Abstract 1089: Two nonselective calcium channels, TRPC3 and TRPC6, play roles in erythropoietin signaling
Authors: Hirschler-Laszkiewicz Iwona M. , Tong Qin , Conrad Kathleen M. , Zhang Wenyi , Chen Shu-Jen , Keefer Kerry A. , Cheung Joseph Y. , Miller Barbara A. 
Published: June 2012

Abstract
The signal transduction pathways of erythropoietin (Epo), a glycoprotein regulating production of red blood cells, have been a model for understanding growth factor stimulated cell proliferation and differentiation. Erythropoietin receptors (Epo-R) are also expressed on non-hematopoietic cells, and recently Epo was reported to have antiapoptotic effects on these cells including a potential role in specific cancers. Two calcium channels were identified which are involved in Epo signaling, TRPC3 and TRPC6. These are homologous, nonselective calcium channels and are members of the canonical transient receptor potential (TRPC) subfamily expressed on human erythroblasts. Both channels have also been implicated in stimulating proliferation of cancer cells; TRPC3 in ovarian cancer, TRPC6 in gliomas, and both channels in breast cancer. Here TRPC3 and TRPC6 function was studied in transfected human embryonic kidney (HEK)-293T cells, and in the human leukemia UT-7/Epo cell line. Measurement of [Ca2+]i was performed on single cells with Digital Video Imaging. TRPC3 is activated by erythropoietin, whereas TRPC6 is not. In fact, TRPC6 can inhibit Epo-induced activation of TRPC3. Domains regulating channel activation were studied here. The TRP domain of TRPC3 is required for its activation, and substitution of five amino acids in the TRPC3 TRP domain with those of TRPC6 significantly reduced the Epo-stimulated increase in [Ca2+]i in TRPC3-expressing cells. Substitution of the same amino acids in the TRPC6 TRP with those of TRPC3 did not enhance the response of TRPC6-expressing cells to Epo stimulation. However, substitution of the distal TRPC6 C-terminus (C2) with that of TRPC3 in addition to substitution of TRPC6 TRP with TRPC3 TRP resulted in a TRPC6 channel with Epo responsiveness similar to TRPC3. In contrast, substitution of the TRPC3 C-terminal leucine zipper motif or TRPC3 phosphorylation sites S681, S708, or S764 with corresponding TRPC6 sequence did not affect TRPC3 Epo-responsiveness. TRPC3, but not TRPC6, and chimeras expressing the distal TRPC3 C terminus showed significantly increased membrane insertion following Epo stimulation. This was associated with substantial cytosketetal localization of TRPC3 and chimeras expressing TRPC3C2 but not TRPC6. These data demonstrate the important role of the TRP domain in specificity of channel responsiveness to agonist stimulation, and the requirement for other sites for optimal response to agonists, such as those found in the distal C-terminus of TRPC3. A few mutations in critical channel sites could have an important effect on proliferation and survival of cells expressing Epo-R.
Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1089. doi:10.1158/1538-7445.AM2011-1089