Reconstitution of R-spondin:LGR4:ZNRF3 adult stem cell growth factor signaling complexes with recombinant proteins produced in Escherichia coli.
R-Spondins are secreted glycoproteins (RSPO1-RSPO4) that have proliferative effects on adult stem cells by potentiating Wnt signaling. RSPO actions are mediated by the leucine-rich repeat (LRR)-containing seven-transmembrane receptors LGR4-LGR6 and the transmembrane E3 ubiquitin ligases ZNRF3 and RNF43. Here, we present a methodology for the bacterial expression and purification of the signaling competent, cysteine-rich Fu1-Fu2 domains of the four human RSPOs, a fragment of the human LGR4 extracellular domain (ECD) containing LRR1-14, and the human ZNRF3 ECD. In a cell-based signaling assay, the nonglycosylated RSPOs enhanced low-dose Wnt3a signaling with potencies comparable to those of mammalian cell-produced RSPOs and RSPO2 and -3 were more potent than RSPO1 and -4. LGR4 LRR1-14 and ZNRF3 ECD inhibited RSPO2-enhanced Wnt3a signaling. The RSPOs bound LGR4 LRR1-14 with nanomolar affinities that decreased in the following order in a time-resolved fluorescence resonance energy transfer (TR-FRET) assay: RSPO4 > RSPO2 > RSPO3 > RSPO1. RSPO-receptor interactions were further characterized with a native gel electrophoretic mobility shift assay, which corroborated the RSPO-LGR4 TR-FRET results and indicated that RSPOs weakly bound ZNRF3 with affinities that decreased in the following order: RSPO2 > RSPO3 > RSPO1. RSPO4:ZNRF3 complexes were not detected. Lastly, ternary RSPO:LGR4:ZNRF3 complexes were detected for RSPO2 and -3. Our results indicate that RSPO and LGR4 N-glycans are dispensable for function, demonstrate RSPO-mediated ternary complex formation, and suggest that the stronger signaling potencies of RSPO2 and -3 result from their strong binding of both receptors. Our unique protein production methodology may provide a cost-effective source of recombinant RSPOs for regenerative medicine applications.
Department of Biochemistry and Molecular Biology, The University of Oklahoma Health Sciences Center , Oklahoma City, Oklahoma 73104, United States.
Heather E
HE
Moad
Augen A
AA
Pioszak
D053687:Adult Stem Cells / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D043562:Receptors, G-Protein-Coupled / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D044767:Ubiquitin-Protein Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D060508:Wnt3 Protein / Q000378:metabolism
D036341:Intercellular Signaling Peptides and Proteins; C411562:LGR4 protein, human; D043562:Receptors, G-Protein-Coupled; D011994:Recombinant Proteins; D060508:Wnt3 Protein; D044767:Ubiquitin-Protein Ligases; C572950:ZNRF3 protein, human