cells contained only hVPAC₂ (Igarashi et al., 2002). We also found, using reverse transcription-polymerase chain reaction, hVPAC₂/PANC1 cells, or hVPAC₂/CHO cells, that only VPAC₂ expression was detected (data not shown).

View larger version (23K): [in this window] [in a new window]   Fig. 1.   Abilities of VIP, [Lys15,Arg16,Leu27]VIP(1-7)GRF(8-27), and Ro 25-1553 to inhibit binding of 125I-labeled peptide to the native human VPAC2 receptor in Sup T1 lymphoblastoma cells, hVPAC2-transfected PANC1 cells, or hVPAC2-transfected CHO cells. Sup T1 human lymphoblastoma cells (top panel), which possess native VPAC2 receptor (2.5 × 106 cells/ml), were incubated for 60 min at 37°C with 75 pM [125I]Ro 25-1553 alone or with the indicated concentrations of unlabeled peptides. Human VPAC2 receptor stably transfected PANC1 cells (0.1 × 106 cells/ml) (middle panel) were incubated for 60 min at room temperature with 75 pM [125I]PACAP(1-27) alone or with the indicated concentrations of unlabeled peptides. Human VPAC2 receptor stably transfected CHO cells (0.2 × 106 cells/ml) (bottom panel) were incubated for 60 min at room temperature with 75 pM [125I]VIP with or without the indicated concentrations of unlabeled peptides. Results are expressed as the percentage of the saturable binding of 125I-labeled peptide observed in the absence of competing peptide. In each experiment, each value was determined in duplicate, and results given are means ± S.E.M. from at least three separate experiments.

View this table: [in this window] [in a new window]   TABLE 1 Abilities of VIP, [Lys15, Arg16, Leu27]VIP(1-7)GRF(8-27), and Ro 25-1553 to interact with human VPAC1 receptor and VPAC2 receptor cells containing native receptor or stably transfected human VPAC1 or VPAC2 Sup T1 human lymphoblastoma cells, hVPAC2/PANC1, and hVPAC2/CHO cells were incubated with 75 pM 125I-labeled peptide and various concentrations of unlabeled VIP and analogs as described in the legend to Fig. 1. T47D breast cancer cells, which natively possess hVPAC1 (Igarashi et al., 2002), hVPAC1/PANC1, and hVPAC1/CHO cells were incubated with 75 pM [125I]VIP and various concentrations of the unlabeled peptides as performed previously (Igarashi et al., 2002). The IC50 was the concentration causing half-maximal inhibition of the saturable binding caused by 1 µM VIP, calculated using the curve-fitting program KaleidaGraph. In each experiment each value was determined in duplicate, and values given are means ± S.E.M. from at least three separate experiments.

Affinity of Single Alanine-Substituted Analogs of VIP for Rat or Human VPAC₂-Containing Cells. To better understand the pharmacophore of VIP for VPAC₂, we performed a scan of VIP by the substitution of each amino acid one at a time with alanine. In general, the alanine scan results were similar in terms of relative affinities to VIP, with each of the cell lines containing hVPAC₂ (Fig. 2; Table 2). Substitution of alanine for Asp³, Phe⁶, Thr⁷, Tyr¹⁰, Tyr²², and Leu²³ caused a >100-fold decrease, and substitution of His¹ or Arg¹² caused a >40-fold decrease in the affinity in every cell line containing hVPAC₂, suggesting that these amino acids were the most important for high affinity interaction at the hVPAC₂ (Fig. 2; Table 2). In 9 of the 26 alanine-substituted analogs, either no change or a decrease in affinity of <5-fold occurred. These included alanine substitution for Ser², Asp⁸, Asn⁹, Gln¹⁶, Val¹⁹, Lys²⁰, Lys²¹, Asn²⁴, and Ser²⁵ (Fig. 2; Table 2). In four positions (Ser², Asp⁸, Asn⁹, and Val¹⁹), alanine substitution caused a small (<2-fold) increase in the affinity over VIP for hVPAC₂ in at least one cell line. There was an excellent correlation (r = 0.965, p < 0.0001) between the IC₅₀ values of the hVPAC₂/PANC1 cells and Sup T₁ cells, as well as the hVPAC₂/CHO cells and the Sup T₁ cells (r = 0.925, p < 0.0001). However, for many analogs, the affinities were greater in hVPAC₂/CHO cells than that for the native hVPAC₂ in Sup T₁ cells or in the hVPAC₂/PANC1 cells. Specifically, for 12 of the 26 alanine-substituted analogs, the affinity varied from 2.0- to 23.3-fold, with a mean of 6.5-fold greater with hVPAC₂/CHO cells than with Sup T₁ cells; and for 9 of the 26 alanine-substituted analogs, the affinity varied from 2.0- to 4.1-fold with a mean of 2.7-fold greater with hVPAC₂/CHO cells than with hVPAC₂/PANC1 cells (Table 2).

View larger version (50K): [in this window] [in a new window]   Fig. 2.   Relative affinities of various single alanine-substituted VIP analogs for native or transfected human (top) or rat (bottom) VPAC2 receptors compared with native VIP. Binding studies to human VPAC2-containing cells were performed (top) using 75 pM 125I-labeled ligand as described in the legend to Fig. 1 and under Materials and Methods. Binding to rVPAC2/CHO cells (0.5 × 106 cells/ml) and rVPAC2/PANC1 cells (0.25 × 106 cells/ml) (bottom) was performed with 75 pM [125I]VIP or [125I]PACAP(1-27), respectively, for 60 min at room temperature. Results are shown with VIP analogs with a single alanine substitution from position 1 on the left to position 28 of VIP on the right. Native VIP has an alanine in positions 4 and 18. Data are expressed as the ratio of the affinity of a given peptide divided by the affinity (IC50) of native VIP calculated from data in Table 2. Less or more potency indicates the amount of decrease or increase, respectively, in affinity that a given alanine substitution caused compared with native VIP. Data are the means from at least three experiments, and in each experiment each point was determined in duplicate.

Affinity of Single D-Amino Acid-Substituted Analogs of VIP for the Human VPAC₂ Receptor. To investigate the importance of the orientation of the amino acid backbone substitution at each position of VIP for determining affinity for the hVPAC₂, studies similar to that performed with alanine-substituted VIP analogs were performed with each amino acid of VIP replaced one at a time with its D-isomer (Fig. 3; Table 3). As seen with the alanine-substituted analogs, the VIP pharmacophore demonstrated by comparing the relative affinity of each D-amino acid-substituted analog to VIP was generally close between the different hVPAC₂ cell types (Fig. 3; Table 3). Only substitution of D-Asn²⁴, D-Leu²⁷, and D-Asn²⁸ caused <5-fold decrease in affinity compared with VIP. In contrast, a decrease >100-fold occurred in all cell lines with substitutions of D-Phe⁶, D-Thr⁷, D-Asp⁸, D-Tyr¹⁰, D-Thr¹¹, D-Arg¹⁴, D-Lys¹⁵, D-Met¹⁷, D-Val¹⁹, D-Lys²¹, D-Tyr²², and D-Ile²⁶. These results demonstrate that the orientation of these amino acids' side chains was particularly important for high affinity VPAC₂ receptor interaction. The other 13 substitutions resulted in a 5- to 99-fold decrease in affinity for the hVPAC₂ (Fig. 3; Table 3). Similar to the alanine substitution, the absolute affinities were generally greater for each D-amino acid-substituted analog in hVPAC₂/CHO cells than for hVPAC₂/PANC1 cells or native hVPAC₂ on Sup T₁ cells. Specifically, 19 of 28 D-amino acid-substituted analogs had 4.2 ± 1.2-fold (range 1.2-26.9) higher affinity for hVPAC₂/CHO cells than for Sup T₁ cells, and 19 of 28 D-amino acid substituted analogs had 3.7 ± 0.7-fold (range 1.2-13.8) higher affinity for Sup T₁ cells than for hVPAC₂/PANC1 cells (Fig. 3; Table 3).

View larger version (61K): [in this window] [in a new window]   Fig. 3.   Relative affinities of various D-amino acid-substituted VIP analogs for native or transfected human (top) or rat (bottom) VPAC2 receptors compared with native VIP. The experimental conditions were the same as those described in the legend to Fig. 2. The IC50 for each peptide was calculated as described in the legend to Fig. 2, and the data were expressed as the ratio of the affinity of a given peptide to that of native VIP using the data in Table 3. Data are the means from at least three experiments, and in each experiment, each point was determined in duplicate.

Efficacy of VIP Analogs with a Single Alanine or D-Amino Acid Substitution to Stimulate cAMP Generation in Human VPAC₂ Stably Transfected Cells. In previous studies, often with tissues containing more than one VIP receptor subtype, various VIP analogs are reported to be partial agonists or antagonists (Gaudin et al., 1996; Moreno et al., 2000). Therefore, to investigate the effect of these amino acid substitutions on efficacy, we determined the ability of each of the VIP analogs with single alanine or D-amino acid to maximally stimulate adenylate cyclase by VPAC₂ receptor activation (Table 4). All VIP analogs were tested at 1 µM concentration in hVPAC₂/PANC1 and hVPAC₂/CHO cells, and those without full efficacy in at least one cell line were tested at higher concentrations. Twenty-five of the 26 alanine-substituted and 25 of the 28 D-amino acid-substituted VIP analogs were full agonists in at least one cell type (Table 4). [D-Val⁵]VIP, [D-Asp⁸]VIP, and [D-Leu²³]VIP were partial agonists causing 60 to 70%, 70 to 80%, and 50 to 70% of the maximal stimulation caused by the maximally effective concentration of VIP (Fig. 4). In contrast, [Ala⁶]VIP, [D-Phe⁶]VIP, [D-Thr⁷]VIP, [D-Thr¹¹]VIP, and [D-Val¹⁹]VIP did not cause maximal stimulation even at concentrations as high as 10 µM; however, this could be explained by the low affinity of these analogs for hVPAC₂ (Table 3; Figs. 3 and 4).

View larger version (23K): [in this window] [in a new window]   Fig. 4.   Comparison of the dose-response curves for VIP and various substituted VIP analogs without full efficacy, to stimulate cAMP accumulation in hVPAC2/CHO cells. The eight VIP analogs (one alanine and seven D-amino acid-substituted analogs) that did not demonstrate the maximal efficacy seen with 1 µM VIP in Table 4 were examined. cAMP generation was determined in hVPAC2/CHO cells (0.05 × 106 cells/well) loaded with [3H]adenine as described under Materials and Methods after 60 min incubation at 37°C with the indicated concentration of VIP or the various VIP analogs. Results are expressed as a percentage of the maximal stimulation of cAMP accumulation caused by 1 µM VIP that was 20.0 ± 3.2-fold over control. In each experiment each value was determined in duplicate and values given are means ± S.E.M. from at least three separate experiments.

Potency of Alanine or D-Amino Acid-Substituted VIP Analogs for Stimulating Adenylate Cyclase through Human VPAC₂ Activation. To compare in detail the potency of the various VIP analogs to activate hVPAC₂ in the different VPAC₂-containing cells with their binding affinities, we determined the dose-response curves for adenylate cyclase stimulation for 22 of the alanine and 18 of the D-amino acid-substituted VIP analogs with different binding affinities (Fig. 5; Table 5). VIP caused a half-maximal stimulation at 4.2 to 6.5 nM (Table 5) in hVPAC₂/PANC1 and CHO cells, which is similar to that reported by others (Gourlet et al., 1997b; Xia et al., 1997; Moreno et al., 2000). In general, the EC₅₀s for the analogs to stimulate cAMP accumulation with hVPAC₂/CHO cells were lower than those with hVPAC₂/PANC1 cells (Table 5). Specifically, with 26 of 34 VIP-substituted analogs in which EC₅₀s could be obtained, the EC₅₀s with hVPAC₂/CHO cells were lower than those with hVPAC₂/PANC1 cells (Fig. 5; Table 5). To determine which hVPAC₂-transfected cells best reflected the potency of the VIP analogs for the native hVPAC₂, we compared the potency of 15 of these analogs and VIP for each hVPAC₂-transfected cell to that for Sup T₁ cells, which natively possess hVPAC₂ (Fig. 6). There was a very close correlation (r = 0.96, p < 0.0001) between the EC₅₀ values for each of these analogs in both the native hVPAC₂ on Sup T₁ cells and hVPAC₂-transfected PANC1 cells. Furthermore, the regression equation comparing the EC₅₀s for these two hVPAC₂ cell types had a slope of 1.02 that was not significantly different from unity (Fig. 6, top). The correlation between the EC₅₀ values for each analog in both the native hVPAC₂ in Sup T₁ cells and hVPAC₂-transfected CHO cells was also good (r = 0.85, p < 0.0001). However, the slope of the regression equation differed markedly between these two types of cells. Specifically, the slope of the regression equation was significantly less than unity (i.e., 0.63) with native and hVPAC₂/CHO cells (p < 0.01) (Fig. 6, bottom).

View larger version (24K): [in this window] [in a new window]   Fig. 5.   Abilities of VIP and various substituted VIP analogs to stimulate cAMP accumulation in various hVPAC2 receptor-containing cells. cAMP stimulation was performed using Sup T1 cells (top panel), hVPAC2/PANC cells (middle panel), or hVPAC2/CHO cells (bottom panel) with or without various concentrations (0.01 nM to 1 µM) of the indicated substituted VIP analogs or native VIP as described under Materials and Methods. Results with four representative analogs with full efficacy to VIP are shown. Maximal stimulation of cAMP accumulation caused by 1 µM VIP was: Sup T1 cells, 7.5 ± 0.9-fold; hVPAC2/PANC1 cells, 49.3 ± 9.8-fold; and hVPAC2/CHO cells, 20.0 ± 3.2-fold over control. In each experiment each value was determined in duplicate and values given are means ± S.E.M. from at least three separate experiments.

View larger version (30K): [in this window] [in a new window]   Fig. 6.   Comparison of the potencies of VIP and various single alanine- or D-amino acid-substituted analogs for stimulating increase in cAMP in Sup T1 cells possessing native human VPAC2 with human VPAC2-transfected PANC1 cells (top panel) or CHO cells (bottom panel). EC50s for each of the hVPAC2-containing cells was determined as described in the legends to Figs. 4 and 5. Data for VIP and 15 VIP analogs are shown ([Ala1]VIP, [Ala2]VIP, [Ala7]VIP, [Ala9]VIP, [Ala10]VIP, [Ala11]VIP, [Ala13]VIP, [Ala16]VIP, [Ala20]VIP, [Ala22]VIP, [Ala27]VIP, [D-Ala4]VIP, [D-Phe6]VIP, [D-Asn24]VIP, and [D-Ser25]VIP). Each point represents data for one analog. The data with EC50s more than 3000 nM were represented as 3000 nM. The best fit and correlation coefficient (r) were calculated by least-squares analysis.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

A number of results in our study demonstrate with VPAC₂, similar to that recently reported with VPAC₁ (Igarashi et al., 2002), that there are significant differences in the VIP pharmacophore for the receptor stably expressed in different cells and that there are important species differences. First, the VIP pharmacophore from alanine or D-amino acid scanning for the native hVPAC₂ on Sup T₁ cells, which contained only hVPAC₂, was almost identical to that revealed by hVPAC₂/PANC1 cells, but differed markedly from hVPAC₂/CHO cells. In a recent study of VPAC₁ (Igarashi et al., 2002), the VIP pharmacophore in stably transfected CHO cells did not faithfully reflect the native hVPAC₁ in T47D human cells, whereas hVPAC₁ in transfected PANC1 cells did. In a recent study of hVPAC₂ (Nicole et al., 2000), as well as in the present study, in general, higher affinities than found in the native receptor are obtained on the receptors transfected into CHO cells. This result was not species-specific because similar results were seen in the present study with rVPAC₂ and in a previous study (Igarashi et al., 2002) with rVPAC₁. However, the extent of increase in affinity with VPAC₂ expression in CHO cells was influenced by the species. The average increase in affinity was 5 times greater with rVPAC₂ in CHO, compared with expression in PANC1 cells, than the gain in affinity of hVPAC₂ in CHO cells over that seen in PANC1 cells. Furthermore, this increase in affinity in CHO-transfected cells is not necessarily constant for each analog; thus, a distortion of the true VIP pharmacophore occurred with both hVPAC₂ in our study and hVPAC₁ in a previous study (Igarashi et al., 2002). At present, the molecular basis for the difference in cell type on affinities for rVPAC₂ and hVPAC₂ is unexplained and could be due to differences in G protein coupling or post-translational modifications. Second, with both D-amino acid and alanine substitution in VIP, there were a number of differences in their effect on the VIP pharmacophore for human and rat VPAC₂. These differences were greater for both alanine- and D-amino acid-substituted analogs for VPAC₁ than for VPAC₂, suggesting that the differences are more related to VPAC receptor subtype rather than differences in differential coupling with high or low affinity states for the different analogs. These results demonstrate that, similar to previous reports with VPAC₁ (Igarashi et al., 2002), it is essential to use transfected systems that reflect the native receptor's VIP pharmacophore for VPAC₂, and that species differences in the VIP pharmacophore of VPAC₂ need to be considered for any analog design.

Alanine scanning demonstrated that the amino acid side chains in VIP that are the most important for determining high affinity for the hVPAC₂ are Phe⁶ = Tyr¹⁰ = Tyr²² > Leu²³  Thr⁷ = Asp³ > Arg¹² > His¹ (Fig. 7). In contrast, the alanine substitution for Ser², Asp⁸, Asn⁹, Gln¹⁶, Val¹⁹, Lys²⁰, Lys²¹, Asn²⁴, and Ser²⁵ caused either no change or only a small decrease (<5-fold) in affinity (Fig. 7), showing that their side chains were not essential for high affinity VPAC₂ interaction. Our results with alanine scanning show similarities to and differences from the few previous studies that have examined the importance of some amino acids in VIP for high affinity hVPAC₂ interaction (Gourlet et al., 1998; Nicole et al., 2000). One important difference we found from one study (Nicole et al., 2000) was that alanine substitution for Tyr²² caused a marked decrease in affinity (>400-fold), whereas it caused only 6.5-fold decrease in the previous study (Nicole et al., 2000). Our results generally agree with a second study (Gourlet et al., 1998), which reported that an Ala²² substitution in VIP caused a 100-fold decrease in affinity for hVPAC₂ and support the proposal (Gourlet et al., 1998) that an aromatic residue at position 22 of VIP was necessary for high affinity for VPAC₂. Another difference from a previous study (Nicole et al., 2000) we found was that alanine substitution for Asp⁸ or Lys²¹ had only a minimal effect on affinity (<2- to 3-fold decrease), whereas they were reported to cause a >300-fold and >60-fold decrease in affinity, respectively (Nicole et al., 2000). These differences are at least partially due to the expression of hVPAC₂ in CHO cells in this previous study (Nicole et al., 2000), which we demonstrate do not reliably reflect the VIP pharmacophore of the native hVPAC₂. Although the general VIP pharmacophore for rVPAC₂ was similar to that for hVPAC₂, there were a number of important differences. In particular, with alanine substitution for His¹, Asn⁹, Lys²⁰, and Ile²⁶, there was a greater effect on the affinity for rVPAC₂ than for hVPAC₂ (p < 0.03).

View larger version (132K): [in this window] [in a new window]   Fig. 7.   VIP pharmacophore for the human VPAC2 receptor. VIP amino acid residues 1 to 28 are shown as two -bends (residues 2-5, 7-10) and a single -helix (residues 11-26) as proposed from conformational, CD, and NMR studies (Fournier et al., 1988; Fry et al., 1989). The most important amino acids revealed by alanine scanning (>50-fold decrease in affinity with alanine substitution) on binding or biological activity are shown in yellow. Amino acid residues having intermediate importance (alanine substitution causes a 5- to 50-fold decrease) are shown in blue. The 9 amino acid residues whose backbone substitutions were not essential for high affinity or potency (substitution causes <5-fold decrease) at the VPAC2 are shown in green.

VIP alanine scanning in hVPAC₂ has some similarities to and differences from that previously reported for hVPAC₁ by us (Igarashi et al., 2002) (Fig. 8, top) and others (Gourlet et al., 1998; Nicole et al., 2000). The two VPAC receptors are similar in VIP pharmacophore in that the side chains of Ser², Asp⁸, Asn⁹, Val¹⁹, Lys²¹, Asn²⁴, and Ser²⁵ are not essential for high affinity for either receptor, whereas the side chains of Asp³, Phe⁶, and Leu²³ are essential for high affinity for both VPAC receptor subtypes. The VPAC receptor subtypes differed in the effects of alanine substitution for Thr⁷, Tyr¹⁰, Thr¹¹, Tyr²², and Leu²⁷, which had a much greater effect on the affinity for hVPAC₂ than for hVPAC₁, and thus, analogs with these alterations might be useful to develop selective hVPAC₁ agonists. Conversely, alanine substitution for Arg¹⁴ and Val¹⁹ had a greater effect on the affinity for hVPAC₁ than for hVPAC₂, and this difference could be useful to make selective hVPAC₂ analogs. Our result with alanine substitution for Val¹⁹ is consistent with a recent study (Moreno et al., 2000) that reported that [Ala¹⁹]VIP had a 1.7-fold higher affinity than VIP at hVPAC₂, while having a 1.5-fold lower affinity than VIP at hVPAC₁.

View larger version (49K): [in this window] [in a new window]   Fig. 8.   Comparison of the effect on binding affinity of a single alanine-substitution in VIP (top) or a D-amino acid substitution in VIP (bottom) for the hVPAC1 and hVPAC2. Data for hVPAC2-transfected PANC1 cells are from Fig. 2 and those for hVPAC1-transfected PANC1 cells are from Igarashi et al. (2002). Data are expressed as the ratio of the affinity of a given peptide divided by the affinity (IC50) of native VIP. Less or more potency indicates the amount of decrease or increase in affinity that a given alanine (top panel) or D-amino acid (bottom panel) substitution caused compared with native VIP, respectively. The affinity of native VIP for human VPAC1/PANC1 human VPAC2/PANC1 cells was 1.6 ± 0.1 nM and 6.9 ± 0.3 nM, respectively. , position of a substitution that caused a 5- to 10-fold decrease in affinity in hVPAC2/PANC1 compared with hVPAC1/PANC1. , position of a substitution that caused >10-fold decrease in affinity in hVPAC2/PANC1 compared with hVPAC1/PANC1. , position of a substitution that caused 5-fold decrease in affinity in hVPAC1/PANC1 compared with hVPAC2/PANC1.

For the first time, the present study, using D-amino acid scanning, provides insight into the importance of the orientation of a given amino acid side chain in VIP in determining the affinity for VPAC₂. Our results demonstrated that the orientations of side chains of Phe⁶, Thr⁷, Asp⁸, Thr¹¹, Met¹⁷, Val¹⁹, and Tyr²² were particularly important for high affinity receptor-ligand interaction in native hVPAC₂. The orientation of side chains of Asn²⁴, Leu²⁷, and Asn²⁸ were of minimal importance for determining affinity, whereas that of the remaining 18 amino acids was of intermediate importance. Similar to the results with alanine scanning discussed above, our results with D-amino acid scanning show some similarities and significant differences between the importance of the different amino acid side chain orientations in VIP for determining high affinity for hVPAC₂ and hVPAC₁ (Fig. 8, bottom). The orientation of the side chains of Phe⁶, Thr⁷, Asp⁸, Thr¹¹, Val¹⁹, Lys²¹, and Tyr²² were particularly important for determining affinity for both receptor subtypes, whereas that of Tyr¹⁰, Thr¹¹, Arg¹⁴, Lys¹⁵, Met¹⁷, Val¹⁹, Tyr²², and Ile²⁶ was more critical for high affinity for hVPAC₂ than for hVPAC₁. In contrast, the orientation of the side chain of Asn²⁴ was much more important for determining high affinity for hVPAC₁ than for hVPAC₂ (Fig. 8, bottom). In a previous study (Gourlet et al., 1998), [D-Ala⁴]VIP was reported to have a 100-fold higher affinity for VPAC₁ than that for VPAC₂; however, we could not confirm this finding.

To determine the effect of a single D-amino acid or alanine replacement in VIP on its ability to activate VPAC₂, we examined the potency and efficacy of each analog to stimulate adenylate cyclase, which is the main intracellular mediator of VPAC₂ (Ulrich et al., 1998; Zhou et al., 1989; Nicole et al., 2000). There are a number of reports of various VIP analogs, including D-amino acid-substituted analogs, functioning as partial agonists or antagonists (Pandol et al., 1986; Goossens et al., 1992; Moreno et al., 2000; Igarashi et al., 2002). There are few data available on VPAC₂. One recent study reported that introducing a D-Phe² and the acylation of the amino terminus by a fatty acid or deleting the amino terminus of Ro 25-1553 derivatives resulted in the development of a partial agonist or antagonist, respectively, for hVPAC₂ (Moreno et al., 2000). In our study, we found that [D-Val⁵]VIP, [D-Asp⁸]VIP, and [D-Leu²³]VIP were partial agonists at hVPAC₂. However, it is unlikely that these analogs could be useful to make antagonists, because each retained moderately high efficacy. In general, we found a close correlation between potency of the substituted VIP analogs for cAMP generation and their affinities for VPAC₂. In terms of potency for VPAC₂ activation, our results from alanine scanning of VIP had a number of differences from those reported in a recent study (Nicole et al., 2000), especially for alanine substitution of His¹, Ser², Thr⁷, Asp⁸, Thr¹¹, and Ile²⁶. In our study, we found that substitution for His¹ caused 100-fold less of a decrease in potency, Ser² 10-fold less, Asp⁸ 60-fold less, and Ile²⁶ 10-fold less than previously reported (Nicole et al., 2000). In contrast, we found that substitution of Thr⁷ caused a 70-fold greater decrease in potency and Thr¹¹ a 22-fold greater decrease than previously reported (Nicole et al., 2000). These differences were likely partially due to the difference in the cell expression systems and illustrate the importance of comparing results to those from cells possessing native receptors, for potency, similar to that discussed above for the assessment of binding affinity.

In conclusion, in the present study we have systematically studied the VIP pharmacophore for rat and human VPAC₂. This has allowed us to establish the importance of a given side chain of VIP and its orientation for determining high affinity interaction with VPAC₂. Our results demonstrate significant species differences and show that the type of cell used for stable expression of VPAC₂ can have a significant effect on the VIP pharmacophore. Our results show a number of important similarities to and differences from that previously reported for VPAC₁ (Nicole et al., 2000; Igarashi et al., 2002). We have also identified a number of amino acids that could likely be replaced to yield simplified VIP analogs that retain high affinity, are receptor subtype-selective, and could possibly be more metabolically stable.