# Unconventional signaling by the R-spondin family of WNT regulators

> **NIH NIH R21** · STANFORD UNIVERSITY · 2020 · $236,550

## Abstract

Project Summary
Unconventional signaling by the R-spondin family of WNT regulators
The four R-spondins (RSPOs1-4) are vertebrate-specific secreted proteins that dramatically amplify signaling
through the WNT/β-catenin pathway, a cell-cell communication system that regulates tissue patterning during
development and regenerative responses in adults. Mutations that damage the RSPO signaling circuit cause
structural birth defects, exemplified by limb truncation, lung hypoplasia and craniofacial malformations seen in
patients with mutations in RSPO2. In adults, RSPOs function as niche-derived signals required for the renewal
of epithelial stem cells in multiple tissues, including the intestine, skin, and bone. RSPOs amplify WNT signals
by simultaneously binding to two receptors: Leucine-rich repeat-containing G-protein coupled receptors (LGRs)
and the ZNRF3/RNF43 transmembrane E3 ubiquitin ligases. RSPOs increase WNT receptor levels only in
specific cell types, such as intestinal stem cells, that express LGR receptors and thus allow the strength of the
WNT signal (a potentially oncogenic signal) to be tightly controlled in time and space. While LGRs had been
considered obligate high-affinity receptors for RSPOs, we made the unexpected discovery (along with other
groups) that RSPOs can amplify WNT signals in the absence of all LGRs (“LGR-independent signaling”) or in
the absence of ZNRF3/RNF43 (“ZNRF3/RNF43-independent signaling”). Distinct from the intensively studied
LGR-mediated signaling in intestinal stem cells, LGR-independent signaling is particularly relevant for RSPO-
related birth defects: it is the dominant mode of signaling during development of the limbs, lungs and
cardiovascular system. Our preliminary results show that alternate, undiscovered receptors must exist that
mediate some cellular responses to RSPOs. To identify these receptors and associated signaling components,
we propose an innovative strategy that combines ligand engineering with genome-wide loss-of-function screens
in cultured cells. In Aim 1, we use chimeric and mutant RSPO ligands in cell and organoid culture systems to
test the hypothesis that Heparan Sulfate Proteoglycans (HSPGs) can function as RSPO co-receptors to
transduce LGR-independent signals. In Aim 2, we use engineered RSPO ligands constrained to signal through
either LGR-independent or ZNRF3/RNF43-independent modes to search for the required genes using CRISPR-
based screens and haploid genetic screens. Successful completion of this project will identify the signaling
machinery that mediates cellular responses to RSPOs in developmental and regenerative contexts, thereby
improving our understanding of WNT-related structural birth defects and providing new strategies to enhance
WNT-regulated regenerative responses in a tissue-selective manner.

## Key facts

- **NIH application ID:** 9958098
- **Project number:** 1R21HD101980-01
- **Recipient organization:** STANFORD UNIVERSITY
- **Principal Investigator:** RAJAT ROHATGI
- **Activity code:** R21 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2020
- **Award amount:** $236,550
- **Award type:** 1
- **Project period:** 2020-03-15 → 2022-02-28

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/9958098

## Citation

> US National Institutes of Health, RePORTER application 9958098, Unconventional signaling by the R-spondin family of WNT regulators (1R21HD101980-01). Retrieved via AI Analytics 2026-05-22 from https://api.ai-analytics.org/grant/nih/9958098. Licensed CC0.

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