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.

Molecular cloning and expression in gonad of Rana rugosa WT1 and Fgf9.

Sry (sex-determining region on the Y chromosome) is required for testicular differentiation in mammals. In addition to Sry, other genes such as WT1, Fgf9, Dax1, Dmrt1 and Sox9 are widely accepted to be involved in the sex determination in vertebrates. However, the roles of these genes during sex determination still remain unclear in amphibians. This study was undertaken to examine the expression of WT1 and Fgf9 in the developing gonad of amphibians. We first isolated the WT1 cDNA from the frog Rana rugosa. Like WT1 in mice, R. rugosa WT1 showed 2 isoforms; i.e., one had an additional 3 amino acids, KTS, included between the third and fourth zinc fingers. However, 17 amino acids in exon 5 of mammalian WT1 could not be found in R. rugosa WT1, which is also the case in turtle and chicken. The mRNA of both isoforms (+KTS, -KTS) was detected in the lung, kidney and testis, but not in the ovary and muscle of adult frogs. The 2 isoforms were expressed first in the embryos at stage 23. Thereafter, the expressions remained constant in the gonad attached to mesonephros of both sexes during sex determination. We next isolated the R. rugosa Fgf9 cDNA encoding 208 amino acids. The amino acid sequence of Fgf9 had similarity greater than 92% with chicken, mouse and human Fgf9s, suggesting that Fgf9 is highly conserved among vertebrate classes. Fgf9 was expressed in the ovary of an adult frog strongly, but in the lung weakly. In contrast, the Fgf9 mRNA was hardly detected in the kidney, testis and muscle. Moreover, Fgf9 did not show a sexually dimorphic expression pattern during sex determination in R. rugosa. The results, taken together, suggest that both WT1 and Fgf9 are expressed in the indifferent gonad prior to sex determination without any difference in the expression between males and females. Thus, it seems unlikely that they are a key factor to initiate the divergence leading to testicular or ovarian differentiation in R. rugosa.