The design, expression, and characterization of human insulin-like growth factor II (IGF-II) mutants specific for either the IGF-II/cation-independent mannose 6-phosphate receptor or IGF-I receptor.
Five mutants of recombinant insulin-like growth factor-II (rIGF-II) that bound with high affinity to either the IGF-II/cation-independent mannose 6-phosphate (IGF-II/CIM6-P) or the IGF-I receptor were prepared by site-directed mutagenic procedures, expressed as fusion proteins in the larva of Bombyx mori or Escherichia coli, purified to homogeneity, renatured, and characterized in terms of their receptor binding affinities and specificities as well as their biological activities. Class I mutants in which Phe26, Tyr27, and Val43 were substituted with Ser, Leu, and Leu, respectively, bound to enriched preparations of rat placental IGF-II/CIM6-P receptors with apparent equilibrium dissociation constants (Kd(app)) that were only slightly greater, i.e. 0.10, 0.05, and 0.06 nM, than that of rIGF-II (0.04 nM) or hIGF-II (0.03 nM). In contrast, replacing Phe26 with Ser resulted in 5- and 20-fold decreases in the affinities of this mutant for highly purified human placental IGF-I and insulin receptors, respectively. The affinities of the two other Class I mutants, [Leu27]- and [Leu43]rIGF-IIs, for these two receptors were reduced 80- to 220-fold. The affinities of Class II mutants, i.e. [Thr48,Ser49,Ile50]- and [Arg54,Arg55] rIGF-IIs, for IGF-I receptors were as potent as rIGF-II; however, they bound very poorly or not at all to the IGF-II/CIM6-P receptor. In the binding study of those mutant rIGF-IIs, IGF-II was observed to have an unexpectedly high affinity for pure human placental insulin receptor preparations. For example, the affinities of hIGF-II, rIGF-II, and two Class II rIGF-II mutants for the insulin receptor were only 3-, 9-, and 5-fold less, respectively, than that of porcine insulin. In two biological assay systems, i.e. the stimulation of DNA synthesis in Balb/c 3T3 cells and glycogen synthesis in HepG2 cells, the Kd(app) of the rIGF-II mutants for the IGF-I receptor but not the IGF-II/CIM6-P receptor correlated with their abilities to produce biological responses.
Sakano
K
K
Molecular Biology Research Laboratory, Daiichi Pharmaceutical Co., Ltd, Tokyo, Japan.
Enjoh
T
T
Numata
F
F
Fujiwara
H
H
Marumoto
Y
Y
Higashihashi
N
N
Sato
Y
Y
Perdue
J F
JF
Fujita-Yamaguchi
Y
Y
D016475:3T3 Cells; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000096:biosynthesis; D004252:DNA Mutational Analysis; D004220:Disulfides / Q000737:chemistry; D005822:Genetic Vectors; D006003:Glycogen / Q000096:biosynthesis; D006801:Humans; D066298:In Vitro Techniques; D007328:Insulin / Q000378:metabolism; D007335:Insulin-Like Growth Factor II / Q000737:chemistry / Q000378:metabolism*; D008024:Ligands; D008113:Liver Neoplasms; D051379:Mice; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D010449:Peptide Mapping; D011485:Protein Binding; D011487:Protein Conformation; D017527:Receptor, IGF Type 2; D011972:Receptor, Insulin / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000378:metabolism*; D017451:Receptors, Somatomedin; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D014407:Tumor Cells, Cultured
D004220:Disulfides; D007328:Insulin; D008024:Ligands; D009841:Oligonucleotides; D017527:Receptor, IGF Type 2; D011956:Receptors, Cell Surface; D017451:Receptors, Somatomedin; D011994:Recombinant Proteins; D007335:Insulin-Like Growth Factor II; D006003:Glycogen; D004247:DNA; D011972:Receptor, Insulin