Figure 6.
PDZ–PDZ interactions did not preclude PDZ–target interactions. (A) Models for the PDZ–PDZ and PDZ–target interaction: PDZ–PDZ interaction precludes binding of targets, PDZ–target binding prevents PDZ-PDZ interaction, PDZ–PDZ and PDZ–target binding can occur simultaneously. (B) TRPL, PDZ3, and PDZ3L formed a ternary complex. COOH-terminal TRPL, MYC-tagged PDZ3 (PDZ3-M) and FLAG-tagged PDZ3 long-form (PDZ3L-F) were expressed in duplicate or triplicate in 293T cells as indicated. Cell lysates were immunoprecipitated with anti-TRPL or anti-MYC antibodies and the Western blots were probed with either anti-MYC (left two panels) or anti-FLAG antibodies (right two panels). (C) PKC, PDZ3, and PDZ3L formed a ternary complex. FLAG-tagged PKC (PKC-F), MYC-tagged PDZ3 (PDZ3-M), MYC-tagged PDZ3 long-form (PDZ3L-M) and FLAG-tagged PDZ3 long-form (PDZ3L-F) were doubly- or triply-expressed in 293T cells as indicated. Cell lysates were immunoprecipitated with anti-MYC antibodies or nonimmune serum and the Western blots were probed with anti-FLAG antibodies. The third panel in B and the fourth panel in C are duplicates. (D) Homomultimerization and target binding were mediated through overlapping but different regions in PDZ3. Plasmids encoding various portions of PDZ3 and linker regions (MYC-tagged) were coexpressed with pPKC-F or pINAD in 293T cells. Coimmunoprecipitations were carried out using anti-MYC antibodies. +, PDZ3 fragments that interacted with PKC or INAD in the assay.