The R-spondin protein family

Abstract

The four vertebrate R-spondin proteins are secreted agonists of the canonical Wnt/β-catenin signaling pathway. These proteins are approximately 35 kDa, and are characterized by two amino-terminal furin-like repeats, which are necessary and sufficient for Wnt signal potentiation, and a thrombospondin domain situated more towards the carboxyl terminus that can bind matrix glycosaminoglycans and/or proteoglycans. Although R-spondins are unable to initiate Wnt signaling, they can potently enhance responses to low-dose Wnt proteins. In humans, rare disruptions of the gene encoding R-spondin1 cause a syndrome of XX sex reversal (phenotypic male), palmoplantar keratosis (a thickening of the palms and soles caused by excess keratin formation) and predisposition to squamous cell carcinoma of the skin. Mutations in the gene encoding R-spondin4 cause anonychia (absence or hypoplasia of nails on fingers and toes). Recently, leucine-rich repeat-containing G-protein-coupled receptor (Lgr)4, Lgr5 and Lgr6, three closely related orphans of the leucine-rich repeat family of G-protein-coupled receptors, have been identified as receptors for R-spondins. Lgr5 and Lgr6 are markers for adult stem cells. Because R-spondins are potent stimulators of adult stem cell proliferation in vivo and in vitro, these findings might guide the therapeutic use of R-spondins in regenerative medicine.

Figure 1

Evolutionary history of R-spondins. Homologs of R-spondin (each contains two Fu domains followed by a thrombospondin protein 1 (TSP1) domain) are shown. Two non-chordate R-spondin sequences with its characteristic domain architecture were detected in the hemichordate acorn worm and the echinodermate sea urchin. These two were used to root the gene phylogeny. The tree clearly shows the two successive whole genome duplications that generated present day R-spondin diversity in vertebrate species such as fish and mammals. Sequences were aligned using MAFFT [80]. The tree was constructed using neighbor-joining as implemented in the clustalx package and visualized using iToL [81,82]. SkRspo is D1LXC5_SACKO from the hemichordate acorn worm Saccoglossus kowalevskii, BfRspo is C3Y1K8_BRAFL from the primitive chordate amphioxus Branchiostoma floridae, and SpRspo is XP_796266.2 from the echinodermate sea urchin Strongylocentrotus purpuratus. Vertebrate protein identifiers used for this tree are as follows: HsRspo4 ENSP00000217260, MmRspo4 ENSMUSP00000041578, DrRspo4 ENSDARP00000123862, DrRspo2 ENSDARP00000100941, HsRspo2 ENSP00000276659, MmRspo2 ENSMUSP00000067325, HsRspo1 ENSP00000348944, MmRspo1 ENSMUSP00000030687, DrRspo1 ENSDARP00000058458, HsRspo3 ENSP00000349131, MmRspo3 ENSMUSP00000090287 and DrRspo3 ENSDARP00000058577.

Figure 2

Protein domain architecture and chromosome location of human R-spondins. Schematic representations are shown for all four human R-spondin proteins. The total lengths of R-spondin1, 2, 3 and 4 are 263, 243, 292 and 234 amino acids, respectively. Three types of domains are detected: two cysteine-rich furin-like repeats, a single thrombospondin domain, and a basic amino-acid-rich domain. The relative protein sequence conservation, as a percentage of identical amino acids, within these domains is indicated. The two furin repeats jointly contain 15 conserved cysteines, conforming to the consensus sequence for this domain in each repeat. Twelve out of 60 amino acid residues are highly conserved in thrombospondin protein 1 (TSP1) domains, six of which are cysteines. Secretion is mediated by an amino-terminal endoplasmic reticulum signal peptide. Putative N-linked glycosylation sites are indicated (N).

Figure 3

Simplified overview of the canonical Wnt signaling pathway. The typical mammalian genome harbors 19 genes encoding Wnt secretory factors and 10 Frizzled (Fzd) genes encoding their receptors. Two low-density lipoprotein receptor-related proteins (Lrp) 5 or 6 act as Fzd co-receptors. Activating combinations of Fzd/Lrp/Wnt initiate signaling activity by silencing the activity of a dedicated β-catenin (βcat) destruction complex. Dvl gene products are instrumental in achieving this. (a) In the absence of Wnt signals, constitutively synthesized cytoplasmic βcat is the immediate target of this complex. Essential components of this complex are two tumor suppressor proteins: Apc (adenomatous polyposis coli) and axin, which act as scaffolds to capture newly synthesized βcat and allow its phosphorylation by the constitutively active kinases casein kinase-1 (Ck1) and glycogen synthase kinase 3 (GSK3), also residing in this complex. (b) The Wnt-binding-induced cytoplasmic accumulation of βcat leads to import into the nucleus and binding to T-cell transcription factor (Tcf)/Lef transcription factors, upon replacement of the transcriptional Groucho repressors. Bipartite Tcf/Lef-βcat complexes are the ultimate effectors of this signaling cascade. A series of secreted antagonists control signaling activity at the level of ligand perception. Secreted Frizzled-related proteins (Sfrp1, 2, 4 and 5), Frzb and Wnt inhibitory factor (Wif) can bind Wnt directly and prevent it from activating their receptors [83-86]. The other Wnt antagonists, Dickkopf 1 (Dkk1) [87] and Wise [88], inhibit by binding to the Lrp co-receptor. R-spondins, also operating at this level, are unique in enhancing Wnt activity. The seven transmembrane Lgr (4, 5 and 6) receptors mediating their action were recently uncovered [48,89,90].

Figure 4

Overview of sex determination in mice. During mouse embryogenesis, bipotential gonads arise from the genital ridges by 10.5 days post-conception (dpc). In somatic cells of XY genital ridges, Sry expression (dark blue line at lower part of figure) starts at 10.5 dpc, reaches a peak at 11.5 dpc and then wanes by 12.5 dpc. A few hours later, Sox9 expression (light blue line at the lower part of the figure) is upregulated to induce differentiation of Sertoli cells. Sox9 expression peaks at 11.5 to 12.5 dpc, continues to be expressed postnatally and is supported by several positive-feedback loops (including fibroblast growth factor 9 (FGF9), prostaglandin D2 (PGD2) and SOX9 itself), and SOX9 subsequently activates many male-specific genes, including the gene encoding anti-Müllerian hormone (Amh). At 12.5 dpc, morphological differences between testis and ovary are evident. In the absence of SRY, genes such as Wnt4, Rspo1 and Foxl2 are expressed in a female-specific manner and induce ovarian development, as characterized by the expression of follistatin and many other ovary-specific genes. FOXL2, forkhead box L2; SOX9, SRY box containing gene 9; SRY, sex-determining region on the chromosome Y. This figure is adapted with permission from [23].