RECOMBINANT HUMAN BETACELLULIN - MOLECULAR-STRUCTURE, BIOLOGICAL-ACTIVITIES, AND RECEPTOR INTERACTION

Soluble forms of human betacellulin (BTC) were purified to homogeneity from the conditioned medium of mouse A9 cells transfected with the BT C precursor cDNA. Three types of soluble BTC, designated BTC-1a, BTC-1 b and BTC-2, were resolved by cation-exchange and size-exclusion colum n chromatography. Physico-chemical analysis has revealed that BTC-1a r epresents the glycosylated, intact molecule composed of 80 amino acid residues (Asp32 to Tyr111 of the precursor molecule). BTC-1b appears t o be a truncated molecule lacking 12 amino acid residues from the amin o 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 wer e essentially identical as judged by their mitogenicity on Balb/c 3T3 fibroblasts. BTC and EGF were equipotent in stimulating Balb/c 3T3 cel l proliferation and rat mesangial cell Ca2+ mobilization as well as in inhibiting the growth of human epidermoid carcinoma A431 cells. BTC a nd EGF antagonized each other with similar dose dependence for binding to A431 cells, indicating that these factors bind the same receptor m olecules with equivalent avidity. The K(d) value of EGF receptor (EGFR ) and BTC is 0.5 nM as determined on Balb/c 3T3 cells. In addition, hu man 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 sit es/cell, and the binding was readily quenched by EGF. These results su ggest that the primary receptor for BTC is EGFR.