Palmitoylation of β4 is required for incorporation of α6β4 in rafts. (A) Constructs encoding wild-type and mutant forms of β4. The β4 tail contains two pairs of type III Fn-like modules (black boxes) interrupted by a connecting segment (gray dotted box). The predicted end of transmembrane domain (TM) and membrane-proximal segment of the tail of β4 and various alanine mutants is shown above. (B) 804G cells expressing wild-type (A) or tail-less (L) β4 and HaCat cells were metabolically labeled with [³⁵S]Met/Cys or [¹²⁵I]IC16 and immunoprecipitated with mAb 3E1. The indicated lane was soaked overnight in 1 M hydroxylamine before fluorography. (C) 804G cells and their derivatives expressing wild-type β4 (A) or the indicated mutant versions of β4 (B, C, E, and L) were metabolically labeled with [³⁵S]Met/Cys or [³H]palmitate and immunoprecipitated with mAb 3E1. (D) 293-T cells expressing wild-type β4 (A) or the indicated mutant forms of β4 (Cys 3, Cys 5, and Cys 6,7) were metabolically labeled with [³⁵S]Met/Cys or [³H]palmitate and immunoprecipitated with mAb 3E1. The ratio of ³H to ³⁵S radioactivity incorporated by mutant forms of β4 was measured by PhosphorImager analysis. (E) 804G cells expressing human wild-type β4 (WT) or palmitoylation-defective β4 (Cys 5) were lysed with Triton X-100 and subjected to sucrose gradient ultracentrifugation. Proteins from each fraction were probed with anti–human β4 N20 or with anti-caveolin N20.

Compartmentalization of α6β4 signaling in lipid rafts. (A) After antibody-mediated ligation of α6β4, HaCat cells were lysed with Triton X-100 and subjected to OptiPrep™ gradient ultracentrifugation. The lipid raft (R) and Triton X-100–soluble (S) fractions were either immunoprecipitated with mAb 3E1 and probed with anti-β4 or anti-pan-Src, or directly probed with antibodies reacting with Yes or Src. The lipid raft and Triton X-100–soluble fractions were also immunoprecipitated with mAb 3E1 and subjected to kinase assay or immunoblotting with anti-phospho Src Y418. (B) PA-JEB keratinocytes expressing wild-type β4 or β4 Cys 5 were plated on dishes coated with mAb 3E1 for the indicated times. Total lysates were probed with anti-phospho Src Y418 or anti-β4 N20. (C) HUVECs transiently transfected with vectors encoding α6β4 were left untreated (C) or treated with the SFK inhibitor PP2 or the EGF-R inhibitor AG1478. Cells were plated on dishes coated with mAb 3E1 for the indicated times or stimulated with EGF. Total proteins were probed with anti-phospho-ERK and anti-ERK-2. (D) HUVECs were transiently transfected with α6 in combination with wild-type β4, phosphorylation-defective β4 (4YF), β4 Cys 5, or tail-less β4 (L). Cells were plated for the indicated times on dishes coated with mAb 3E1. Blotting was as in B. (E) PA-JEB keratinocytes stably transduced with empty vector (LZRS), wild-type β4, and β4 Cys 5 were deprived of mitogens, detached, and plated on coverslips coated with purified laminin-5 or incubated in suspension with mAb 3E1, and were then plated on coverslips coated with anti–mouse IgGs. After 24 h of incubation with EGF, the cells were subjected to anti-BrdU staining. The data represent the average and SDs of values obtained from three experiments and are expressed as percentage of rescue.

Incorporation of α6β4 in lipid rafts. (A) After lysis in Triton X-100, HaCat cells were fractionated by sucrose gradient ultracentrifugation. Equal aliquots were blotted with antibodies to β4, transferrin receptor (Trf-R), tubulin, Yes, and caveolin-1 (Cav-1). The percentage of β4 recovered from each fraction was estimated by densitometry. (B) HaCat cells were left untreated or treated with 0.2% saponin or 10 mM MβCD before lysis and ultracentrifugation. Fractions were probed with anti-β4. (C) HaCat cells were incubated in suspension with mAb W6.32 (anti-MHC) or 3E1 (anti-β4) followed by anti–mouse IgGs, or were treated with anti–mouse IgGs only. Cells were lysed in Triton X-100 and subjected to OptiPrep™ gradient ultracentrifugation. Blotting was with anti-β4.

Palmitoylation of β4 is not necessary for α6β4-mediated adhesion and assembly of hemidesmosomes. (A) 293-T cells were left untransfected (NT) or transfected with α6 in combination with wild-type β4 or β4 Cys 5, and were then plated on laminin-5 matrix at 4°C. Three experiments yielded essentially identical results. (B) Immortalized β4-deficient PA-JEB keratinocytes were left untransfected (NT) or transiently transfected with α6 in combination with wild-type β4 or β4 Cys 5. Double-immunofluorescent staining with anti-β4 and anti–HD-1/plectin indicates that wild-type β4 and β4 Cys 5 are both recruited to the basal cell surface and colocalize with plectin in hemidesmosome-like adhesions. In untransfected cells, plectin codistributes instead with the actin cytoskeleton. (C) PA-JEB keratinocytes transduced with empty vector (LZRS), wild-type β4, or β4 Cys 5 were plated in the presence of anti-α3 mAb PIB5 on laminin-5 matrix for 30 min at 37°C. Results are expressed as percentages of the adhesion of control transfectants in the absence of the inhibitory mAb. (D) PA-JEB keratinocytes stably transduced with empty vector (LZRS), wild-type β4, or β4 Cys 5 were plated on laminin-5 matrix for 48 h and then treated with trypsin/EDTA. The number of cells that had detached by the indicated times was determined by direct counting. Four experiments yielded essentially the same result.