Engineering High-Potency R-spondin Adult Stem Cell Growth Factors

Abstract

Secreted R-spondin proteins (RSPOs1-4) function as adult stem cell growth factors by potentiating Wnt signaling. Simultaneous binding of distinct regions of the RSPO Fu1-Fu2 domain module to the extracellular domains (ECDs) of the LGR4 G protein-coupled receptor and the ZNRF3 transmembrane E3 ubiquitin ligase regulates Wnt receptor availability. Here, we examine the molecular basis for the differing signaling strengths of RSPOs1-4 using purified RSPO Fu1-Fu2, LGR4 ECD, and ZNRF3 ECD proteins in Wnt signaling and receptor binding assays, and we engineer novel high-potency RSPOs. RSPO2/3/4 had similar signaling potencies that were stronger than that of RSPO1, whereas RSPO1/2/3 had similar efficacies that were greater than that of RSPO4. The RSPOs bound LGR4 with affinity rank order RSPO4 . RSPO2/3 . RSPO1 and ZNRF3 with affinity rank order RSPO2/3 . . RSPO1 .RSPO4. An RSPO2-4 chimera combining RSPO2 ZNRF3 binding with RSPO4 LGR4 binding was a "Superspondin" that exhibited enhanced ternary complex formation and 10-fold stronger signaling potency than RSPO2 and efficacy equivalent to RSPO2. An RSPO4-1 chimera combining RSPO4 ZNRF3 binding with RSPO1LGR4 binding was a "Poorspondin" that exhibited signaling potency similar to RSPO1 and efficacy equivalent to RSPO4. Conferring increased ZNRF3 binding upon RSPO4 with amino acid substitutions L56F, I58L, and I63M enhanced its signaling potency and efficacy. Our results reveal the molecular basis for RSPOs1-4 activity differences and suggest that signaling potency is determined by ternary complex formation ability, whereas efficacy depends on ZNRF3 recruitment. High-potency RSPOs may be of value for regenerative medicine and/or therapeutic applications.

Calcitonin and Amylin Receptor Peptide Interaction Mechanisms

“…For this reason also the IC 50 values from competition assays were not converted to K I values, although we have previously observed that under conditions similar to those used here IC 50 values from the competition assays provide reasonable estimates of the true affinities (29). All competitor peptides were checked for nonspecific inhibition by testing their ability to inhibit the Alpha-LISA signal obtained with an MBP-RSPO2 fusion protein and biotinylated ZNRF3 ECD, which are unrelated to CTR and RAMP1/2⅐CTR complexes (38). None of the peptides exhibited significant inhibition in the control assay at the concentrations used here (data not shown).…”

Crystal structure of R-spondin 2 in complex with the ectodomains of its receptors LGR5 and ZNRF3

“…We provide here detailed insights into the recognition of Rspo2 by LGR5. These insights can inform the development of potency-enhanced artificial Rspo molecules ( Moad and Pioszak, 2013; Warner et al, 2015 ) for example, to enhance tissue regeneration. Conversely, our mapping of the interaction determinants can also facilitate the design of Rspo antagonistic binding molecules, with potential applications in fighting Wnt driven cancers.…”

“…A plethora of crystal structures of Rspo alone and in complex with LGR4 or -5, ZNRF3 or RNF43 were published in 2013 ( Wang et al, 2013; Chen et al, 2013; Zebisch et al, 2013; Peng et al, 2013a; Peng et al, 2013b ), defining key elements of the LGR–Rspo–E3 complex only two years after the identification of this elaborate mechanism for potentiating Wnt signalling. All LGR complex structures published to date ( Xu et al, 2013; Wang et al, 2013; Chen et al, 2013; Peng et al, 2013a ) contain Rspo1 as binding partner rather than one of the more potent Rspo2 or -3 ligands ( Kim et al, 2008; Zebisch et al, 2013; Warner et al, 2015 ). One paper described the crystal structure of a ternary LGR5–RSPO1–RNF43 complex that displayed a clear 1:1:1 stoichiometry ( Chen et al, 2013 ).…”

PDGFRα + pericryptal stromal cells are the critical source of Wnts and RSPO3 for murine intestinal stem cells in vivo

“…The function of RSPOs was first uncovered in a screen for Wnt pathway activators in Xenopus , where RSPO2 markedly increased sensitivity to Wnt ligands ( 8 ). Subsequent studies established that all RSPOs can sensitize cells to Wnts, albeit with varying potency ( 9 – 11 ). While all RSPOs are secreted, the more Wnt-active RSPO2 and RSPO3 are tightly cell-surface associated via binding to syndecans ( 12 ).…”