Diffuse pharmacophoric domains of vasoactive intestinal peptide (VIP) and further insights into the interaction of VIP with the N-terminal ectodomain of human VPAC1 receptor by photoaffinity labeling with [Bpa6]-VIP.

The widespread 28-amino acid neuropeptide vasoactive intestinal peptide (VIP) exerts its many biological effects through interaction with serpentine class II G protein-coupled receptors named VPAC receptors. We previously provided evidence for a physical contact between the side chain at position 22 of VIP and the N-terminal ectodomain of the hVPAC1 receptor (Tan, Y. V., Couvineau, A., Van Rampelbergh, J., and Laburthe, M. (2003) J. Biol. Chem. 278, 36531-36536). We explored here the contact site between hVPAC1 receptor and the side chain at position 6 of VIP by photoaffinity labeling. The photoreactive para-benzoyl-l-Phe (Bpa) was substituted for Phe(6) in VIP resulting in [Bpa(6)]-VIP, which was shown to be a hVPAC1 receptor agonist in Chinese hamster ovary cells stably expressing the recombinant receptor. After obtaining the covalent (125)I-[Bpa(6)-VIP].hVPAC1 receptor complex, it was sequentially cleaved by cyanogen bromide, peptide N-glycosidase F, endopeptidase Glu-C, and trypsin, and the cleavage products were analyzed by electrophoresis. The data demonstrated that (125)I-[Bpa(6)-VIP] were covalently attached to the short 104-108 fragment within the N-terminal ectodomain of the receptor. The data were confirmed by creation of a receptor mutant with new CNBr cleavage site. In a three-dimensional model of the receptor N-terminal ectodomain, this fragment was located on one edge of the putative VIP-binding groove and was adjacent to the fragment covalently attached to the side chain at position 22 of VIP. Altogether these data showed that the central part of VIP, at least between Phe(6) and Tyr(22), interacts with the N-terminal ectodomain of the hVPAC1 receptor.

The neuropeptide vasoactive intestinal peptide (VIP) 1 is present in both central and peripheral nervous systems as well as in immune cells (1). It controls a large array of biological functions in the brain and peripheral organs (1) and was shown recently to exert potent anti-inflammatory actions (2). The two cloned VIP receptors also bind with high affinity another neuropeptide, the pituitary adenylyl cyclase-activating peptide, and have been named VPAC 2 receptors thereby (3). They are class II G protein-coupled receptor-like receptors for all peptides structurally related to VIP and also receptors for parathyroid hormone, calcitonin, and corticotropin-releasing factor (3).
The VPAC1 receptor is prototypic of class II G protein-coupled receptors and has been extensively studied by molecular biology techniques including site-directed mutagenesis and molecular chimerism (for review see Ref. 3). These studies made it possible to delineate the receptor domains involved in high affinity VIP binding (3), selectivity toward some natural peptide agonists (4,5) and also activation of adenylyl cyclase (6). With respect to VIP binding, it appeared that the N-terminal ectodomain of the receptor plays a crucial role, although it is not sufficient to ensure high affinity (3). A three-dimensional model of the N-terminal ectodomain of the hVPAC1 receptor has been developed suggesting the existence of a VIP-binding groove within this domain (7). Despite these extensive studies of the structure-function relationship of hVPAC1 receptor, the physical sites of interaction between VIP and its receptors had remained elusive until recent photoaffinity showing labeling experiments that the side chain of position 22 of VIP is in direct contact with one edge of the putative binding groove in the N-terminal ectodomain (8).
It is well known that VIP has diffuse pharmacophoric domains, with the amino acid residues important for biological activity being distributed along the whole 28-amino acid peptide chain (9). In this context, we further explored the contact sites between VIP and the hVPAC1 receptor by incorporating a photoactivable benzophenone group on the side chain at position 6 of VIP. This was done by substituting para-benzoyl-L-Phe (Bpa) for phenylalanine 6. This site of incorporation was selected for several reasons: (i) Phe 6 is important for the biological activity of VIP (9) and is strictly conserved in all natural hormones structurally related to VIP (1). (ii) The substitution of Bpa for phenylalanine keeps an aromatic residue, and we expected that, even though Phe 6 is important for biological activity, the [Bpa 6 ]-VIP probe should keep reasonable affinity for the receptor. (iii) Position 6 and the previously explored position 22 (8) are at the two ends of the central ␣-helical domain of VIP (9,10). We report here that the amino acid in position 6 of VIP is in the environment of the short sequence 104 -108 within the N-terminal ectodomain of the receptor when the photoaffinity probe is bound to the receptor.

EXPERIMENTAL PROCEDURES
Materials-Restriction enzymes and culture medium were obtained from Invitrogen. Eucaryotic expression vector was from Clontech (Palo Alto, CA). DNA Sequenase kit and radioactive reagents were from Amersham Biosciences. The site-directed mutagenesis kit was from Promega (Charbonnières, France). Synthetic oligonucleotides were from Invitrogen. The human VPAC1 receptor cDNA was cloned in our laboratory (11). A receptor construct containing an inserted FLAG sequence between Ala 30 and Ala 31 and fused in the C-terminal position with the green fluorescent protein (12) was used in all studies. It has the same phenotype as the wild-type receptor with respect to VIP binding and activation of adenylyl cyclase (12). This construct or derived mutants were stably transfected in CHO cells as described (9). The CHO-F7 clone expressing the human VPAC1 construct (B max , 5 pmol/mg protein; K d , 0.6 nM VIP) was used in most experiments. VIP and the photolabile VIP probe, [Ala 17 , para-benzoyl-L-Phe 6 ]-VIP-28 referred to as [Bpa 6 ]-VIP were obtained by custom synthesis from Neosystem (Strasbourg, France). 125 I-VIP, 125 I-[Bpa 6 -VIP], and 125 I-cAMP were prepared and purified in our laboratory as previously described (9). All of the highly purified chemicals used were from Sigma.
Site-directed Mutagenesis-Development of a new hVPAC1 receptor mutant that incorporated an additional site for CNBr cleavage in a key position was necessary for the current work consisting of substitution of methionine for Phe 93 . Five other mutants were also constructed in which residues within the 104 -108 receptor sequence were individually replaced by alanine. Oligonucleotide-directed mutagenesis was performed as previously described (4). Identification of the desired mutations was obtained by direct sequencing of the mutated region (4). The mutants were stably transfected in CHO cells as described (4).
Cell Culture and Membrane Preparation-The CHO-F7 cells expressing the wild-type VPAC1 receptor construct and CHO cells expressing stably transfected receptor mutant were utilized as sources of receptor for this study and were cultured in Ham's F-12 medium supplemented with 10% decomplemented fetal calf serum, 100 units/ml penicillin G, and 100 g/ml streptomycin in a humidified atmosphere containing 95% air and 5% CO 2 at 37°C. The cells were grown to confluency. After removing the culture medium, the attached cells were washed two times with phosphate-buffered saline and then harvested with a rubber policeman and centrifuged for 10 min at 3,000 ϫ g. The cell pellet was exposed for 30 min at 4°C to hypoosmotic 5 mM HEPES buffer, pH 7.4, and the membranes were obtained as described (9) and stored at Ϫ80°C until use. The protein content was measured by the procedure of Bradford (13) with bovine serum albumin as standard.
Ligand Binding and Adenylyl Cyclase Activity Assays-These assays were used for characterization of the newly synthesized photolabile probe [Bpa 6 ]-VIP. The ligand binding assay was as described (9). Briefly, the membranes (100 g of proteins/ml) derived from transfected CHO cells were incubated for 1 h at 30°C with 0.05 nM 125 I-VIP in the presence of increasing concentrations of VIP or [Bpa 6 ]-VIP, in 20 mM HEPES buffer, pH 7.4, containing 2% (w/v) bovine serum albumin. The reaction was stopped by transferring the incubation medium to an excess of ice-cold buffer. Bound and free peptides were separated by centrifugation (14,000 ϫ g for 10 min), and the membrane pellets were washed two times with 10% (w/v) sucrose in ice-cold 20 mM HEPES. The radioactivity was then assayed in a ␥-counter. Specific binding was calculated as the difference between the amount of 125 I-VIP bound in the absence (total binding) and the presence (nonspecific binding) of 1 M unlabeled VIP. The concentration of peptides that elicited halfmaximal inhibition of specific 125 I-VIP binding (K i ) was determined by computer analysis. Adenylyl cyclase activity in cell membranes was assayed in the presence of increasing concentrations of VIP or [Bpa 6 ]-VIP as described (9). Dose-response curves were fitted, and the concentrations of peptides giving half-maximal responses (EC 50 ) were calculated using the Prism software suite (GraphPad Software, San Diego, CA).
Receptor Photoaffinity Labeling-Transfected cells were incubated in darkness with 10 nM 125 I-[Bpa 6 -VIP] in 10 ml of 20 mM HEPES buffer, pH 7.4, containing 0.2% (w/v) ovalbumin, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM tosyl-L-lysine chloromethyl ketone. After 1 h of incubation at room temperature, the cells were pelleted by centrifugation, and the pellets were resuspended in 4 ml of 20 mM HEPES with 1 mM phenylmethylsulfonyl fluoride and 0.1 mM tosyl-L-lysine chloromethyl ketone. 1 ml of labeled cells were then added to each well of a six-well tissue culture plate. This was photolyzed ( ϭ 365 nm) on ice at a distance of ϳ2 cm. After 40 min of UV exposure, the cells were collected into individual 1.5-ml Eppendorf tubes, washed two times with 10 mM HEPES containing 25 mM glycine, 75 mM NaCl buffer, pH 2.5, and washed one time with 20 mM HEPES. The photolabeled receptors were then analyzed directly by electrophoresis or after chemical or enzymatic cleavage.
Chemical Cleavage of Photoaffinity-labeled VPAC1 Receptor Constructs-The photolabeled receptors in cells were incubated overnight in darkness with 10 mg/ml CNBr in 80% formic acid at room temperature. The CNBr present in the reaction mixture was then removed by Waters C18 Sep-Pak purification. After extensive washing of the Sep-Pak cartridge with 40% acetonitrile in 0.1% trifluoroacetic acid, CNBrgenerated peptide fragments were eluted with 2 ml of 100% acetonitrile. The fractions were counted for radioactivity and evaporated under vacuum (Speed-Vac). The resulting material was either analyzed directly by electrophoresis or after enzymatic treatments (see below). After migration, labeled bands were visualized by autoradiography. For further enzymatic treatments the bands were cut out, electroeluted (model 422 electroeluter; Bio-Rad), and lyophilized.
Enzymatic Treatments-For protein deglycosylation, the material obtained after CNBr treatment was incubated for 2 h at 37°C with PNGase F (3 units/ml) as described (14). Endopeptidase Glu-C digestion of proteins in this material was obtained by incubation for 6 h at 25°C in 25 mM ammonium carbonate, 0.01% SDS, 5% acetonitrile, pH 7.8, as described (15). Trypsin cleavage of proteins was performed for 2 h at 37°C in 0.1 M MOPS buffer, pH 7.0.
Electrophoresis and Autoradiography-Products of cleavage were resolved on NuPAGE 4 -12% Bis-Tris Gel using MES SDS running buffer system from Invitrogen performed according to the method of Laemmli (16) under reducing conditions in the presence of 20 mM dithiothreitol. After electrophoresis, the gels were dried and exposed to x-ray film for 3-10 days with intensifying screens at Ϫ80°C as described (14). The apparent molecular masses of radiolabeled receptor fragments were determined by interpolation on a plot of the mobility of the protein molecular weight markers colored with Rainbow TM from Amersham Biosciences or protein ladder prestained with Benchmark TM from Invitrogen versus the log values of their masses.

RESULTS AND DISCUSSION
In this study, the synthetic [Bpa 6 ]-VIP photoaffinity probe that incorporated the photolabile residue para-benzoyl-L-Phe to replace Phe 6 of VIP was first evaluated for its ability to interact specifically with the human VPAC1 receptor stably expressed in CHO-F7 cells. [Bpa 6 ]-VIP has an efficacy similar to that of native VIP in stimulating adenylyl cyclase activity but a lower potency with EC 50 values of 1.10 and 0.05 nM, respectively ( Fig. 1). In the binding assay, [Bpa 6 ]-VIP was also less potent than native VIP in inhibiting 125 I-VIP binding with K i values of 36 and 0.6 nM, respectively. These data indicate that the [Bpa 6 ]-VIP probe is a low potency VPAC1 receptor agonist which is instrumental for photoaffinity labeling of the receptor because its affinity is not too much decreased. The fact that the addition of a para-benzoyl group on Phe 6 of VIP results in a decrease of affinity for VIP receptor is in agreement with the important role of Phe 6 in VIP activity as previously demonstrated by alanine scanning of the neuropeptide (9). As stated under "Materials," the photoaffinity probe is actually Ala 17 in addition to Bpa 6 . The replacement of Met 17 by Ala was employed to simplify subsequent analysis following CNBr cleavage (see below) and was previously shown to keep intact the binding affinity for the hVPAC1 receptor (9).
After incubation of CHO-F7 cells with the radioiodinated 125 I-[Bpa 6 -VIP] probe, labeled cells were exposed to UV to create a covalent bond between the probe and the receptor. SDS-PAGE analysis of proteins revealed the existence of a single labeled band that completely disappeared when an excess of cold VIP was co-incubated with the radioiodinated probe (Fig. 2). This band migrated at a molecular mass of 95 kDa (Fig.  2) in good agreement with the expected mass (92 kDa) considering the previously characterized (14) mass of the glycosylated VPAC1 receptor (64 kDa), the presence of the green fluorescent protein (25 kDa) at C terminus of the receptor, and the covalently attached [Bpa 6 ]-VIP (3 kDa) in the complex. The 95-kDa labeled band shifted to a 70-kDa band after deglycosylation with PNGase F (Fig. 2) in consonance with the presence of the 9-kDa carbohydrate moiety on each of the three consensus N-glycosylation sites in the N-terminal extracellular domain of the hVPAC1 receptor (14). Altogether, these data indicated that the 125 I-[Bpa 6 -VIP] photoaffinity probe was able to efficiently and specifically label the human VPAC1 receptor.
We proceeded with our study with the identification of the VPAC1 receptor domain to which 125 I-[Bpa 6 -VIP] is covalently attached. For that purpose the 95-kDa band corresponding to 125 I-[Bpa 6 -VIP]⅐hVPAC 1 R complex was subjected to a series of chemical and enzymatic cleavages. Cyanogen bromide cleavage of the 95-kDa band generated a single 30-kDa labeled band (Fig. 3). This band was further shifted to 11 kDa after treatment with PNGase F (Fig. 3), indicating that it is heavily glycosylated, most probably at two consensus N-glycosylation sites. Considering the molecular mass of the attached probe (3 kDa) and the probable presence of the 9-kDa carbohydrate moiety on each of the N-glycosylation sites (14), the best can-didate corresponding to the covalent attachment of 125 I-[Bpa 6 -VIP] is represented by the Trp 67 -Met 137 glycosylated receptor fragment (Table I) (Table I). To further validate the Trp 67 -Met 137 receptor fragment as the region of interaction with the 125 I-[Bpa 6 -VIP] probe, we created a new CNBr cleavage site by substituting a methionine for Phe 93 by site-directed mutagenesis. This site was chosen because it stands between the two N-glycosylation sites on Asn 69 or Asn 100 . The mutant F93M was constructed and stably expressed in CHO cells. It bound VIP with an affinity similar to that of the wild-type receptor (Fig. 4). The K d for VIP was 1.5 and 0.6 nM for the F93M mutant and wild-type receptor, respectively. After incubation of the F93M mutant with 125 I-[Bpa 6 -VIP] and CNBr treatment of proteins, a 18-kDa labeled band was observed instead of a 30-kDa band for the wild-type receptor (Fig. 5). The 12-kDa mass difference observed between the wild-type receptor and mutant cannot be simply accounted for by the shortening of the peptide fragment e.g. 2.6 kDa for the 67-93 sequence or 4.3 kDa for the 94 -137 sequence. It is consistent the removal of the 9-kDa carbohydrate moiety known to be linked to Asn 69 and Asn 100 (Ref. 14 and Fig. 3). Moreover, as shown in Fig. 5, the deglycosylation of the 18-kDa labeled band with PNGase F generated a fragment migrating at an approximate molecular mass of 9 kDa, corresponding to the removal of a single carbohydrate moiety. Although these experiments clearly confirmed that the 67-137 receptor fragment was the site of covalent binding of the 125 I-[Bpa 6 -VIP] probe, alone they did not allow to determine which one of the 67-93 or 94 -137 fragment was labeled in the F93M receptor mutant. To discriminate between the two fragments, the 18-kDa labeled band (see above) was electroeluted and incubated with trypsin. After trypsin treatment, the labeled material migrated as a narrow 4-kDa band (Fig. 5). Considering that the 125 I-[Bpa 6 -VIP] probe itself is cleaved by trypsin (Table II), generating the 1.4 kDa 1-12 VIP fragment bearing both the para-benzoyl-L-Phe 6 and the 125 I-Tyr 10 , the only receptor fragment compatible with the labeling of a 4-kDa band is fragment 104 -127 (Table I), whose theoretical mass is 2.6 kDa. Indeed, the mass of this fragment (2.6 kDa) plus the mass of the cleaved probe (1.4 kDa) corresponds to the 4-kDa estimated for the labeled band after trypsin cleavage (Fig. 5). All of the other receptor fragments generated by trypsin cleavage (Table I) are much too small to be compatible with the experimental data.
To further delineate the site of covalent binding of the affin- ity probe, the 30-kDa CNBr-generated fragments (Fig. 3) obtained after photoaffinity labeling of the wild-type receptor was digested with endopeptidase Glu-C (Fig. 3), which under our experimental conditions cleaves proteins at the C-terminal side of Glu residues with the notable exception of the Glu-Pro sequence (15). Endopeptidase Glu-C treatment of the 30-kDa band generated a 25-kDa band representing the receptor segment 67-108 that contains two N-glycosylation sites on Asn 69 and Asn 100 (Table I). Accordingly, this band was further shifted to 8 kDa upon treatment with PNGase F (Fig. 3). These data were thus consistent with the covalent binding of the 125 I-[Bpa 6 -VIP] probe to the Trp 67 -Glu 108 glycosylated receptor fragment (Table I). Then trypsin, which cleaves at the C-terminal side of Lys and Arg residues, was used to further narrow the domain of covalent binding of the [Bpa 6 ]-VIP probe. As shown in Fig. 3, trypsin digestion of the 30-kDa band resulted in a nonglycosylated fragment migrating at 4 kDa. The mass of trypsin-generated product strongly supported the possibility that the Ser 104 -Lys 127 fragment is actually the site of attachment of [Bpa 6 ]-VIP (Table I) as demonstrated above with the F93M receptor mutant (Fig. 5). Finally, the CNBr-generated 30-kDa band was sequentially digested with PNGase F, endopeptidase Glu-C, and trypsin. This sequential treatment generated a 2-kDa labeled band. This fragment was compatible with the covalent attachment of the probe to the Ser 104 -Glu 108 sequence of the hVPAC1 receptor. Indeed, its mass of 2 kDa was very similar to the mass of the receptor fragment (0.55 kDa) plus the mass of the trypsin-cleaved probe (1.4 kDa; see above and Table II).
Our data show that there is a spatial approximation between Phe 6 of VIP and the 104 -108 sequence within the N-terminal ectodomain of the human VPAC1 receptor. In a previous complete alanine scanning of VIP (9), the residues comprising VIP have been characterized into one of three different categories. Phe 6 was placed in category 2, which is defined as residues whose substitution into alanine is associated with a significant decrease of the binding affinity and biological potency of the corresponding analog as well as a change of the predicted   6 -VIP]-labeled hVPAC1 receptor fragment generated by CNBr cleavage a Theoritical mass of the receptor fragment calculated from the aminoacid sequence. The N-glycosylation is not taken into account. b Glycosylation of the hVPAC1 receptor has been shown to occur on three consensus N-glycosylation sites including Asn 58 , Asn 69 , and Asn 100 (14). ϩ indicates the presence of a glycosylation site occupied by a 9-kDa carbohydrate moiety (14) in the receptor fragment.
c Large italic N indicates Asn residues that are N-glycosylation sites. structure as compared with native VIP (9). Consequently, we speculated that the decreased affinity of the F6A VIP mutant was due to its altered structure, although the possible direct involvement of the category 2 residues, including Phe 6 , in VIP binding to receptor cannot be ruled out. In this context, we tried to identify the individual residue, or residues, in the short 104 -108 receptor fragment that may interact with residue 6 of VIP by individually mutating to alanine the five amino acids of the fragment. As shown in Table III 3 From this data, we can suggest that Cys 105 in the receptor is a candidate for interacting with Phe 6 in VIP. However, we should be careful with this conclusion because Cys 105 is one of the six cysteine residues that are strictly conserved in the N-terminal ectodomain of all class II G protein-coupled receptors and are involved in the formation of disulfide bridges that are crucial for receptor affinity (3). In consonance with our data, the C105A mutant failed to be labeled by the photoaffinity 125 I-[Bpa 6 -VIP] probe (not shown). The present data identified the N-terminal ectodomain of the hVPAC1 receptor as the contact region between receptor and the side chain at position 6 of VIP. This is in good agreement with previous mutagenesis studies suggesting that the N-terminal ectodomain is a major site of VIP binding (reviewed in Ref. 3). Indeed, mutagenesis showed that the individual mutation of 12 amino acids into alanine between Glu 36 and Pro 115 resulted in important or total loss of VIP binding (3, 7, 18 -20). It is quite interesting to note that this large segment revealed by mutagenesis studies encompasses the short Ser 104 -Glu 108 sequence of the receptor shown here by photoaffinity labeling to be the contact region of the side chain at position 6 of VIP. We previously developed a structural model of the N-terminal ectodomain of the VPAC1 receptor by sequence homology with a yeast lipase (7). This model showed that residues that are important for VIP binding, as demonstrated by mutagenesis, are gathered around an electronegative groove with an outspanning shell of three tryptophan residues at one end (7). In this context, the fact that the present study demonstrated that 125 I-[Bpa 6 -VIP] covalently attached to the Ser 104 -Glu 108 segment of the hVPAC1 receptor is most interesting. Indeed, this segment is located on one edge of the putative binding groove (Fig. 6). Another most meaningful aspect of the present study is    (8). This is illustrated in Fig. 6, which highlights the position of the two contact segments and also tentatively positions VIP in the binding groove taking into account the previous modelization (9) and NMR (23) studies of the peptide, which supported the idea that VIP has a central ␣-helix with a kink and less structured C-and N-terminal ends. Our data (present paper and Ref. 8) are consistent with a model in which the central part of VIP, at least between Phe 6 and Tyr 22 , lies in the binding groove identified in the N-terminal ectodomain of the hVPAC1 receptor (Fig. 6). This model is also consistent with the idea that crucial basic residues present in the central part of VIP including Arg 14 , Lys 15 , and Lys 21 (9) may interact with the important acidic residues in the electronegative groove in the N-terminal ectodomain of the receptor (7). Development of new photoaffinity probes with Bpa substitutions at other positions between Phe 6 and Tyr 22 of VIP should be done to further strengthen this model. The previous evidence for diffuse pharmacophoric domains of VIP that extend along the whole 28amino acid peptide chain (9) also indicates that new photoaffinity probes should be developed with Bpa substitutions at chain termini of VIP. This is more especially relevant for the VIP N-terminal domain, which, based on indirect evidence, is thought to interact with a still poorly characterized binding site on the core of the receptor (3,21). The hypothesis that the VIP-binding cleft could reside between the hVPAC1 receptor N-terminal ectodomain for the central part of VIP (this paper and Ref. 8) and the receptor body for the N-terminal domain of VIP is reminiscent of the situation described for some other class II peptide receptors (22)(23)(24)(25).
In conclusion, the results of the photoaffinity labeling study of the hVPAC1 receptor with the 125 I-[Bpa 6 -VIP] probe provide a narrow 104 -108 contact region, possibly Cys 105 , between the receptor N-terminal ectodomain and the side chain at position 6 of VIP. Together with a previous photoaffinity labeling study (8), these data indicate that the central part of VIP lies in a binding groove within the N-terminal ectodomain of the hVPAC1 receptor. Additional studies with new photoaffinity probes of VIP are needed to further delineate the sites of interaction between the central part of VIP and this binding groove for which a structural model has been already developed (3,7) and also to identify new anchor points of the N-terminal domain of VIP in the receptor core. Such studies are currently in progress in our laboratory.