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. Author manuscript; available in PMC: 2010 Sep 6.
Published in final edited form as: Cell. 2008 Oct 9;135(2):334–342. doi: 10.1016/j.cell.2008.10.002

Figure 3. Lrp4 binds selectively and with high affinity to neural isoforms of Agrin.

Figure 3

(A) The graph shows the gates for distinguishing cells expressing: Lrp4-mCherry, MuSK-GFP, Lrp4-mCherry plus MuSK-GFP, as well as parental BaF3 cells, as measured by flow cytometry. (B) The histograms depict binding of Cy5-Agrin B8 to the gated populations of cells shown in (A) at 100 nM Agrin. Cy5-Agrin B8 bound preferentially to cells expressing Lrp4-mCherry or Lrp4-mCherry plus MuSK-GFP and weakly to cells expressing MuSK-GFP alone. (C) Lrp4-expressing cells bound Agrin B8 with ~100-fold higher affinity (~6 nM) than Agrin B0. Binding between Agrin B8 and Lrp4 is strengthened ~20-fold in the presence of Ca++. The inset shows binding at the lower concentrations of Agrin. The 95 kd and 50 kd forms of Agrin B8 bound similarly to cells expressing Lrp4 or Lrp4 and MuSK (data not presented). Agrin binding to parental cells was minimal (B) and was subtracted from all values. (D) Co-expression of MuSK-GFP potentiates binding of Cy5-Agrin B8 to cells expressing Lrp4-mCherry; addition of the soluble ectodomain from MuSK (2 µM) also enhances binding of Cy5-Agrin B8 to Lrp4-mCherry expressing cells.