Figure 6.

Interaction of PTPσ with Nrxn1 is regulated by alternative splicing in the Ig domain of PTPσ and PTPσ complexes with Nrxn1α in vivo. A, Representative images of cell-surface binding assays. HEK293T cells expressing HA-tagged Nlgn1 or the indicated PTPσ splice variants were incubated with 10 μg/ml of control IgC, or the indicated Ig-Nrxn1 fusion proteins, and then analyzed by double-immunofluorescence imaging of Ig-fusion proteins (red) and HA antibodies (green). Scale bar, 10 μm. B, Quantification of the average red intensities of transfected HEK293T cells in A. n indicates the number of cells as follows: Ig-Nrxn1α^-SS4/Nlgn1, n = 37; Ig-Nrxn1α^-SS4/PTPσ^A+B+, n = 29; Ig-Nrxn1α^-SS4/PTPσ^A+B–, n = 26; Ig-Nrxn1α^-SS4/PTPσ^A–B+, n = 27; Ig-Nrxn1α^-SS4/PTPσ^A–B–, n = 35; Ig-Nrxn1α^+SS4/Nlgn1, n = 31; Ig-Nrxn1α^+SS4/PTPσ^A+B+, n = 27; Ig-Nrxn1α^+SS4/PTPσ^A+B–, n = 25; Ig-Nrxn1α^+SS4/PTPσ^A–B+, n = 27; Ig-Nrxn1α^+SS4/PTPσ^A–B–, n = 24; Ig-Nrxn1β^-SS4/Nlgn1, n = 31; Ig-Nrxn1β^-SS4/PTPσ^A+B+, n = 26; Ig-Nrxn1β-SS4/PTPσ^A+B–, n = 27; Ig-Nrxn1β-SS4/PTPσ^A–B+, n = 28; Ig-Nrxn1β^-SS4/PTPσ^A+B–, n = 33; Ig-Nrxn1β^+SS4/Nlgn1, n = 22; Ig-Nrxn1β^+SS4/PTPσ^A+B+, n = 23; Ig-Nrxn1β^+SS4/PTPσ^A+B–, n = 26; Ig-Nrxn1β^+SS4/PTPσ^A–B+, n = 28; Ig-Nrxn1β^+SS4/PTPσ^A–B–, n = 25; IgC/Nlgn1, n = 16; IgC/PTPσ^A+B+, n = 15; IgC/PTPσ^A+B–, n = 17; IgC/PTPσ^A–B+, n = 19; and IgC/PTPσ^A–B–, n = 16. C, In vivo pulldown assays. Recombinant Ig-Nrxn1α^-SS4 or Ig-Nrxn1β^-SS4 proteins were immobilized using protein A-Sepharose and incubated with rat synaptosomal lysates. Immunoblotting was performed using antibodies against PTPσ, PTPδ, TrkC, and Nlgn2. D, Purified recombinant HA-PTPσ Ig1-3 proteins were incubated with purified Ig-Nrxn1α^-SS4 proteins, as indicated. Precipitates obtained using protein A-Sepharose were analyzed by immunoblotting with HA antibodies. *Positions of Fc fusion proteins used for binding assays, as revealed in parallel Ponceau S-stained membranes (right). Input, 2%. E, Coimmunoprecipitation experiment in mouse brains demonstrating that PTPσ forms complexes with Nrxn1α. Crude synaptosomal fractions of adult mouse brains were immunoprecipitated with anti-Nrxn1α antibody and immunoblotted with anti-PTPσ. An equal amount of rabbit IgG was used as a negative control. Input, 2%. F, G, Saturation binding of Ig-Nrxn1α^-SS4 (F) or Ig-Nrxn1β^-SS4 (G) to PTPσ expressed in HEK293T cells. Inset, Scatchard plot generated by linear regression of the data, with the K_d calculated from three or four independent experiments. Data are mean ± SEM.