Recombinant retroviruses (RV) have been widely used as vectors for clinical gene therapy of malignant brain tumors. Because of the very limited stability of these vectors in vivo, RV producing cells (VPC) are routinely used for intratumoral RV release. The host immune system, however, recognizes the intratumorally grafted allogeneic or xenogeneic VPC, and mounts an immune response against them. Humoral and cellular immune responses eventually result in reduction of VPC numbers and in limited success of RV mediated gene therapy approaches. This study presents a non-pharmacological and spatially limited approach for protection of VPC grafted in the CNS against destruction by host immune responses. Murine fibroblast-derived VPC expressing herpes-simplex-virus type I thymidine kinase (HSV-tk) were genetically modified to co-express a human Fas ligand (CD95L) deletion mutant (DeltaFasL) resistant to enzymatic cleavage and shedding. Direct interactions between Fas (CD95) on lymphocytes and DeltaFasL on VPC upon cell-cell contact rapidly caused apoptosis in lymphocytes. In addition, cultured malignant brain tumor cells (U87, LN18, LN229) transduced with DeltaFasL-RV were rendered apoptotic by Fas/DeltaFasL interaction. DeltaFasL-expressing VPC grafted in a 9L rat brain tumor model survived in significantly higher numbers compared with control VPC, and did not cause an increase in neutrophil infiltration of tumors. Gene therapy of tumor bearing animals grafted with the modified DeltaFasL-VPC and given the prodrug Ganciclovir resulted in significantly increased survival rates compared to treatment with control VPC and Ganciclovir. In conclusion, prolonged intratumoral presence of DeltaFasL-VPC seems to be a direct consequence of the expression of the membrane-bound mutant FasL, and may result in increased total RV output and improved tumor transduction with RV.

In the present study, we have analyzed a previously identified constitutively active pituitary adenylate cyclase activating polypeptide (PACAP) type I (PAC1) receptor with a deletion of the single amino acid residue Glu261 (Y.-J. Cao. G. Gimpl. F. Fahrenholz, A mutation of second intracellular loop of pituitary adenylate cyclase activating polypeptide type I receptor confers constitutive receptor activation. FEES Lett. 469 11000)). This glutamic acid residue is highly conserved within the second intracellular loop of class IT G protein-coupled receptors and may thus be of importance for many members of this receptor class. To explore the molecular characteristics of this mutant receptor, we performed photoaffinity labeling using previously defined photoreactive PACAP analogues. In COS cells, the PAC1 receptor was expressed in two differently glycosylated forms: a M-r 75 000 and a M-r 55 000 form. According to partial deglycosylation, at least three carbohydrate chains may exist in the rat PAC1 receptor expressed in COS cells. The constitutively active PAC1 receptor was expressed at the surface of COS-7 cells at the same density as the wild-type receptor. With respect to the different photoreactive PACAP analogues, the labeling specificity was the same for the wild-type versus mutant receptor. I-125-[Lys(15)(pBz(2))]-PACAP-27 and I-125-[Bpa(22)]-PACAP-27 were efficiently incorporated into each of the receptors, whereas I-125-[Bpa(6)]-PACAP-27 labeled each of the receptors only to a negligible extent. This suggests that both receptors have the same or at least a very similar hormone binding site which is in close contact to Tyr(22) and Lys(15) located in the carboxy-terminal a-helical region of the PACAP-27 molecule. However, in comparison with the wild-type PAC1 receptor. the constitutively active receptor showed a markedly (approx. 6-8-fold) enhanced photoaffinity labeling efficiency in particular of the high glycosylated form. The enzymatically deglycosylated rat PAC1 receptor was efficiently labeled by photoreactive PACAP analogues. In contrast, nonglycosylated PAC1 receptors produced by tunicamycin treatment of the transfected COS-7 cells showed a 30-fold lower affinity for PACAP-27 and were capable of signal transduction with 30-50-fold lower potency as compared with the glycosylated PAC 1 receptors. (C) 2001 Published by Elsevier Science B.V.

Pituitary adenylate cyclase activating polypeptide type I receptor (PACAPr) belongs to the novel subfamily of the G-protein coupled receptors with a long extracellular N-terminus, which functions as a major binding site for the PACAP. Three different N-terminal fragments of rat PACAPr were overexpressed in Escherichia coli and purified using His-tags or maltose-binding protein as anchors for affinity chromatography. The purified and refolded proteins were used for the production and screening of monoclonal antibodies (MAbs) to PACAPr. Fifteen hybridoma cell lines producing MAbs specific to PACAPr were generated and characterized. Epitope analysis by competitive enzyme-linked immunoadsorbent assay (ELISA) indicated the presence of two groups of overlapping epitopes in the N-terminal fragment of PACAPr. Reactivity of MAbs with SDS-denaturated and native rat PACAPr was demonstrated by immunoblotting and flow cytometric analysis using transiently transfected COS cells and stably transfected CHO cells expressing rat PACAPr. Each antibody was examined by immunoblotting for the ability to cross react with the human PACAPr in human neuroblastoma NB-OK cells and most of them were shown to recognize human PACAPr as effectively as rat PACAPr. MAbs against the N-terminal extracellular domain of PACAPr can be used for the immunochemical study of the receptor-ligand interaction and for the investigation of PACAPr distribution in normal and tumor tissues.

A bstract : Structure‐function studies and photoaffinity labeling experiments were performed to identify residues and domains of PACAP involved in the interaction with PACAP receptors. For this purpose, a series of photoreactive analogues of PACAP(1‐27) containing a photoreactive benzophenone (BP) residue in different peptide structural domains were utilized to analyze the interaction of PACAP(1‐27) with pig PACAP type 1 receptors. Five PACAP derivatives were created with a photoreactive amino acid in the following peptide domains: either the disordered N‐terminal or the helical C‐terminal domain or a short loop region within the C‐terminal helical domain of the peptide. Their receptor binding properties and efficiencies were tested on pig brain PACAP receptors. The results indicate the importance of the helical C‐terminal domain of PACAP(1‐27) for receptor binding affinity. Monoiodination of the photoreactive analogues did not change their binding affinities. Experiments with pig brain membranes demonstrated that the 125 I‐labeled photoreactive analogues specifically label a protein band of Mr66,000. The efficiency of photoreactive labeling differed for the various analogues. These findings suggest that Tyr 22 and Lys 15 in PACAP(1‐27) are located in or close to the hormone binding site of the PACAP type 1 receptor. The results provide evidence that the α‐helical C‐terminal region of PACAP is directly involved in receptor binding.

To identify residues and domains of the peptide hormone pituitary adenylate-cyclase-activating polypeptide (PACAP) that interact with the type I receptor, two photoreactive analogues of PACAP-(1-27)-peptide were synthesized using solid-phase peptide synthesis. Phe6 or Tyr22 within the PACAP sequence were replaced by p-benzoyl-L-phenylalanine (Bz-Phe) thus creating two PACAP derivatives with a photoreactive amino acid in either the disordered N-terminal or the helical C-terminal part of the peptide. The ligand-binding properties and the efficiencies of these peptide analogues as photolabels were tested for pig brain PACAP receptors. [Bz-Phe6]-PACAP-(1-27)-peptide (Kd 1.3 nM) retained the high binding affinity of PACAP-(1-27)-peptide (Kd 0.5 nM), wheras Bz-Phe substitution of Tyr22 reduced the affinity about tenfold (Kd 4.4 nM) thus demonstrating the importance of Tyr22 for receptor binding. Monoiodination of the photoreactive analogues did not change the binding affinity of the photoreactive analogues. Photoaffinity labeling using pig brain membrane demonstrated that the 125I-labeled photoreactive analogues specifically label a 66000-Mr protein band. Photoaffinity labeling of the rat brain PACAP receptor expressed in COS cells resulted in two specifically photolabeled proteins: a major band of Mr 58000 and a minor band of Mr 78000. By treatment of photolabeled membranes with N-glycosidase F, both of the polypeptide bands were converted to a single polypeptide band of Mr 54000, which corresponds to the deglycosylated PACAP receptor. Despite its lower receptor affinity, [Bz-Phe22]-PACAP-(1-27)-peptide labeled the PACAP type I receptor in pig brain membranes and the rat receptor expressed in COS cells with much higher efficiency (20-fold for the pig receptor) than [Bz-Phe6]-PACAP-(1-27)-peptide. These findings suggest that Tyr22 in PACAP-(1-27)-peptide is located in or close to the hormone-binding site of the PACAP type I receptor. The results provide evidence that the alpha-helical C-terminal region of PACAP is directly involved in receptor binding.

The PACAP receptor represents a member of a novel subfamily of G-protein coupled receptors with a common structurally conserved extracellular domain of about 150 amino acids. We have addressed the question whether this extracellular amino-terminus of the PACAP type I receptor can solely function as a PACAP binding domain. For that purpose a cDNA was constructed that encodes the membrane-anchored amino-terminus of the rat PACAP receptor including the decapeptide epitope EQKLISEEDL for immunodetection. COS-7 cells were transfected with this cDNA and a comparable construct of the wild-type receptor. Binding analysis showed that the amino-terminal fragment of the PACAP receptor bound PACAP with high-affinity (Kd=3.8 nM; Bmax=12.8 pmol/mg protein). In comparison to the full-length receptor (Kd=0.2 nM; Bmax=1.96 pmol/mg protein) its affinity was reduced by a factor of about 20. The results suggest that the amino-terminus of the PACAP receptor functions as the major binding site for its ligand.

In pig brain a type I receptor for pituitary adenylate cyclase activating polypeptide (PACAP) has been identified and structurally characterized. Scatchard analysis of the equilibrium binding data indicates a single class of binding sites with Kd of 0.50 nM and Bmax of 2.52 pmol/mg. The receptors could be efficiently solubilized with 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1- propanesulfonate (Chapso) without altering their ligand binding parameters (Kd = 0.60 nM; Bmax = 2.46 pmol/mg). In both preparations the ligand-receptor complex was identified as a 68 kDa polypeptide by affinity crosslinking with 125I-PACAP-27 and disuccinimidyl suberate. Employment of nonreducing conditions decreased the electrophoretic mobility of the complex to a 60 kDa species suggesting that the native PACAP receptor exists in a compact conformation stabilized by intramolecular disulfide bridges. Disulfide bonds are also important for the ligand binding activity of the PACAP receptor since pretreatment of the membranes with dithiothreitol led to complete inhibition of PACAP binding and to 61% dissociation of the ligand-receptor complex. Among different sulfhydryl selective reagents tested, p-chloromercuriphenylsulfonic acid was found to inhibit PACAP binding to its receptor in a dose-dependent fashion (IC50 = 0.5 mM) suggesting that one or more sulfhydryl groups are located close to the ligand binding domain of the receptor. Desialylation of the affinity-labeled PACAP receptor with neuraminidase revealed a 61 kDa protein, whereas deglycosylation with N-glycosidase F decreased the M(r) to 52,000. Chromatography on a series of lectin agaroses showed the highest affinity for wheat germ agglutinin (WGA). WGA was also effective in dose-dependent inhibition of PACAP binding activity. Our results characterize the pig PACAP receptor as a tri- or tetraantennary non- or low fucosylated complex type glycoprotein with a protein core of about 49 kDa containing several terminal sialyl residues.