Biochemical characterization of the vanilloid receptor 1 expressed in a dorsal root ganglia derived cell line.
The vanilloid receptor VR1 is an ion channel predominantly expressed by primary sensory neurons involved in nociception. Here we describe its biochemical properties and assess the subcellular localization, the glycosylation state and the quaternary structure of VR1 expressed in HEK293 cells and in the DRG-derived cell line F-11 (N18TG2 mouse neuroblastoma x rat dorsal root ganglia, hybridoma). VR1 was found to be glycosylated in both cell types. Of the five potential N-glycosylation sites, the predicted transient receptor potential channel-like transmembrane folding proposes N604 is localized extracellularly. We used site-directed mutagenesis to mutate the Asn at position 604 to Thr. This mutated VR1 was not glycosylated, confirming the extracellular location of N604 and its role as the exclusive site of glycosylation of the VR1 protein. VR1 occured in high molecular mass complexes as assessed by blue native PAGE. In the presence of limited amounts of SDS dimers, trimers and tetramers of VR1 were observed, consistent with the predicted tetrameric quaternary structure of the receptor. Cross-linking with dimethyladipimidate yielded almost exclusively dimers. Whereas VR1 localized both to the plasma membrane and to intracellular membranes in HEK293 cells, it localized predominantly to the plasma membrane in F-11 cells. Using confocal laserscanning microscopy, we observed an enrichment of anti-VR1 immunoreactivity in neurite-like structures of F-11 cells. In the light of conflicting literature data on biochemical characteristics of VR1, our data suggest that dorsal root ganglion-derived F-11 cells provide a powerful experimental system for the study of VR1 biochemistry.
Jahnel
R
R
Arbeitsgruppe Neurochemie and Arbeitsgruppe Molekulare Medizin, Institut für Chemie-Biochemie, Freie Universität Berlin, Germany.
Dreger
M
M
Gillen
C
C
Bender
O
O
Kurreck
J
J
Hucho
F
F
D000818:Animals; D001216:Asparagine / Q000235:genetics; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D005727:Ganglia, Spinal / Q000166:cytology / Q000502:physiology*; D006023:Glycoproteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006031:Glycosylation; D051379:Mice; D016297:Mutagenesis, Site-Directed; D011489:Protein Denaturation; D017510:Protein Folding; D051381:Rats; D011955:Receptors, Drug / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014162:Transfection
D006023:Glycoproteins; D011955:Receptors, Drug; D001216:Asparagine