title: Understanding the cellular function of TRPV2 channel through generation of specific monoclonal antibodies.
abstract: Transient receptor potential vanilloid 2 (TRPV2) is a Ca(2+)-permeable nonselective cation channel proposed to play a critical role in a wide array of cellular processes. Although TRPV2 surface expression was originally determined to be sensitive to growth factor signaling, regulated trafficking of TRPV2 has remained controversial. TRPV2 has proven difficult to study due to the lack of specific pharmacological tools to modulate channel activity; therefore, most studies of the cellular function of TRPV2 rely on immuno-detection techniques. Polyclonal antibodies against TRPV2 have not been properly validated and characterized, which may contribute to conflicting results regarding its function in the cell. Here, we developed monoclonal antibodies using full-length TRPV2 as an antigen. Extensive characterization of these antibodies and comparison to commonly used commercially available TRPV2 antibodies revealed that while monoclonal antibodies generated in our laboratory were suitable for detection of endogenous TRPV2 by western blot, immunoprecipitation and immunocytochemistry, the commercially available polyclonal antibodies we tested were not able to recognize endogenous TRPV2. We used our newly generated and validated TRPV2 antibodies to determine the effects of insulin-like growth factor 1 (IGF-1) on TRPV2 surface expression in heterologous and endogenous expression systems. We found that IGF-1 had little to no effect on trafficking and plasma membrane expression of TRPV2. Overall, these new TRPV2 monoclonal antibodies served to dispel the controversy of the effects of IGF-1 on TRPV2 plasma membrane expression and will clarify the role TRPV2 plays in cellular function. Furthermore, our strategy of using full-length tetrameric TRP channels may allow for the generation of antibodies against other TRP channels of unclear function.
authors.affiliation: Department of Physiology & Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio, United States of America ; Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, United States of America.
authors.forename: Matthew R
authors.initials: MR
authors.lastname: Cohen
authors.affiliation: Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, United States of America.
authors.forename: Kevin W
authors.initials: KW
authors.lastname: Huynh
authors.affiliation: Monoclonal Antibody Core Facility, Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, Oregon, United States of America.
authors.forename: Daniel
authors.initials: D
authors.lastname: Cawley
authors.affiliation: Department of Physiology & Biophysics, School of Medicine, Case Western Reserve University, Cleveland, Ohio, United States of America ; Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, United States of America.
authors.forename: Vera Y
authors.initials: VY
authors.lastname: Moiseenkova-Bell
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D005786:Gene Expression Regulation / Q000187:drug effects; D006367:HeLa Cells; D006801:Humans; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D008297:Male; D051379:Mice; D009206:Myocardium / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000378:metabolism; D050916:TRPV Cation Channels / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism*
chemical_list: D000911:Antibodies, Monoclonal; D011994:Recombinant Proteins; D050916:TRPV Cation Channels; C476722:Trpv2 protein, rat; D007334:Insulin-Like Growth Factor I