A betacellulin mutant promotes differentiation of pancreatic acinar AR42J cells into insulin-producing cells with low affinity of binding to ErbB1.
Betacellulin (BTC) is one of the members of the epidermal growth factor (EGF) ligand family of ErbB receptor tyrosine kinases. It is a differentiation factor as well as a potent mitogen. BTC promotes the differentiation of pancreatic acinar-derived AR42J cells into insulin-producing cells. It independently and preferentially binds to two type I tyrosine kinase receptors, the EGF receptor (ErbB1) and ErbB4. However, the physiochemical characteristics of BTC that are responsible for its preferential binding to these two receptors have not been fully defined. In this study, to investigate the essential amino acid residues of BTC for binding to the two receptors, we introduced point mutations into the EGF domain of BTC employing error-prone PCR. The receptor binding abilities of 190 mutants expressed in Escherichia coli were assessed by enzyme immunoassay. Replacement of the glutamic acid residue at position 88 with a lysine residue in BTC was found to produce a significant loss of affinity for binding to ErbB1, while the affinity of binding to ErbB4 was unchanged. In addition, the mutant of BTC-E/88/K showed less growth-promoting activity on BALB/c 3T3 cells compared with that of the wild-type BTC protein. Interestingly, the BTC mutant protein promoted differentiation of pancreatic acinar AR42J cells at a high frequency into insulin-producing cells compared with AR42J cells that were treated with wild-type BTC protein. These results indicate the possibility of designing BTC mutants, which have an activity of inducing differentiation only, without facilitating growth promotion.
Nagaoka
Tadahiro
T
Department of Medical Bioengineering, Graduate School of Natural Science and Technology, Okayama University, 3-1-1 Tsushima-Naka, Okayama 700-8530, Japan.
Fukuda
Takayuki
T
Hashizume
Toshihiro
T
Nishiyama
Tomoko
T
Tada
Hiroko
H
Yamada
Hidenori
H
Salomon
David S
DS
Yamada
Satoko
S
Kojima
Itaru
I
Seno
Masaharu
M
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D066259:Betacellulin; D002454:Cell Differentiation / Q000187:drug effects; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D066246:ErbB Receptors / Q000378:metabolism*; D006801:Humans; D007142:Immunoglobulin gamma-Chains / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology*; D036341:Intercellular Signaling Peptides and Proteins / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D008934:Mitogens / Q000378:metabolism; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000378:metabolism*; D009154:Mutation / Q000235:genetics; D046790:Pancreas, Exocrine / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D011485:Protein Binding / Q000187:drug effects; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D019163:Reducing Agents / Q000494:pharmacology; D012995:Solubility / Q000187:drug effects
C587851:BTC protein, human; D066259:Betacellulin; C587852:Btc protein, mouse; D007142:Immunoglobulin gamma-Chains; D036341:Intercellular Signaling Peptides and Proteins; D008934:Mitogens; D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D019163:Reducing Agents; D066246:ErbB Receptors