Understanding the cellular function of TRPV2 channel through generation of specific monoclonal antibodies
Authors: Daniel Cawley
Matthew R Cohen
Vera Y Moiseenkova-Bell
Kevin W Huynh
Journal: PLoS ONE (2013)
Discipline: Multidisciplinary
Keywords: TRPV2; monoclonal antibodies; immuno-detection techniques; growth factor signaling; trafficking
Affiliations: 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., Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, United States of America., Monoclonal Antibody Core Facility, Vaccine and Gene Therapy Institute, Oregon Health and Science University, Beaverton, Oregon, United States of America.

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. 

Retracted: No

PubPeer comments:
 Volatile impurities and not the purported pheromone activates the Odorant Receptor 7a. The main findings and conclusions are therefore suspected to be an artifact. https: www.biorxiv.org content 10.1101 503169v1 , 
 #1 Our paper presents behavioral evidence that food-odors stimulate *Drosophila* males to secrete a deposited aggregation pheromone.u00a0 Based on extraction of surfaces containing this food-odor-induced pheromone and GC-MS, this pheromone was identified as 9-tricosene. Our work further suggested that 9-tricosene stimulates ab4A (Or7a).u00a0 Mutation of Or7a, and ablation of Or7a-expressing neurons, abolished the behavioral responses to the naturally produced food-odor-induced pheromone, indicating that Or7a is involved in its detection and behavior. The preprint highlighted here presents evidence that samples of 9-tricosene acquired from Sigma contain trace amounts of E-2-hexenal, which is also a potent stimulator of Or7a. The preprint suggests that it was these E-2-hexenal impurities, not 9-tricosene, that were found to stimulate Or7a.u00a0 However, as we showed in our publication, levels of 9-tricosene on males are only enriched upon food-odor stimulation, which was not examined in the preprint. In addition, the preprint lacks evidence that 9-tricosene does not activate Or7a. As described in the paper, in addition to Sigma 9-tricosene, we also tested two additional independent sources of 9-tricosene not examined in the preprint (TCI America Cat#T1242, AK Scientific Cat#M691) that also strongly activated Or7a. While it is possible that olfactory recordings using Sigma 9-tricosene represent a mixture of 9-tricosene and E2-hexenal induced responses, the main findings of the paper remain. ,