Abstract 422: Extracellular Domain of the (pro)renin Receptor Participates in Dimerization

The (pro)renin receptor [(P)RR] is a single transmembrane protein with a large N-terminal extracellular domain and a short C-terminal cytoplasmic domain. Three molecular forms of (P)RR are known: a full-length form, a C-terminal truncated soluble form [s(P)RR], and an N-terminal truncated transmembrane form (M8-9 fragment). It has been reported that (P)RR forms homodimer, but the region participating in dimerization is unknown. Here we report that the extracellular domain of (P)RR participate in dimerization. Using secretory glutathione S-transferase (GST) as a fusion tag, we developed an in vivo pull-down assay to investigate (P)RR dimerization in the cell. GST-tagged extracellular domain (amino acid residues 17-304) of human (P)RR [GST-ECD], as well as full-length (P)RR [GST-Full] and M8-9 fragment [GST-M8-9], was retained in the cell, while GST alone was efficiently secreted into the culture medium when transiently expressed in COS-7 cells. Consistent with this observation, immunofluorescence microscopy showed prominent localization of GST-ECD to the ER. In vivo GST pull-down experiments revealed that GST-ECD as well as GST-Full bound FLAG-tagged full-length (P)RR, whereas GST-M8-9 bound it little, if any, when transiently coexpressed in HEK293T cells. To investigate whether s(P)RR can form heterodimer with full-length (P)RR, we established a CHO cell line stably expressing human (P)RR with C-terminal 10His-tag. All three forms of (P)RR were detected in the cell lysate and only s(P)RR was detected in the culture medium. Full-length (P)RR and s(P)RR were prominently localized to the ER as observed by immunofluorescence microscopy. Pull down experiment using His-tag affinity resin showed coprecipitation of s(P)RR with full-length (P)RR. These results indicate that the extracellular domain of (P)RR participates in dimerization.