Dual expression of mouse and rat VRL-1 in the dorsal root ganglion derived cell line F-11 and biochemical analysis of VRL-1 after heterologous expression Ricarda Jahnel 1, *, Olaf Bender 1, *, Lisa M. Mu¨ nter 1 , Mathias Dreger 1 , Clemens Gillen 2 and Ferdinand Hucho 1 1 Arbeitsgruppe Neurochemie, Institut fu ¨ r Chemie-Biochemie, Freie Universita ¨ t Berlin, Germany; 2 Gru ¨ nenthal GmbH, Aachen, Germany The vanilloid-like TRP-channel VRL-1 (TRPV2) is a non- selective cation channel expressed by primary sensory neurons and non-neuronal tissues [Caterina, M.J., Rosen, T.A., Tominaga, M., Brake, A.J and Julius, D. (1999) Nature 398, 436–441]. It is one of the six members of the vanilloid-like TRP-channel family which is now termed the TRPV family [Montell, G., Birnbaumer, L., Flockerzi, V., Bindels, R.J., Brutford, E.A., Caterina, M.J., Clapham, D.E., Harteneck, C., Heller, S., Julius, D., Kojima, I., Mori, Y., Penner, R., Prawitt, D., Scharenberg, A.M., Schultz, G., Shimizu, N. and Zhu, M.X. (2002) Mol. Cell 2, 229–231]. As it is a temperature-gated channel, VRL-1 appears to be functionally related to VR1. In contrast to VR1, VRL-1 is activated at a higher temperature threshold and it does not respond to capsaicin or protons. Here we describe the expression of VRL-1 in the rat dorsal root ganglion-derived cell line F-11, a hybridoma of mouse neuroblastoma (N18TG2) and rat dorsal root ganglion cells. We found by RT-PCR that F-11 cells express not only the rat VRL-1, but also its mouse orthologue in a single cell. The F-11 parental cell line N18TG2 also expressed murine VRL-1. Due to its neuronal character, the DRG-derived F-11 cell line provides an experimental system for the study of VRL-1 biochemistry. However, one has to be aware that both the mouse and the rat protein are expressed simultaneously. Furthermore we cloned VRL-1 from rat brain and analyzed its glycosylation and localization in comparison to the endogenously expressed protein in F-11 cells. In contrast to the endogenous VRL-1 the overexpressed protein is glycosylated. Similar to VR1 the glycosylation is N-linked as shown by an deglycosyla- tion assay. Immunofluorescence analysis of the endo- genous VRL-1 in F-11 cells gives only weak signals in the cytoplasm whereas the overexpressed rat VRL-1 appears mainly at the plasma membrane. Keywords: TRPV2; F-11; glycosylation; localization; single cell RT-PCR. The vanilloid receptor 1 (VR1 or TRPV1) is a cation channel predominantly expressed by primary sensory neu- rons involved in nociception. Caterina et al.[1]isolateda cDNA encoding the homologous VRL-1 by searching an EST database for sequences related to VR1. According to the new nomenclature, VRL-1 is now referred to as TRPV2 [2]. The rat cDNA (Acc. No. AF129113) encodes a 761- amino acid VRL-1 protein, which is 82% identical to the mouse protein of 756 amino acids (Acc. No. NM_011706), and 78% identical to the human VRL-1 protein (Acc. No. NM_016113) of 764 amino acids. Computer analysis of the sequence predicts six transmembrane sequences, a pore- loop, a cytoplasmic N-terminal sequence with three anky- rin-repeat domains, and a cytoplasmic C-terminal sequence. Functional analysis showed that VRL-1 does not respond to capsaicin, low pH or moderate heat, but is activated at high temperatures, with a threshold at about 52 °C [1]. Immunocytochemical experiments revealed that VRL-1 is expressed in medium- to large-diameter dorsal root ganglion neurons and in the spinal cord, specifically in Lissauer’s tract and the dorsal horn [1]. Ichikawa et al.[3]detected VRL-1 immunoreactivity in 14% of the trigeminal ganglia cell bodies. Furthermore Stenholm et al. [4] found VRL-1 in the tooth pulp-innervating neurons and the gingival neurons. Co-expression with VR1 was rarely observed. Northern blot analysis revealed expression of an approxi- mately 2.5-kb VRL-1 transcript in rat sensory ganglia and spinal cord, as well as in lung, spleen, intestine, and multiple brain subregions [1]. This led to the speculation that non- neuronal tissues expressing VRL-1 probably respond to stimuli other than heat. An endogenous ligand for VRL-1 still has to be identified. One property of the murine VRL-1 which Kanzaki et al. [5] had originally identified as a Ca 2+ -permeable channel (GRC) from mouse spleen is the regulation by insulin-like growth factor (IGF-1). IGF-1 causes the translocation of GRC to the plasma membrane where it forms a constitu- tively active channel at room temperature. However, it still Correspondence to F. Hucho, Institut fu ¨ r Chemie-Biochemie, Freie Universita ¨ t Berlin, Thielallee 63, 14195 Berlin, Germany. Fax: + 49 0308385 3753, Tel.: + 49 0308385 5545, E-mail: hucho@chemie.fu-berlin.de Abbreviations:DAPI,4¢-6-diamidino-2-phenylindole-2HCl; DRG, dorsal root ganglion; GRC, growth-factor-regulated channel; TRP, transient receptor potential; VR1, vanilloid receptor type 1 (TRPV1); m/rVRL-1, mouse/rat vanilloid receptor-like protein 1 (TRPV2). *Note: Both authors contributed equally to this work. (Received 17 July 2003, revised 21 August 2003, accepted 1 September 2003) Eur. J. Biochem. 270, 4264–4271 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03811.x needs to be investigated how this relates to the rat and human VRL-1. We previously investigated biochemical characteristics of the capsaicin sensitive Vanilloid receptor VR1 transiently transfected in the rat dorsal root ganglia derived cell line F-11, a hybridoma of mouse neuroblastoma and rat dorsal root ganglion cells [6]. VR1 expressed in a neuronal cell system should be more related to the in vivo situation than fibroblast- or kidney-derived cell lines. VR1 was not endogenously expressed in F-11 cells. These cells, formerly generated and characterized by Platika et al. [7] and Francel et al. [8], have been proven useful models for authentic DRG cells. They showed that several features of differen- tiated DRG cells are present in F-11 cells, e.g. release of substance P, the presence of l-andd-opioid receptors, receptors for prostaglandin and bradykinin and L -type calcium channels. Recently, Bender et al. [9] isolated a rat VRL-1 mRNA from F-11 cells identical to that observed in dorsal root ganglia extracts. They suggested, that the F-11 cells most likely are derived from medium-sized Ad-fibers. Here we show that the F-11 cells not only express the rat VRL-1, but also the mouse VRL-1 and that the mouse variant is derived from the F-11 parental cell line N18TG2. This finding is of interest when VRL-1 is functionally studied in these cells. Additionally in this study we cloned VRL-1 from total RNA isolated from rat brain and analyzed the glycosylation and localization of the endo- genous VRL-1 in F-11 cells compared to VRL-1 over- expressed in the same cell line. Experimental procedures Reverse transcription of rat brain, F-11 and N18TG2 total RNA Total RNA of rat brain, F-11 and N18TG2 cells was prepared using TRIzol Reagent (Invitrogen) according to the instruction of the manufacturer. 1 lgtotalRNAwas incubated for 10 min at 65 °C with 500 ng Oligo(dT)- Primer and chilled on ice. First strand synthesis was performed in a 20-lL reaction with 200 U Superscript II reverse transcriptase in the presence of 40 U RNAse Out, 500 l M each dNTP and 5 m M dithiothreitol (all reagents from Invitrogen) at 42 °C for 60 min followed by an inactivation step at 70 °Cfor15min.Asacontrol each sample was additionally treated without reverse transcriptase. VRL-1 specific PCR To portray rat VRL-1 in overlapping PCR fragments in F-11 cells PCR primers were designed according to the rat VRL-1 sequence (Acc. No. AF129113). rVRL-1–34F: 5¢-CTGGAGACTTCCGATGGAGA-3¢ and rVRL-1–568R: 5¢-CATCCGCTCCATTCTCTACC-3¢ to obtain fragment A with 534 bp; rVRL-1–549F: 5¢-GGTAGAGAATGGAGCGGATG-3¢ and rVRL-1– 1205R: 5¢-ACCAAGTAGCAGGCGAAGTT-3¢ to obtain fragment B with 656 bp; VRL-1/1061F: 5¢-ACTCGGTGC TGGAGATCATC-3¢ and VRL-1/1897R: 5¢-TGAGAAG GACGTAGGCCAAC-3¢ to obtain fragment C with 836 bp; rVRL-1–1618F: 5¢-TTCCTGCTGGTCTACCTG GT-3¢ and rVRL-1–2251R: 5¢-CTTCCTCTGAGGCACT GTTC-3¢ to obtain fragment D with 633 bp. All primers were purchased from MWG Biotech AG. PCR was performed with rat brain, N18TG2 and F-11 cDNA as template. Two microlitres RT reaction was used in 25 lL PCR amplifications using Taq-DNA-Polymerase (Invitrogen) after heating the sample to 95 °C for 3 min. PCR conditions were 94 °C,30s;57°C,30s;72°C,30s for 35 cycle and an additional elongation step at 72 °C for 7 min. PCR was analyzed by electrophoresis in a 0.9% agarose gel. Single cell RT-PCR of F-11 cells F-11 cells were diluted in 5 · RT first strand buffer to a concentration of 1 cell per 4 lL and checked under the microscope in a 96-well plate. Only single cells were used for directly reverse transcription and PCR. First strand synthe- sis was performed in a 20-lL reaction with 200 U Super- script II reverse transcriptase in the presence of 40 U RNAse Out and 5 m M dithiothreitol (all reagents from Invitrogen) at 42 °C for 60 min followed by an inactivation step at 70 °C for 15 min. As a control each sample was additionally treated without reverse transcriptase. PCR was performed as described above for 40 instead of 35 cycles. Non-denaturing PAGE PCR products in a nondenaturing sample buffer were loaded on a 6% nondenaturing PAGE (buffered by tris- borate-EDTA). The gel was stained in an ethidiumbromide bath for 20 min and visualized under UV light. Bands were eluted by the crush and soak method [10] and DNA purified by Phenol/Chloroform extraction. PCR fragments were ligated into the pCRII-TOPO vector (Invitrogen) and sequenced. The accuracy of sequences from the open reading frame was checked by DNA sequencing of both strands. The resulting sequences of specific bands were identical to GenBank sequence Acc. No. AF129113 or Acc. No. NM_011706. Cloning of rVRL-1 Rat vanilloid receptor like-protein 1 (rVRL-1) was cloned from total rat brain cDNA. Reverse-transcription poly- merase chain reaction (RT-PCR) was carried out with specific rVRL-1 forward and reverse primers designed using the GenBank sequence AF129113. Primers corresponding to nucleotides 330–353 and 2587–2610 (rVRL-1_F: 5¢-AT GACTTCAGCCTCCAGCCCCCCA-3¢ and rVRL-1_R: 5¢-GGGACTGGAGGACCTGAAGGGGCA-3¢, respect- ively) were used to clone the open reading frame of rVRL-1 in frame with GFP into the pcDNA3.1/CT-GFP-TOPO vector (Invitrogen). A 2.5 lL RT reaction was used in 25 lL PCR amplifications containing 4% dimethylsulfox- ide using Pfu Polymerase (Life Technologies). After heating thesampleto95°C for 3 min PCR conditions were: 95 °C, 30 s; 57 °C,30s;72°C, 5 min for 35 cycles and an additional elongation step at 72 °C for 7 min. To create 5¢A-overhang for TOPO cloning the reaction was incubated with Taq-DNA-Polymerase (Promega) for 20 min at 72 °C. PCR was analyzed by electrophoresis in a 1% agarose gel Ó FEBS 2003 Co-expression of mouse and rat VRL-1 in F-11 cells (Eur. J. Biochem. 270) 4265 for the presence of specific 2.3 kb rVRL-1 band. The band was excised from the gel and purified with UltraClean DNA Purification Kit (MOBIO Laboratories, Inc.) using the manufacturer’s instructions. Four microlitres rVRL-1 cDNA was ligated into 1 lL pcDNA3.1/CT-GFP-TOPO vector in the presence of 1 lL salt solution for 5 min at RT. The accuracy of the entire open reading frame was checked by DNA sequencing of both strands. To obtain the full length rVRL-1 protein without GFP, site-directed mutagenesis of pcDNA3.1/rVRL-1-CT-GFP was performed using the QuikChange TM Site-Directed Mutagenesis Kit (Stratagene). Two primers were designed to introduce the codon for P761 and a stop codon to prevent GFP expression: mutVRL-1_F (5¢-TCAGGTCCTCCAG TCCCCCTAAGGGCAATTCTGCAGAT-3¢)andmut- VRL-1_R (5¢-ATCTGCAGAATTGCCCTTAGGGGGA CTGGAGGACCTGA-3¢). The five introduced nucleotides are shown in bold, they generate a new codon for P761 and a stop codon. The accuracy of the resulting new plasmid pcDNA3.1/rVRL-1 was confirmed by DNA sequencing of both strands. Cell culture and transfection of F-11 cells F-11 cells (obtained from M.C. Fishman, Cardiovascular Research Center, Massachusetts General Hospital, Charles- town, MA, USA) were cultured in Ham’s F-12 media with Glutamax-I (Life Technologies), supplemented with 20% fetal calf serum (GibcoBRL), 2% hypoxanthin/thymidine/ aminopterin supplement (Biochrom KG), 100 lgÆmL )1 streptomycin, and 100 lgÆmL )1 penicillin at 37 °C in a humidified atmosphere with 5% CO 2 on plastic tissue culture grade flasks (Nunc). N18TG2 cells (DSMZ ACC 103) were cultured in Dulbecco’s MEM, supplemented with 10% fetal bovine serum (GibcoBRL), 100 l M 6-thioguanine (Sigma), 100 lgÆmL )1 streptomycin, and 100 lgÆmL )1 penicillin at 37 °C in a humidified atmosphere with 5% CO 2 on plastic tissue culture grade flasks (Nunc). F-11 cells were transiently transfected with the pcDNA3.1/rVRL-1 or pcDNA3.1/rVRL-1-CT-GFP plas- mid using LipofectAMINE Plus (Life Technologies) according to the procedure recommended by the manufac- turer. Plasmids were constructed as described above. Indirect immunofluorescence was performed as described previously [6]. For detection of VRL-1 anti-VRL-1 poly- clonal rabbit IgG (Oncogene, 1 : 100) was used as first and Cy 2 -conjugated goat anti-(rabbit IgG) Ig (Jackson Immuno research Laboratories) as second antibody. Nuclei were stained with 0.0004% 4¢-6-diamidino-2-phenylindole-2HCl (DAPI) for 3 min before mounting. Immunostaining was visualized using fluorescence microscopy. Preparation of F-11 cell homogenate and particulate Non-transfected or transfected cells from culture dishes were washed once with cold NaCl/P i and harvested in NaCl/P i . The following steps were carried out at 4 °C. The cells were gently centrifuged for 5 min at 500 g and then resuspended in sucrose/Tris/MgSO 4 (0.25 M sucrose, 50 m M Tris-Cl, pH 7.4, 5 m M MgSO 4 ) containing one protease inhibitor cocktail tablet for 50 mL buffer (Complete TM , Boehringer Mannheim) and 1 m M phenyl- methanesulfonyl fluoride. Cells were homogenized in a glass homogenizer. The homogenate was frozen in liquid nitrogen and stored at )70 °C until use. For particulate preparation the homogenate was centrifuged for 20 min at 40 000 g. The resulting pellet was resuspended in 0.25 M sucrose/Tris/MgSO 4 and frozen as described above. Western blot analysis Aliquots (20 lg) of F-11 cell homogenate or particulate in loading buffer were subjected to SDS/PAGE (10%). Bands of protein were electroblotted onto nitrocellulose mem- branes (Schleicher and Schuell). Membranes were soaked for2hinTBST(20m M Tris buffer, pH 7.5, 150 m M NaCl, 0.1% Tween 20) with 5% nonfat dry milk and subsequently incubated for 1 h with anti-VRL-1 polyclonal rabbit IgG (Oncogene, 1 : 100) with and without 5 lgÆmL )1 blocking peptide (KNSASEEDHLPLQVLQSP-COOH) in TBST with 5% nonfat dry milk. After washing, blots were incubated for 1 h with a goat anti-(rabbit-IgG) Ig coupled to horseradish-peroxidase (Dianova, 1 : 1000). For detec- tion of the VRL-1-GFP fusion protein anti-GFP polyclonal goat IgG (Abcam, 1 : 5000) was used as first and mouse anti-(goat-IgG) Ig coupled to horseradish-peroxidase (Pierce, 1 : 1000) as second antibody. Immunoreactive bands were visualized by enhanced chemoluminescence detection methods (ECL Western Blotting Detection Reagents, Amersham Pharmacia Biotech). Deglycosylation with N-glycanase and endoglycosidase H Deglycosylation with PNGase F and endoglycosidase H (NEB) was performed in a volume of 50 lL with 100 lgof F-11 cell particulate according to the manufacturer’s instruc- tions. Non transfected and rVRL-1 or rVRL-1-CT-GFP transfected F-11 cells were used. 30 lg of a membrane preparation from the electric organ of Torpedo californica containing mainly nicotinic acetylcholine receptor (nAChR) was used as a positive control. After incubation the probes were separated by electrophoresis on a 10% Laemmli-SDS gel. Western blot analysis was performed as described above. Results A construct coding for a VRL-1-GFP fusion protein was prepared by cloning the open reading frame of rVRL-1 from rat brain RNA without the last codon for amino acid P761 and the stop codon into pcDNA3.1/CT-GFP-TOPO vec- tor. The resulting sequence was identical to GenBank sequence Acc. No. AF129113 with four single nucleotide exchanges in codons at amino acid 82 [573 AGT fi 573 AGG], amino acid 367 [1428 CCG fi CTG], amino acid 397 [1518 TTC fi TTT] and amino acid 462 [1713 TTT fi TTC]. These differences lead to two changes in the amino-acid sequence in VRL-1, namely S82R and P367L. The other two differences are silent. To obtain the full length rVRL-1 protein without GFP site-directed mutagenesis of pcDNA3.1/rVRL-1-CT-GFP was per- formed in a way that the codon for P761 and a stop codon was introduced (pcDNA3.1/rVRL-1). 4266 R. Jahnel et al.(Eur. J. Biochem. 270) Ó FEBS 2003 VRL-1 is expressed in F-11 cells We probed an F-11 cell homogenate after SDS/PAGE and electroblotting with a polyclonal rabbit antibody directed against the C-terminus of rat VRL-1. It is important to mention that this antibody also detects the mouse variant [11]. The anti-VRL-1 antibody specifically detected a protein band at  80 kDa (Fig. 1). This band was not detected when 5 lgÆmL )1 of the immunization peptide was added to the primary antibody reaction to block the VRL-1- antigen-specific antibody. The anti-VRL-1 immunoreactive protein band at  80 kDa is in agreement with the calculated molecular mass of rat VRL-1. Mouse and Rat VRL-1 are both expressed in F-11 cells To prove that the full length VRL-1 mRNA is present in F-11 cells we examined the rat VRL-1 corresponding to Acc. No. AF129113 in overlapping PCR fragments after reverse transcription of F-11 total RNA (fragment A: 34– 568 bp ¼ 534 bp, fragment B: 549–1205 bp ¼ 656 bp, fragment C: 1061–1897 bp ¼ 836 bp and fragment D: 1618–1897 bp ¼ 633 bp). Even though there was only one band visible in the agarose gel for each PCR product, sequence analysis of different cloning experiments yielded either the rat or sometimes the mouse VRL-1 sequence. To prove the possible expression of mouse VRL-1 in F-11 cells, we tested the PCR products on a 6% nondenaturing polyacrylamide gel and found that fragment A, B and D clearly separated into two bands from which the upper band always corresponded to a rat brain control (Fig. 2). Both bands from F-11 fragment B were cloned into pCRII-Topo vector (Invitrogen). Sequence analysis revealed that the upper band always corresponds to rat VRL-1 mRNA, whereas the lower band was identical with the mouse VRL-1 mRNA (Acc. No. NM_011706). Graphi- cal alignment of the open reading frames of rat VRL-1 (Acc. No. AF129113) and mouse VRL-1 (Acc. No. NM_011706) shows that rat and mouse VRL-1 share 92% sequence identity. The ORF length of rat VRL-1 is 2286 bp whereas the mouse VRL-1 has 2271 bp. This results in a protein of 761 amino acids for rVRL-1 and of 756 amino acids for mVRL-1 which cannot be separated by SDS/PAGE. The primers we chose to examine rat VRL-1 in F-11 cells were also capable of amplifying the mouse orthologue (Fig. 3). Consequently we examined the F-11 parental cell line N18TG2 and found mouse VRL-1 expression due to its origin (mouse neuroblastoma) (Fig. 4). As controls, rat brain and F-11 cDNA were used. On a 6% polyacrylamide gel fragments A, B and D of F-11 cells again separated into two bands. The slower migrating band always corresponded to the rat brain control, and the faster migrating band to the N18TG2 control (Fig. 5). The existence of both rat and mouse VRL-1 in the same cell was confirmed by F-11 single cell RT-PCR. In all analyzed cells (n ¼ 15) expression of both VRL-1 species was found. Separation of the RT-PCR products of fragment D on a 6% nondenaturing polyacrylamide gel resulted in two bands for each single F-11 cell (Fig. 6) and subsequent sequence analysis of the upper and lower fragment from one single cell confirmed the previous results. Localization of heterologously expressed vs. endogenous VRL-1 in F-11 To assess the subcellular localization of the VRL-1 receptor, we imaged rVRL-1 and rVRL-1-GFP heterologously expressed in F-11 cells in comparison with the endogenous protein by fluorescence microscopy. Indirect immuno- fluorescence of the endogenous VRL-1 visualized with Fig. 1. VRL-1 Western blot of F-11 cell homogenate. Anti-VRL-1 polyclonal rabbit IgG detected a band at  80kDainF-11cell homogenate. This band was completely abolished with 5 lgÆmL )1 blocking peptide corresponding to the C-terminus of VRL-1 (KNSASEEDHLPLQVLQSP-COOH). Fig. 2. Non-denaturing PAGE of overlapping PCR products from F-11 and rat brain. PCR from F-11 cDNA using rat VRL-1 specific primers result in two bands for fragments A, B and D, whereas PCR from rat brain cDNA only result in one band. The slower migrating F-11 cDNA fragments (F11) always correspond to the rat brain (rb) cDNA control. Both F-11 bands from fragment B were eluted from the gel and cloned into the pCRII-TOPO vector. Sequence analysis clearly showed that the faster migrating band corresponded to mouse VRL-1 (Acc. No. NM_011706) whereas the slower migrating band corres- ponded to rat VRL-1 (Acc. No. AF129113). Ó FEBS 2003 Co-expression of mouse and rat VRL-1 in F-11 cells (Eur. J. Biochem. 270) 4267 Fig. 3. Graphical alignment of the open reading frames of rat VRL-1 (Acc. No. AF129113) and mouse VRL-1 (Acc. No. NM_011706). Rat and mouse VRL-1 cDNA share a homology of 92%. The ORF length of rat VRL-1 is 2286 bp whereas the mouse VRL-1 has 2271 bp. This results in a 761 amino-acid protein for rVRL-1 and 756 amino-acid protein for mVRL-1. Colored arrows indicate primer sequences to amplify overlapping PCR fragments of rat VRL-1 (yellow to obtain fragment A with 534 bp, green for fragment B with 656 bp, orange for fragment C with 836 b and cyan for fragment D with 633 bp). Fig. 4. RT-PCR of fragment A. RT-PCR with rat VRL-1 specific primer for fragment A (34–568 bp ¼ 534 bp) yielded not only a product from RNA of F-11 (F11) and rat brain (rb) but also of the F- 11 parental cell line N18TG2. This product could only be derived from the mouse variant. +/–, with/without reverse transcriptase. Fig. 5. Non-denaturing PAGE of overlapping PCR products from F-11, rat brain and N18TG2. F-11 PCR products (F11) A, B and D separated into two bands, whereas PCR products from rat brain (rb) or N18TG2 cells (N) only result in one band. The slower migrating of the two F-11 PCR bands always corresponded to the rat brain control, the faster migrating band to the N18TG2 control. In case of C the PCR product from rat brain appeared lower than that of F-11 and N18TG2. 4268 R. Jahnel et al.(Eur. J. Biochem. 270) Ó FEBS 2003 anti-VRL-1 polyclonal antibody gives only weak signals in the cytoplasm of F-11 cells (Fig. 7A,B). In contrast rVRL-1 transiently expressed in F-11 cells showed up predominantly at the plasma membrane and neurite-like extensions (Fig. 7C,D). When rVRL-1-GFP of transiently transfected F-11 cells was imaged by the GFP fluorescence, some of it also localizes to the plasma membrane but most of the fusion protein appeared on intracellular membranes (ER). rVRL-1 is a glycoprotein In lysates of pcDNA3.1/rVRL-1 transfected F-11 cells, anti- VRL-1 immunoreactivity appeared as multiple bands which is characteristic for glycoproteins. To confirm this post- translational modification of VRL-1, we incubated cell lysates from transfected cells with glycosidases as described previously [6]. Deglycosylation of rVRL-1 was probed by anti-VRL-1 Western blotting. Incubation with Endo H abolished the upper band of the anti-VRL-1 immunoreac- tivity doublet band at > 80/84 kDa. The lower band of the doublet at 80 kDa increased proportionally in intensity, but the diffuse anti-VRL1 immunoreactivity at 97 kDa was unaffected (Fig. 8A). Neither the 97 kDa nor the 84 kDa anti-VRL-1 immunoreactivity band were observed subse- quent to incubation of the rVRL-1-containing cell lysates with PNGase F; only the 80 kDa band could be observed. Our data indicate that both high mannose-type glycosyla- tion and complex glycosylation of rVRL-1 occurs. These findings are in agreement with previous investigations on rVR1 [6]. Identical observations were made with pcDNA3.1/ rVRL-1-CT-GFP transfected F-11 cells. Anti-GFP (Fig. 8B) and anti-VRL-1 (Fig. 8C) Western blots of F-11 cells show an analogous pattern of the rVRL-1 glycopro- tein, when the  30 kDa molecular weight of GFP is taken into account. The main rVRL-1-GFP band appeared at  110 kDa, the upper band at 114 kDa and the diffuse band at 128 kDa. The endogenous VRL-1 can also be observed at  80 kDa in the anti-VRL-1 Western blot and does not appear to be glycosylated. Lower bands are due to unspecific reaction of the anti-VRL-1 antibody. Discussion F-11 cells were first generated by Platika et al. [7] as a model to analyze the properties of single neurons. Four cell lines (F-11 A–D) showed properties characteristic of DRG neurons, such as action potentials, extensive neurite-like processes and expression of neuronal gangliosides. Even Fig. 6. Non-denaturing PAGE of single cell RT-PCR fragment D. Single F-11 cells (sc1–5) show the two band pattern for fragment D. The upper correspond to the rat brain (rb) cDNA control, the lower band to the N18TG2 (N) cDNA control. Both fragments from a single F-11 cell (sc1) were cloned and sequenced. Fig. 7. Fluorescent images of VRL-1 and VRL-1-GFP in F-11 cells. Indirect immunofluorescence with anti-VRL-1 polyclonal antibody (A–D). Endogenous VRL-1 in F-11 cells only gives week signals in the cytoplasm (A,B) whereas VRL-1 in transfected F-11 cells (C,D) shows predomi- nantly plasma membrane localization. GFP fluorescence of VRL-1-GFP transfected F-11 cells (E,F). Here VRL-1-GFP is located at the plasma- membrane but part of the protein remains at intracellular membranes (ER). Overlay images with DAPI-stain for nuclei are shown. Ó FEBS 2003 Co-expression of mouse and rat VRL-1 in F-11 cells (Eur. J. Biochem. 270) 4269 though F-11 cells have varying numbers of mouse and rat chromosomes, they show a remarkable homogeneity of neuronal features. Three rat and mouse isoenzymes [nucleo- side phosphorylase (NP), peptidase B (PB), mannose phosphate isomerase (MPI)] were shown to be expressed in all four stable F-11 cell lines A–D. Unique expression of mouse PB and MPI was observed in line C [7]. As shown by Mevel-Ninio and Weiss [12] it cannot be excluded that some gene expression might be due to selective activation of a previously silent gene from the neuroblastoma cell line by the DRG. They observed this phenomenon in other cell fusion experiments. Depending on growth conditions these fusion cells alter their morphology to a neuronal phenotype, exhibiting many long neurite-like extensions. There are several differential features of DRG cells present in F-11 cells such as l-and d-opioid receptors, receptors for prostaglandin and brady- kinin, and voltage-sensitive calcium channels. Also F-11 cells synthesize and release substance P [8]. Here we show that F-11 cells express another receptor expected in DRG neurons. They express the vanilloid-like TRP channel VRL-1 as shown by Western blot analysis. Bender et al. [9] isolated a rat VRL-1 mRNA from F-11 cells identical to that observed in dorsal root ganglion extracts and suggested that the F-11 cells most likely are derived from medium- sized Ad-fibers. Surprisingly when performing PCR with rat VRL-1 specific primers we detected two products instead of one, from which the slower migrating band corresponded to a rat brain control. Sequence analysis of both bands clearly showed that the faster migrating band corresponds to mouse VRL-1 (Acc. No. NM_011706) whereas the slower migrating band corresponds to rat VRL-1 (Acc. No. AF129113). Our findings that the mouse and rat VRL-1 are both expressed in individual F-11 cells fits well with this result. In F-11 parental cell line N18TG2 we detected the expression of mouse VRL-1 due to its murine origin. Therefore we propose that in the F-11 fusion cell no selective activation of a previously silent gene from the neuroblastoma cell line by the DRG cell occurs. Rat and mouse VRL-1 cDNA are 92% identical. On protein level five amino acids located near the N-terminus are missing in the rat VRL-1 as compared to its mouse orthologue. Unfortunately it is not possible to distinguish between the two on the protein level using commercially available antibodies. Due to the expression of the endo- genous protein, the N18TG2 cells are appropriate for functional investigations of the mouse VRL-1. The DRG- derived F-11 cell line provides an additional powerful experimental system for functional studies of VRL-1. However, one has to be aware that both the mouse and the rat protein are expressed. In a comparative study we here show that in F-11 cells the heterologously expressed rVRL-1 is located at the plasma membrane and in neurite-like structures whereas the endogenous protein appears only in the cytoplasm, prob- ably at intracellular membranes. When GFP is fused to the C-terminus of rVRL-1 a portion is observed at the plasma membrane, but is associated with intracellular membranes, likely the endoplasmic reticulum. Another notable differ- ence could be observed when we looked at the glycosylation status of VRL-1. Surprisingly we observed no evidence of glycosylation of the endogenous VRL-1 at  80 kDa in the anti-VRL-1 Western blot, whereas the overexpressed rVRL-1wasshowntobeglycosylatedinthesamecells. Obviously, we only observe an endogenous VRL-1 variant in F-11 cells, which for unknown reasons is retained in intracellular compartments in an unglycosylated state. Because in Western blot and immunofluorescence experi- ments the amount of VRL-1 is very low, this might represent an immature protein. One way to prove whether Fig. 8. Deglycosylation with N-glycanase and endoglycosidase H of VRL-1 and VRL-1-GFP transfected F-11 cells. (A) Anti-VRL-1 Westernblot (WB) of VRL-1 transfected F-11 cells. Anti-VRL-1 immunoreactivity appeared as multiple bands in lysates of transfected F-11 cells which is characteristic for a glycoprotein. Incubation of cell lysates with Endo H abolished the upper band of the anti-VRL-1 immunoreactivity doublet band at  80/84 kDa, the diffuse anti-VR1 immunoreactivity at  97 kDa was unaffected. After incubation with N-Gly, neither the  97 kDa nor the  84 kDa anti-VRL-1 immunoreactivity band were observed, only the 80 kDa band showed up with strong intensity. (B) and (C) Anti-GFP and anti-VRL-1 Western blot of F-11 cells expression VRL-1-GFP. The same band pattern of the VRL-1 glycoprotein as in (A) is observed, with the difference that GFP shifts the molecular weight  30 kDa higher. The endogenous VRL-1 can be observed at  80 kDa in (C) and is not glycosylated. Lower bands are unspecific reaction of the anti-VRL-1 antibody. 4270 R. Jahnel et al.(Eur. J. Biochem. 270) Ó FEBS 2003 the m-VRL-1 and/or r-VRL-1 protein is expressed signifi- cantly by untransfected F-11 cells would be to overexpress the mVRL-1 and look for glycosylation, but at present the mouse clone is not available to us. Whether the glycosyla- tion is responsible for membrane targeting of VRL-1 and can be induced by signaling molecules which activate endogenous VRL-1 in F-11 cells still needs to be investi- gated. Our data indicate that both N-linked high mannose-type glycosylation and complex glycosylation, i.e. endoglycosi- dase H-resistant glycosylation of rVRL-1 occur. These findings are similar to previous investigations on rVR1, where the same glycosylation pattern was found [6]. In order to determine the glycosylation sites of rVRL-1 and mVRL-1 we used the PROSITE prediction tool (http://www.exp asy.org/tools/scanprosite; Gattika et al. [13]) for the detec- tion of Asn-X-Ser/Thr Asn-glycosylation consensus motif within the VRL-1 primary structure. Three potential Asn- glycosylation sites were detected for rVRL-1, namely 63–66 NTSA, 571–574 NNST and 572–575 NSTV. With respect to the membrane topology predicted by the program TMHMM (http://www.cbs.dtu.dk/services/TMHMM-2.0; [14]) only N571 and/or N572 are located extracellularly and can be glycosylated. An alignment of rVR1 and rVRL-1 shows that these Asn-glycosylation sites are located in an analog- ous position to the Asn-glycosylation site in rVR1, between the fifth transmembrane sequence and the pore loop. Only one potential Asn-glycosylation site was detected for mVRL-1, namely 567–570 NTTV, which also is located extracellularly. We were previously able to confirm N604 as the only glycosylation site in rVR1 by site-directed muta- genesis [6]. Acknowledgements We would like to thank Dr Erik Wade (Gru ¨ nenthal GmbH) and Dr Chris Weise for critical reading of the manuscript. Many thanks to Doris Kru ¨ ck for her technical assistance with cell culture. 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