The binding ability analysis of the normal VLDL receptor and its mutant

Abstract

The ligand-binding domain of VLDL receptor contains eight imperfectly similar repeats. To discuss the contribution of each repeat to ligand binding, the RT-PCR technique was used to clone the VLDLR-cDNA from the heart muscle of Chinese people. Two recombinants were further constructed, which contained the full-length cDNA of VLDLR and the mutant lacking repeats 1–5. CHO cell line was transfected with two recombinants. The expression of VLDLR gene could be detected by RT-PCR from the CHO cells transfected with pCD-VR. The results of binding experiments showed that the ability of the CHO cells transfected with the full-length cDNA of VLDL-R binding Dil-labeled β-VLDL was higher than that of the CHO cells transfected with the mutant. Our findings indicated that human VLDL-R gene could be expressed effectively on CHO cells, and the receptor was almost inactivated when repeatsl-5 were deleted.

Keywords

VLDL receptor / gene expression / β-VLDL / CHO cells

References

[1] MehtaK D, ChenW J, GoldsteinJ L, et al. . The low density lipoprotein receptor in Xenopus laevis. I. Five domains that resemble the human receptor. J Biol Chem, 1991, 26610406-10406

[2] YamamotoT, DavisC G, BrownMS, et al. . The human LDL receptor: A cysteine-rich protein with multiple aulsequences in its mRNA. Cell, 1984, 3927-27

[3] YamamotoT, BishopR W, BrownM S, et al. . Deletion in cysteine-rich region of LDL receptor impedes transport to cell surface in WHHL rabbit. Science, 1986, 2321230-1230

[4] LeeL Y, MohlerW A, SchaferB L, et al. . Nucleotide sequence of the rat low density lipoprotein receptor cDNA. Nucleic Acids Res, 1989, 171259-1259

[5] TakahashiS, KawarabayasiY, NakaiT, et al. . Rabbit very low density lipoprotein receptor: a low density lipoprotein receptor like protein with distinct ligand specificity. Proc Natl Acad Sci USA, 1992, 899252-9252

[6] EsserV, LimbirdL E, BrownM S, et al. . Muational analysis of the ligand binding domain of the low density lipoprotein recetpor. Biol Chem, 1988, 26313283-13283

[7] RusseltD W, BrownM S, GoldsteinJ L. Different combinations of cysteine-rich repeats mediate binding of low density lipoprotein receptor to two different proteins. J Biol Chemm, 1989, 26421682-21682

[8] SakaiJ, HoshinoA, TakahashiS, et al. . J Biol Chem, 1994, 2692173-2173