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J. Biol. Chem., Vol. 278, Issue 38, 36531-36536, September 19, 2003
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From the INSERM U410, Neuroendocrinologie et Biologie Cellulaire Digestives, Institut National de la Santé et de la Recherche Médicale, Faculté de Médecine Xavier Bichat, Paris F-75018, France
Received for publication, May 7, 2003 , and in revised form, June 13, 2003.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The VPAC1 receptor is a prototypical class II peptide receptor that has been extensively studied by site-directed mutagenesis and molecular chimerism with respect to characterization of VIP binding domains (for review see Ref. 6), the existence of selectivity filters for low affinity agonists (10, 11) and identification of cytoplasmic domains required for activation of adenylyl cyclase (12). These studies showed that the N-terminal extracellular domain of hVPAC1 receptor is necessary although not sufficient for high affinity VIP binding (6). Moreover, microdomains consisting of small clusters of amino acids located in the N-terminal ectodomain (11) or at the junction of extra loop 1 and transmembrane helix 3 (10) were characterized as selectivity filters restricting access of VIP-related peptides to the receptor. A three-dimensional model of a large part of the N-terminal ectodomain of hVPAC1 receptor has been recently developed (13). Despite these extensive studies of the structure-function relationship of hVPAC1 receptor, we do not yet know the physical sites of interaction between VIP and its receptor.
In this context, we developed a photoaffinity probe of VIP by substituting para-benzoyl-L-Phe (Bpa) for tyrosine 22. This site of incorporation of the benzophenone group, a photoactivable group of choice for high efficiency covalent labeling of proteins (14), was selected for several reasons. (i) Tyr22 is present in the central part of the 28-amino acid VIP, a domain that has been shown previously by alanine scanning to contain several amino acids that are crucial for VIP binding (15). (ii) Alanine scanning showed that Tyr22 itself is not important for the biological activity of VIP (15), and we expected that incorporation of Bpa in this position would result in a high affinity probe. (iii) The substitution of Bpa for tyrosine keeps an aromatic residue. We report here the development and characterization of the 125I-[Bpa22-VIP] photoaffinity probe for covalent labeling of the hVPAC1 receptor and provide evidence that VIP is in direct contact with the N-terminal ectodomain of the receptor. Moreover, we show that amino acid in position 22 of VIP is in the environment of the short sequence 109–120 within this ectodomain when VIP is bound to the receptor.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Site-directed Mutagenesis and Cell Transfection—Oligonucleotide-directed mutagenesis was performed as previously described (10). Identification of the desired mutations was obtained by direct sequencing of the mutated region (10). Mutants were stably transfected in CHO cells as described previously (15).
Cell Culture and Membrane Preparation—The CHO-F7 cells expressing the wild-type VPAC1 receptor construct and the CHO cells expressing stably transfected receptor mutants were cultured in Ham's F-12 medium supplemented with 10% decomplemented fetal calf serum and 100 units/ml penicillin G and 100 µg/ml streptomycin in a humidified atmosphere containing 95% air and 5% CO2 at 37 °C. Cells were grown to confluency. After removing the culture medium, attached cells were washed twice with phosphate-buffered saline and then harvested with a rubber policeman and centrifuged for 10 min at 3,000 x g. The cell pellet was exposed for 30 min at 4 °C to hypoosmotic 5 mM HEPES buffer, pH 7.4, and membranes were obtained as described previously (15) and stored at –80 °C until use. Protein content was measured by the procedure of Bradford (19) with bovine serum albumin as standard.
Ligand Binding Assay and Measurement of Adenylyl Cyclase Activity—Ligand binding assay was as described previously (15). Membranes (100 µg of proteins/ml) derived from transfected CHO cells were incubated for 1 h at 30 °C with 0.05 nM [125I]VIP in the presence of increasing concentrations of VIP or Bpa22-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 x g for 10 min), and membrane pellets were washed twice 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 [125I]VIP bound in the absence (total binding) and the presence (nonspecific binding) of 1 µM unlabeled VIP. Binding data were analyzed using the LIGAND computer program. The concentration of peptides that elicited half-maximal inhibition of specific [125I]VIP binding (Ki) was determined by computer analysis. Adenylyl cyclase activity in membranes from clone F7 cells was assayed in the presence of increasing concentrations of VIP or Bpa22-VIP as described previously (15). Dose-response curves were fitted, and concentrations of peptides giving half-maximal responses (EC50) were calculated using the Prism software suite (GraphPad Software, San Diego, CA).
Photoaffinity Labeling—Transfected cells were incubated in darkness with 10 nM 125I-[Bpa22-VIP] in 10 ml of 20 mM HEPES buffer, pH 7.4, containing 0.2% (w/v) ovalbumin, 1 mM phenylmethylsulfonyl fluoride, and 0.1 mM TLCK. After a 1-h incubation at room temperature, cells were pelleted by centrifugation and the pellets resuspended in 4 ml of 20 mM HEPES with 1 mM phenylmethylsulfonyl fluoride and 0.1 mM TLCK. 1 ml of labeled cells was then added to each well of a six-well tissue culture plate, and the photoreaction (
= 365 nm) was carried out on ice at a distance of 
2 cm. After 40-min UV exposure, cells were collected into individual 1.5-ml Eppendorf tubes and washed twice with 10 mM HEPES containing 25 mM glycine, 75 mM NaCl buffer, pH 2.5, and 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. Cyanogen bromide 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, CNBr-generated peptide fragments were eluted with 2 ml of 100% acetonitrile. 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).
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 previously (20). V8 endoproteinase digestion of proteins in this material was obtained by incubation for 6 h at 25 °C in 25 mM ammonium bicarbonate, 0.01% SDS, 5% acetonitrile, pH 7.8, as described previously (21).
Electrophoresis and Autoradiography—Samples were analyzed by Tricine/SDS-PAGE performed according to the method of Schägger and Von Jagow (22) under reducing conditions in the presence of 0.2 M dithiothreitol. The apparent molecular masses of radiolabeled receptor fragments were determined by interpolation on a plot of the mobility of the broad range protein markers from BioLabs and the RainbowTM colored protein molecular weight markers from Amersham Biosciences versus the log values of their masses. After electrophoresis, gels were dried and exposed to x-ray film for 3–10 days with intensifying screens at –80 °C as described previously (20).
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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To further delineate the site of covalent binding of the affinity probe, the 30- and 11-kDa (deglycosylated) CNBr-generated fragments (Fig. 2) were digested with endoproteinase V8, which under our experimental conditions cleaves proteins at the C-terminal side of Glu residues with the notable exception of Glu-Pro sequence (21). As expected, considering the presence of Glu residues in the Trp67-Met137 receptor fragment, this cleavage further decreased the apparent molecular mass of the labeled band (Fig. 2). V8 endoproteinase treatment of the 30-kDa band generated a 6-kDa band (Fig. 2). Identical data were obtained by V8 endoproteinase treatment of the 11-kDa band. Given the mass of generated products and the absence of further effect of PNGase F (data not shown) on the migration of the labeled products after V8 endoproteinase digestion, the data support the covalent attachment of the 125I-[Bpa22-VIP] probe to the Gly109-Glu133 receptor fragment (see Table I).
To further validate the Gly109-Glu133 fragment as the site of attachment of Bpa22-VIP and to precisely determine which subdomain is concerned, we created new CNBr cleavage sites in this sequence by substituting methionine for Ile120 (Table I). The I120M hVPAC1 receptor mutant was constructed and stably expressed in CHO cells. It bound VIP with an affinity identical (Ki = 0.6 nM) to that of the wild-type receptor (Fig. 3). The I120M mutant expressed in CHO cells was photolabeled with 125I-[Bpa22-VIP], yielding a 95-kDa band upon electrophoresis (data not shown) as observed for the wild-type receptor (Fig. 2). The I120M mutant was then cleaved by CNBr and deglycosylated by PNGase F without or with further cleavage by V8 endoproteinase (Fig. 5). After CNBr treatment, the I120M mutant yielded a 9-kDa band as compared with a 11-kDa band for the wild-type receptor. Although the exact determination of such low molecular masses is clearly difficult by electrophoresis, it is crucial to point out that the band obtained with the mutant is systematically shifted as compared with that obtained for the wild-type receptor over four experiments. This finding strongly supported that the Gly109-Glu133 fragment is actually the site of attachment of Bpa22-VIP. Further cleavage with V8 endoproteinase yielded a 5-kDa band, which was consistently shifted over four experiments as compared with the 6-kDa band observed for the wild-type receptor (Fig. 5). Altogether, these data are consistent with the covalent attachment of 125I-[Bpa22-VIP] to the Gly109-Ile120 sequence of the hVPAC1 receptor.
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Since we have previously developed a three-dimensional model for a large part of the N-terminal ectodomain of the hVPAC1 receptor (13), the location of the Gly109-Ile120 sequence was analyzed and is highlighted in Fig. 6. It is of great interest to consider that this short sequence is within or contiguous to the proposed VIP binding groove (13), which contains amino acid residues previously shown by site-directed mutagenesis (6) to be important for VIP binding (Fig. 6).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The photolabile VIP derivative with substitution of Bpa for tyrosine at position 22 of VIP is a high potency full VIP agonist as shown by its ability to stimulate adenylyl cyclase activity in CHO expressing recombinant hVPAC1 receptor. This could be expected from previous alanine scanning studies, which showed that Tyr22 is not important for VIP binding to hVPAC1 receptor and subsequent adenylyl cyclase activation (15). On this basis, it is reasonable to assume that the photoaffinity probe adopts a binding mode similar to that taken up by native VIP. The proposed covalent attachment of 125I-[Bpa22-VIP] to the Gly109-Ile120 segment within the N-terminal ectodomain of the hVPAC1 receptor (see Fig. 6) is unambiguously consistent with the photoaffinity labeling results. Following UV irradiation of the complex formed by the receptor construct fused in the C-terminal position with the green fluorescent protein and 125I-[Bpa22-VIP], covalent labeling of a glycoprotein of 95 kDa was observed (see Fig. 2). Upon cleavage with CNBr, the labeled material was shifted to 30 kDa. Extensive studies of hVPAC1 receptor glycosylation have shown that the receptor expressed in COS cells or CHO cells is glycosylated with three N-glycosylation sites on Asn59, Asn68, and Asn100 in the N-terminal ectodomain, each site being occupied by 9-kDa carbohydrates (20, 23). The fact that the 30-kDa band was further shifted to 11 kDa after digestion with PNGase F clearly demonstrated that the labeled receptor fragment was heavily glycosylated, most probably, on two N-glycosylation sites. Altogether, these data were consistent with the labeling of the large Trp67-Met137 receptor fragment, which contains two glycosylation sites on Asn69 and Asn100 (see Table I). Further cleavage with V8 endoproteinase and development of new CNBr cleavage sites by substituting methionine for native amino acids at four different positions in receptor mutants provided clear evidence for the covalent attachment of 125I-[Bpa22-VIP] to the Gly109-Ile120 segment of the receptor (see Figs. 4 and 5).
Photoaffinity labeling identifies receptor domains in close proximity to the bound photoligand. The other privileged approach to molecular delineation of ligand-receptor interaction, site-directed mutagenesis, identifies domains necessary for function that are not necessarily proximal to the site of that function (24). As far as the VIP-hVPAC1 receptor interaction is concerned, there is not only a good agreement between photoaffinity labeling and mutagenesis experiments but also an exquisite complementarity as discussed further on. (i) The covalent attachment of 125I-[Bpa22-VIP] to the N-terminal ectodomain of the hVPAC1 receptor is consistent with previous experiments in which the receptor has been truncated or mutated. Indeed, deletion of the N-terminal ectodomain of hVPAC1 receptor abolishes its ability to bind VIP (6). Moreover, site-directed mutagenesis identified many residues within the N-terminal ectodomain as crucial or important for VIP binding including Glu36, Ser64, Met66, Trp67, Asp68, Trp73, Pro74, Pro87, Phe90, Gly109, Trp110, or Pro115 (6, 13, 25–27). These residues have been assumed to be positioned in a tertiary structure (see below) probably stabilized by the formation of three disulfide bonds formed between six cysteine residues, which are strictly conserved in class II G protein-coupled receptors (7). Indeed, mutation of all of the six cysteines clearly abolishes VIP binding to the hVPAC1 receptor (28). (ii) Site-directed mutagenesis showed that residues that are crucial for VIP binding are dispersed along the primary sequence of the N-terminal ectodomain of hVPAC1 receptor (6). A structural model of a large part of this domain has been constructed by sequence homology with a yeast lipase whose structure was available in the Protein Data Bank (13). This model confirmed the existence of a signal sequence at the N terminus (see Fig. 6) and showed most importantly that residues that are important for VIP binding are gathered around an electronegative groove with an outspanning shell of three tryptophan residues at one end (13). In this context, the fact that 125I-[Bpa22-VIP] covalently attached to the Gly109-Ile120 segment of the hVPAC1 receptor is quite meaningful. Indeed, this segment is located on one side of the groove and encompasses several crucial residues (6, 13) including Gly109, Trp110, and Pro115 (see Fig. 6). In that respect, the present study provides the first experimental basis for the current view of VIP binding to the hVPAC1 receptor. Indeed, it has been suggested that the central part of VIP with its crucial basic residues (15) may interact with the acidic residues in the electronegative groove in the N-terminal ectodomain of the receptor (6, 13). This finding fits well with the fact that Bpa22 in the photoaffinity probe is adjacent to the crucial basic residue Lys21 of VIP (15). The C-terminal part of VIP may also interact with the N-terminal ectodomain of the receptor, whereas it has been suggested that the N-terminal domain of VIP interacts with a still poorly characterized binding domain on the core of the receptor that may consist of, at least, the first and second extracellular loops and the upper part of the second transmembrane helix as viewed from the outside of the cell (6). In particular, based on dual mutations in VIP and hVPAC1 receptor, it was suggested that Asp3 side chain of VIP penetrates in the transmembrane domain in proximity to basic amino acids from transmembrane helix 2 (29). The hypothesis that peptide-binding cleft could reside between the hVPAC1 receptor N-terminal ectodomain and the receptor body is reminiscent of the situation described for two other class II receptor, the parathyroid hormone receptor (30, 31) and the secretin receptor (32). Development of new photoaffinity probes with Bpa substitutions at the N and C termini of VIP are clearly needed to further delineate the sites of interaction of the different part of this 28-amino acid neuropeptide with its receptors.
In conclusion, the results of the photoaffinity labeling study of the hVPAC1 receptor provides a contact region between the receptor and the side chain at position 22 of VIP. This represents the first direct evidence for physical proximity between VIP and the N-terminal ectodomain of its receptor in the VIP-receptor complex. The compatibility between the structural model of the N-terminal ectodomain of hVPAC1 receptor (6, 13) and the present photoaffinity cross-linking study further validates this model and paves the way of future docking of VIP in its receptor. Additional studies with new photoaffinity probes of VIP are clearly needed to identify anchor points in hVPAC1 receptor for interaction with other parts of the long 28-amino acid VIP. Such studies are currently in progress in our laboratory.
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FOOTNOTES |
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Ph.D. student at the University Paris 11.
Recipient of a postdoctoral fellowship (Poste Vert) from INSERM.
¶ To whom correspondence should be addressed: INSERM U410, Faculté de Médecine Xavier Bichat, 75018 Paris, France. Tel.: 33-1-44-85-61-30; Fax: 33-1-44-85-61-24; E-mail: laburthe{at}bichat.inserm.fr.
1 The abbreviations used are: VIP, vasoactive intestinal peptide; Bpa, p-benzoyl-L-phenylalanine; CHO, Chinese hamster ovary cells; PNGase F, peptide N-glycosidase F; TLCK, N
-p-tosyl-L-lysine chloromethyl ketone; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine; TM, transmembrane; h, human; VPAC, VIP pituitary adenylate cyclase-activating peptide receptor; LNB, long N-terminal type B.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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) versus native VIP (•) in stimulating adenylyl cyclase activity in CHO-F7 cell membranes expressing wild-type hVPAC1 receptor. The data are expressed as percentage of maximal stimulation above basal obtained with 1 µM native VIP. Basal enzyme activity and enzyme activities stimulated by 1 µM VIP and 1 µM Bpa22-VIP are 8.0 ± 0.2, 30.2 ± 1.0, and 28.7 ± 1.2 pmol of cAMP formed/min/mg protein. All of the data are means ± S.E. of three experiments. The error bars are smaller than the symbols.
); R99M mutant (
); E108M (
); and I120M mutant (
