title: The transient receptor potential (TRP) channel TRPC3 TRP domain and AMP-activated protein kinase binding site are required for TRPC3 activation by erythropoietin.
abstract: Modulation of intracellular calcium ([Ca(2+)](i)) by erythropoietin (Epo) is an important signaling pathway controlling erythroid proliferation and differentiation. Transient receptor potential (TRP) channels TRPC3 and homologous TRPC6 are expressed on normal human erythroid precursors, but Epo stimulates an increase in [Ca(2+)](i) through TRPC3 but not TRPC6. Here, the role of specific domains in the different responsiveness of TRPC3 and TRPC6 to erythropoietin was explored. TRPC3 and TRPC6 TRP domains differ in seven amino acids. Substitution of five amino acids (DDKPS) in the TRPC3 TRP domain with those of TRPC6 (EERVN) abolished the Epo-stimulated increase in [Ca(2+)](i). Substitution of EERVN in TRPC6 TRP domain with DDKPS in TRPC3 did not confer Epo responsiveness. However, substitution of TRPC6 TRP with DDKPS from TRPC3 TRP, as well as swapping the TRPC6 distal C terminus (C2) with that of TRPC3, resulted in a chimeric TRPC6 channel with Epo responsiveness similar to TRPC3. Substitution of TRPC6 with TRPC3 TRP and the putative TRPC3 C-terminal AMP-activated protein kinase (AMPK) binding site straddling TRPC3 C1/C2 also resulted in TRPC6 activation. In contrast, substitution of the TRPC3 C-terminal leucine zipper motif or TRPC3 phosphorylation sites Ser-681, Ser-708, or Ser-764 with TRPC6 sequence did not affect TRPC3 Epo responsiveness. TRPC3, but not TRPC6, and TRPC6 chimeras expressing TRPC3 C2 showed significantly increased plasma membrane insertion following Epo stimulation and substantial cytoskeletal association. The TRPC3 TRP domain, distal C terminus (C2), and AMPK binding site are critical elements that confer Epo responsiveness. In particular, the TRPC3 C2 and AMPK site are essential for association of TRPC3 with the cytoskeleton and increased channel translocation to the cell surface in response to Epo stimulation.
authors.affiliation: Departments of Pediatrics, Hershey, Pennsylvania 17033.
authors.forename: Iwona
authors.initials: I
authors.lastname: Hirschler-Laszkiewicz
authors.affiliation: Departments of Pediatrics, Hershey, Pennsylvania 17033.
authors.forename: Qin
authors.initials: Q
authors.lastname: Tong
authors.affiliation: Departments of Pediatrics, Hershey, Pennsylvania 17033.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Waybill
authors.affiliation: Departments of Pediatrics, Hershey, Pennsylvania 17033.
authors.forename: Kathleen
authors.initials: K
authors.lastname: Conrad
authors.affiliation: Departments of Pediatrics, Hershey, Pennsylvania 17033.
authors.forename: Kerry
authors.initials: K
authors.lastname: Keefer
authors.affiliation: Departments of Pediatrics, Hershey, Pennsylvania 17033.
authors.forename: Wenyi
authors.initials: W
authors.lastname: Zhang
authors.affiliation: Departments of Pediatrics, Hershey, Pennsylvania 17033.
authors.forename: Shu-Jen
authors.initials: SJ
authors.lastname: Chen
authors.affiliation: Department of Medicine, Jefferson Medical College, Philadelphia, Pennsylvania 19107.
authors.forename: Joseph Y
authors.initials: JY
authors.lastname: Cheung
authors.affiliation: Departments of Pediatrics, Hershey, Pennsylvania 17033; Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, Pennsylvania 17033. Electronic address: bmiller3@psu.edu.
authors.forename: Barbara A
authors.initials: BA
authors.lastname: Miller
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000737:chemistry* / Q000378:metabolism; D020816:Amino Acid Motifs; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D002454:Cell Differentiation; D002460:Cell Line; D049109:Cell Proliferation; D004912:Erythrocytes / Q000166:cytology; D004921:Erythropoietin / Q000378:metabolism; D006801:Humans; D017434:Protein Structure, Tertiary; D017467:Receptors, Erythropoietin / Q000378:metabolism*; D015398:Signal Transduction; D050052:TRPC Cation Channels / Q000378:metabolism*; D000074287:TRPC6 Cation Channel
chemical_list: D017467:Receptors, Erythropoietin; D050052:TRPC Cation Channels; C104603:TRPC3 cation channel; D000074287:TRPC6 Cation Channel; C117032:TRPC6 protein, human; D004921:Erythropoietin; D055372:AMP-Activated Protein Kinases; D002118:Calcium