CDB15:0000146 BTC — EGFR

ABSTRACT: Soluble forms of human betacellulin (BTC) were purified to homogeneity from the conditioned medium of mouse A9 cells transfected with the BTC precursor cDNA. Three types of soluble BTC, designated BTC-1a, BTC-1b and BTC-2, were resolved by cation-exchange and size-exclusion column chromatography. Physicochemical analysis has revealed that BTC-1a represents the glycosylated, intact molecule composed of 80 amino acid residues (Asp32 to Tyr111 of the precursor molecule). BTC-1b appears to be a truncated molecule lacking 12 amino acid residues from the amino terminus of BTC-1a. BTC-2 was found to be a 50-amino acid molecule (Arg62 to Tyr111) that corresponds to the epidermal growth factor (EGF) structural unit. The biological activities of these BTC molecules were essentially identical as judged by their mitogenicity on Balb/c 3T3 fibroblasts. BTC and EGF were equipotent in stimulating Balb/c 3T3 cell proliferation and rat mesangial cell Ca2+ mobilization as well as in inhibiting the growth of human epidermoid carcinoma A431 cells. BTC and EGF antagonized each other with similar dose dependence for binding to A431 cells, indicating that these factors bind the same receptor molecules with equivalent avidity. The Kd value of EGF receptor (EGFR) and BTC is 0.5 nM as determined on Balb/c 3T3 cells. In addition, human mammary carcinoma MDA-MB-453 cells, which express multiple members of the EGFR family, were found to possess 2.7 x 10(3) BTC binding sites/cell, and the binding was readily quenched by EGF. These results suggest that the primary receptor for BTC is EGFR.

ABSTRACT: Betacellulin (BTC) is a member of the EGF ligand family that directly binds to both EGFR and HER4 and induces the growth of certain epithelial cell types. Fusion proteins composed of the terminal 48 or 50 amino acids of mature betacellulin and a binding defective form of Pseudomonas exotoxin (BTC-TX48 and BTC-TX50, respectively), have been produced. BTC-TX50 induced tyrosine phosphorylation of both EGFR and HER4, whereas BTC-TX48 induced phosphorylation of HER4 but to a much lesser extent EGFR, indicating that the presence of two additional amino acid residues, Arg62 and Lys63, contribute to full kinase activity. BTC-TX50 was up to 300-fold more active at inhibiting protein synthesis than BTC-TX48 on cell lines expressing EGFR, most likely due to the >tenfold higher affinity of BTC-TX50. MDA-MB-453 breast carcinoma cells which express HER4 but not EGFR, were not sensitive to either BTC-TX form. These data indicate that despite the ability of BTC-TX to bind and phosphorylate HER4, it was only cytotoxic to cells expressing EGFR. The inability of BTC-TX to kill cells was likely due to its failure to internalize through HER4.