Figure 1: Identification of an asprosin-interacting receptor in the mouse brain.
(a) Dual-label immunofluorenscence RNAscope hybridization showing relative overlap between AgRP (red) and Olfr734 (green) expressing cells in the arcuate (ARC) nucleus of wild-type (WT) mice. Scale bars, 100 μm. 3V, 3rd ventricle.
(b) qPCR results showing relative mRNA levels of Olfr734 (left) from visually enriched tdTOMATO expressing neurons of AgRP-cre mice and (right) cre-responsive ribotag enriched transcripts from AgRP neurons of AgRP-cre mice.
(c) Schematic overview of asprosin immunoprecitation (IP; left) of brain tissue incubated with recombinant asprosin or recombinant GFP before mass spectrometry (MS) analysis, (right) overlap of candidate asprosin-interacting proteins from 3 repeats of IP/MS. 58 candidate proteins, including Ptprd appeared in all 3 repeats.
(d) Dual-label immunofluorenscence RNAscope hybridization showing relative overlap between AgRP (red) and Ptprd (green) expressing cells in the ARC nucleus of WT mice. Scale bars, 100 μm.
(e) qPCR results showing relative mRNA levels of Ptprd (left) from visually enriched tdTOMATO expressing neurons of AgRP-cre mice and (right) cre-responsive ribotag enriched transcripts from AgRP neurons of AgRP-cre mice.
(f) Reciprocal in vitro immunoprecipitation of recombinant GST-asprosin and extracellular domain of his-tagged PTPRD (PTPRD-6his), with mouse IgG and rabbit IgG as negative controls.
(g-i) Quantification of the binding affinity between Asprosin and PTPRD by Microscale thermophoresis (k; MST; Kd of 4.2 ±10.2 nM), Bio-layer interferometry (l; BLI; Kd of 57.0 ±1.83 nM), and by Surface plasmon resonance analysis (m; SPR; Kd of 36.8 ±5.7 nM). Data are represented as mean ± SEM.