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As a component of the renin-angiotensin system, the (pro)renin receptor [(P)RR] activates prorenin along with intracellular signaling pathways. In this study, the glutathione S-transferase-fused extracellular domain of (P)RR expressed in mammalian cells was recovered in the detergent phase in detergent-based two-phase separation experiments, and intracellular localization was observed by immunocytochemistry, suggesting retention inside the cell through stable membrane association.

The (pro)renin receptor [(P)RR] induces the catalytic activation of prorenin, as well as the activation of the mitogen-activated protein kinase (MAPK) signaling pathway; as such, it plays an important regulatory role in the renin-angiotensin system. (P)RR is known to form a homodimer, but the region participating in its dimerization is unknown. Using glutathione S-transferase (GST) as a carrier protein and a GST pull-down assay, we investigated the interaction of several (P)RR constructs with full-length (FL) (P)RR in mammalian cells. GST fusion proteins with FL (P)RR (GST-FL), the C-terminal M8-9 fragment (GST-M8-9), the extracellular domain (ECD) of (P)RR (GST-ECD), and the (P)RR ECD with a deletion of 32 amino acids encoded by exon 4 (GST-ECDd4) were retained intracellularly, whereas GST alone was efficiently secreted into the culture medium when transiently expressed in COS-7 cells. Immunofluorescence microscopy showed prominent localization of GST-ECD to the endoplasmic reticulum. The GST pull-down analysis revealed that GST-FL, GST-ECD, and GST-ECDd4 bound FLAG-tagged FL (P)RR, whereas GST-M8-9 showed little or no binding when transiently co-expressed in HEK293T cells. Furthermore, pull-down analysis using His-tag affinity resin showed co-precipitation of soluble (P)RR with FL (P)RR from a stable CHO cell line expressing FL h(P)RR with a C-terminal decahistidine tag. These results indicate that the (P)RR ECD 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.