Participation of the extracellular domain in (pro)renin receptor dimerization |
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Authors: | Chiharu Suzuki-Nakagawa Misa Nishimura Tomoko Tsukamoto Sho Aoyama Akio Ebihara Fumiaki Suzuki Tsutomu Nakagawa |
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Institution: | 1. The United Graduate School of Agricultural Sciences, Gifu University, Gifu, Japan;2. Department of Applied Life Science, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan |
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Abstract: | 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. |
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Keywords: | ECD extracellular domain ER endoplasmic reticulum FL full-length (P)RR (pro)renin receptor s(P)RR soluble (pro)renin receptor |
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