Four vertebrate R-spondin proteins are secretory agonists of the classical Wnt / β-catenin signaling pathway. These proteins are approximately 35 kDa and are characterized by two amino-terminal furin-like repeats necessary and sufficient for Wnt signalling enhancement, and one closer to the carboxyl terminus that can bind to matrix glycosaminoglycans and / or proteoglycans Thrombospondin Domain. Although R-spondins cannot initiate Wnt signaling, they can effectively enhance the response to low-dose Wnt proteins. In humans, the rare disruption of the gene encoding R-spondin1 causes XX reversal syndrome (phenotype males), palm palm keratosis (palm and plantar thickening due to excessive keratin formation), and susceptible to squamous cell carcinoma of the skin. Mutations in the gene encoding R-spondin4 can cause onychomycosis (missing or stunted nails on the fingers and toes). Recently, leucine repeat-rich G protein-coupled receptor Lgr 4, Lgr5 and Lgr6 have been identified as three closely related orphans of leucine repeat-rich G protein-coupled receptor. Lgr5 and Lgr6 are markers of adult stem cells. Since R-spondins are effective stimulators of adult stem cell proliferation in vivo and in vitro, these findings may guide the therapeutic use of R-spondins in regenerative medicine.
The four R-spondin proteins share a common domain structure. The amino-terminal endoplasmic reticulum signal peptide ensures entry into the secretory pathway. The processed mature protein has two cysteine-rich furin-like repeats at the amino terminus. The TSR-1 central domain is followed by a region with a large number of basic amino acids at the carboxyl terminus (Figure 1). Two furin-like repeats near the amino terminus are related to domains found in furin, a member of the subtilisin-like preprotein-converting enzyme family. Although the function of this domain in furin is unknown, it is ubiquitous in many important growth factor receptors (such as EGF, insulin, HGF, and neurotrophic factors), suggesting that it plays an important role in function.
Figure 1. Protein domain architecture and chromosome location of human R-spondins.
Localization and function
Extensive functional analysis of R-spondin protein using Wnt reporter gene detection in 293T cells was found to be related to the classic Wnt / β-catenin pathway (Figure 2). The latter plays a central role in cell proliferation, differentiation, and stem cell maintenance. When the secreted proteins of the Wnt family bind to the Frizzled (Fzd) receptor and low-density lipoprotein receptor-related protein 5 or 6 (LRP5 / 6) co-receptors, activity is initiated. At this level, the pathway is controlled by a series of extracellular antagonists (Figure 3). R-spondins have a unique synergy with Wnt proteins. Therefore, R-spondin activation has been shown to be sensitive to the presence of the extracellular Wnt inhibitor Dickkopf-1 (DKK1), and overexpression of any known intracellular component of this pathway does not induce synergy. Protein domain analysis showed that furin repeats were essential and sufficient to mediate Wnt enhancement by R-spondins. In vivo experiments demonstrating this Wnt enhancement phenomenon were performed in early frog embryos. At eight cell stages, depletion of Rspondin2 in one blastomere results in a decrease in nodular ganglia and muscle cells at the injection site. Depletion of the gastric nodule prevents the transcription of myoD and myf5 genes and subsequently impairs muscle development. At this stage of development in chicks and mammals, manipulation of Wnt activity significantly phenotypes these effects. Rspondins-enhanced Wnt pathway enhancement has also been seen in experimentally induced tumors. The breast tumor virus integration site was found in both the gene of the Wnt family members and the gene of R-spondin2, which explained the persistently high level of Wnt activity in the tumor.
Figure 2. Simplifed overview of the canonical Wnt signaling pathway.