EXPRESSION OF EPIDERMAL GROWTH-FACTOR RECEPTOR IN FETAL MOUSE SUBMANDIBULAR-GLAND DETECTED BY A BIOTINYLTYRAMIDE-BASED CATALYZED SIGNAL AMPLIFICATION METHOD

Branching morphogenesis of the fetal mouse submandibular gland (SMG) c an be modulated in vitro by stimulation or inhibition of the epidermal growth factor receptor (EGFR). Because the mRNAs for EGF and EGFR are detectable in RNA of SMG rudiments isolated directly from fetuses, th e EGF system probably operates physiologically as a regulator of SMG m orphogenesis. However, neither EGFR protein nor its precise cellular l ocalization has been characterized in the fetal SMG. Here we show EGFR protein in fetal mouse SMC by immunoprecipitation, affinity labeling, ligand-induced autophosphorylation, and immunohistochemistry. SMGs fr om E16 fetuses (day of vaginal plug = E0) were labeled with [S-35]-cys teine/methionine and homogenized. After addition of specific antibody to EGFR, the immunoprecipitate was isolated, resolved by polyacrylamid e gel electrophoresis, and detected by autoradiography. A single band of 170 kD was detected, corresponding to the EGFR protein. Affinity la beling with [I-125]-EGF of the membrane fraction of E18 SMG also revea led a prominent band at 170 kD, showing that this EGFR protein can bin d specifically to its ligand. Incubation of SMG membranes from E18 fet uses with EGF in the presence of [gamma-P-32]-ATP, followed by immunop recipitation with anti-phosphotyrosine antibody also showed a single b and at 170 kD, demonstrating autophosphorylation of the EGFR in respon se to binding of its ligand. Immunohistochemical localization of the c ellular sites of EGFR in the fetal SMG required use of a catalyzed sig nal amplification procedure, with biotinyltyramide as the amplifying a gent. EGFR was localized predominantly, if not exclusively, in cell me mbranes of epithelial cells of the rudiment, whereas staining of mesen chymal cells was equivocal. Staining was strongest on duct cells, acid weak on cells of the end-pieces. These findings clearly show that a f unctional EGFR protein is expressed in fetal SMG chiefly, if not exclu sively, on epithelial cells.