Participation of the extracellular domain in (pro)renin receptor dimerization.
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 United Graduate School of Agricultural Sciences, Gifu University, Gifu, Japan.
Chiharu
C
Suzuki-Nakagawa
Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan.
Misa
M
Nishimura
Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan.
Tomoko
T
Tsukamoto
Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan.
Sho
S
Aoyama
The United Graduate School of Agricultural Sciences, Gifu University, Gifu, Japan; Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan.
Akio
A
Ebihara
The United Graduate School of Agricultural Sciences, Gifu University, Gifu, Japan; Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan.
Fumiaki
F
Suzuki
The United Graduate School of Agricultural Sciences, Gifu University, Gifu, Japan; Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan. Electronic address: nakagawa@gifu-u.ac.jp.
Tsutomu
T
Nakagawa
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002522:Chlorocebus aethiops; D003412:Cricetulus; D057809:HEK293 Cells; D006801:Humans; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary; D011956:Receptors, Cell Surface / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000032:analysis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012084:Renin-Angiotensin System; D014162:Transfection; D025262:Vacuolar Proton-Translocating ATPases / Q000032:analysis / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000093342:Prorenin Receptor
D011956:Receptors, Cell Surface; D011993:Recombinant Fusion Proteins; D025262:Vacuolar Proton-Translocating ATPases; C458319:ATP6AP2 protein, human; D000093342:Prorenin Receptor
(Pro)renin receptor; ATP6AP2; Dimerization; Extracellular domain; Retention