Participation of the extracellular domain in (pro)renin receptor dimerization

• Published in: Biochemical and biophysical research communications 2014-02, Vol.444 (4), p.461-466
• Main Authors: Suzuki-Nakagawa, Chiharu, Nishimura, Misa, Tsukamoto, Tomoko, Aoyama, Sho, Ebihara, Akio, Suzuki, Fumiaki, Nakagawa, Tsutomu
• Format: Article
• Language: English
• Subjects: (Pro)renin receptor; 60 APPLIED LIFE SCIENCES; AMINO ACIDS; ANGIOTENSIN; Animals; ATP6AP2; Cercopithecus aethiops; CHO CELLS; COPRECIPITATION; COS Cells; Cricetulus; DIMERIZATION; ENDOPLASMIC RETICULUM; Extracellular domain; GLUTATHIONE; HEK293 Cells; Humans; MICROSCOPY; Protein Multimerization; Protein Structure, Tertiary; RECEPTORS; Receptors, Cell Surface - analysis; Receptors, Cell Surface - chemistry; Receptors, Cell Surface - genetics; Receptors, Cell Surface - metabolism; Recombinant Fusion Proteins - analysis; Recombinant Fusion Proteins - chemistry; Recombinant Fusion Proteins - genetics; Recombinant Fusion Proteins - metabolism; RENIN; Renin-Angiotensin System; RETENTION; SECRETION; Transfection; TRANSIENTS; Vacuolar Proton-Translocating ATPases - analysis; Vacuolar Proton-Translocating ATPases - chemistry; Vacuolar Proton-Translocating ATPases - genetics; Vacuolar Proton-Translocating ATPases - metabolism
• ISSN: 0006-291X; 1090-2104
• DOI: 10.1016/j.bbrc.2014.01.073

•The (pro)renin receptor [(P)RR] is a regulator of the renin–angiotensin system.•The region responsible for (P)RR dimerization was investigated.•(P)RR extracellular domain constructs were retained intracellularly.•The extracellular domain of (P)RR is responsible for its dimerization.•Novel insight into the regulatory mechanism of soluble (P)RR secretion is provided. 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.