R-Spondin 3 (RSPO3, roof plate-specific spondin3), also called cysteine-rich and single thrombospondin domain containing-1 (Cristin 1), is a ~31kD secreted protein that shares ~40% amino acid identity with the other three R-Spondin family members (1, 2). All are positive modulators of Wnt /b-catenin signaling, but each has a distinct expression pattern (1-4). Like other R-spondins, R-Spondin3 contains two adjacent cysteine-rich furin-like domais (aa33-135) with one potential N-glycosylationsite (aa36) , followed by a thrombospondin (TSP-1) motif (aa147-207) and a region rich in basic residues (aa211-269). Only the furin-like domains are needed for b-catenin stabilization (2). Within aa21-209, human R-Spondin 3 shares 93%, 92%, 97%, 96% and 92% aa identity with mouse, rat, equine, bovine and canine R-Spondin 3, respectively. Potential isoforms of 279 and 297 aa diverge at aa 210 and 276, respectively (5). Mouse R-Spondin 3 is critical for development of the placental labyrinthine layer, probably by promoting VEGF expression and thus vascular development (6, 7). It is also essential for expression of the placenta-specific transcription factor, Gcm1. In the mouse embryo, R-Spondin 3 is often expressed by or located near endothelial cells (6). It is found in the roof plate, tail , somites, otic vesicles, cephalic mesoderm, truncus arteriosus, atrioventricular canal of the developing heart, and strongly but transiently in developing limbs (4, 7). R-Spondins regulate Wnt/b-catein by competing with the Wnt antagonist DKK-1 for binding to the Wnt co-receptors LRP-6 and Kremen, reducing their DKK-1-mediated internalization (8, 9). Reports differ on whether R-Spondins bind LRP-6 directly (8-10). R-Spondin 3 has also been identified as an oncogene (11).
Biological Activity:Measured by its ability to induce activation of b-catenin response in a Topflash Luciferase assay using HEK293T human embryonic kidney cells. The ED50 for this effect is typically 0.5-2ng/ml
Endotoxin: ≤1EU/ug (LAL)
Storage and Stability:Lyophilized powder may be stored at -20°C. Reconstitute with sterile buffer or ddH2O. Aliquot and store at -20°C. Reconstituted product is stable for 6 months at -20°C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.
