Figure 4.

The CNKSR1 PDZ domain binds IGSF4C, whose PDZ motif mediates cell adhesion. (A) Dendogram of the five-member nectin-like immunoglobulin family based on CLUSTALW protein sequence alignment showing conserved Class II PDZ-binding motifs and annotation of those family members documented to interact with PDZ domain-encoding proteins. (B) CNKSR1 interacts with the PDZ domain of IGSF4C. (Upper panel) Schematic representations of Myc-tagged CNKSR1 constructs used to verify and map interaction between CNKSR1 and IGSF4C; (middle panel) along with GFP-tagged IGSF4C, HEK293T cells were transfected with constructs encoding Myc-tagged wild-type CNKSR1 (Myc-WT) or Myc-tagged CNKSR1 constructs encoding various CNKSR1 domains. Cell lysates were immunoprecipitated with anti-Myc antibody and then blotted with anti-GFP to detect interactions. In the immunoprecipitates, heavy chains are indicated (HC); (bottom panel) total lysates were immunoblotted with anti-Myc antibody to confirm equal expression of constructs. (C) BRET assays confirm the interaction of CNKSR1 with IGSF4C in intact cells. GFP-CNKSR1 was transfected into HEK293T cells with one of three constructs: luciferase alone, luciferase fused to the IGSF4C cytoplasmic domain (IGSF4C-Luc), or luciferase fused to the IGSF4C extracellular domain (Luc-IGSF4C). Forty-eight hours after transfection, the cells were detached and subjected to BRET analysis. Shown are the results of three independent experiments with average bioluminescence resonance energy transfer and standard error. (Asterisks) Significant increase (t-test; P < 0.01) in BRET ratio upon expression of cytoplasmic domain-tagged IGSF4C compared with luciferase alone. (D) Cell aggregation activity of IGSF4C. HEK293 cells transfected with GFP (black diamonds), IGSF4C-GFP (black squares), or IGSF4C (AAA)-GFP (lacking the PDZ motif, white triangles) were treated with trypsin in the presence of EDTA and then dispersed by pipetting to obtain a single-cell suspension. Each single-cell suspension was rotated in Ca²⁺- and Mg²⁺-free HBSS containing 5 mM EDTA for 30 and 60 min. The degree of aggregation of cells was represented by the ratio of the total particle number at time t of incubation (Nt) to the initial particle number (No). Similar results were obtained when cell suspensions were rotated in HBSS containing Ca²⁺ and Mg²⁺ or Ca²⁺- and Mg²⁺-free HBSS.