Fig. 2. Wildtype and mutant Myc-CRB3 constructs expressed in MDCK cells. (A) Sequence alignment of the intracellular portion of Crumbs homologues from indicated organisms. There are two conserved motifs: the juxtamembrane region predicted to bind a protein of the FERM superfamily and the extreme C-terminal PDZ-binding motif. Conserved residues in these two regions are shown in bold. The three most conserved residues in the FERM-binding region were all mutated to alanines (also shown in bold) in the Myc-CRB3 FERMmut construct. The Myc-CRB3ERLI protein lacks the PDZ-binding motif. Not shown is the Myc-CRB3ND sequence where the intracellular portion of CRB3 is unchanged; instead, the extracellular N-glycosylation site is mutated as described previously (Makarova et al., 2002). (B) MDCK stable cell lines expressing wild-type Myc-CRB3 and the indicated Myc-CRB3 mutant proteins were grown for 24 hours in normal calcium media and then co-immunostained with anti-Myc and anti-ß-catenin antibodies. The ß-catenin is used as a marker of the lateral membrane. (C) Lysates were prepared from the stable cell lines used in (B). Anti-Myc immunoprecipitates were resolved by SDS-PAGE. The various Myc-CRB3 proteins and co-precipitated endogenous Pals1 were visualized by blotting with anti-CRB3 and anti-Pals1 antibodies, respectively. (D) Increasing amounts of GST-CRB3 and GSTCRB3ERLI fusion proteins were resolved by SDS-PAGE and immunoblotted as indicated.