ABSTRACT
We have previously described the presence of two (high- and low-affinity) HDL binding sites on the hepatoma cell line (HepG2) (R. Barbaras, X. Collet, H. Chap, and B. Perret (1994) Biochemistry 33, 2335-2340]. Moreover, apoA-I, the major HDL apolipoprotein, interacts with these two binding sites, while lipid-free apoA-I binds only to the high-affinity sites. Using tryptic HDL fragments and HepG2 cell monolayers as an "affinity matrix," we identified an apoA-I peptide of 16 amino acids, spanning between residues 62 and 77, as a ligand domain. The corresponding synthetic peptide displays high-affinity (K(d) approximately 10(-7) M) and low-capacity (B(max) 8 pmol/mg of cell protein) binding components. Competition experiments with this peptide, using (125)I-labeled free apoA-I as a ligand, show that this binding corresponds to the high-affinity binding sites already described. In conclusion, we identified the apoA-I 62-77 region as a specific high-affinity ligand domain of HDL on HepG2 cells.
Subject(s)
Apolipoprotein A-I/metabolism , Amino Acid Sequence , Animals , Apolipoprotein A-I/chemistry , Apolipoprotein A-I/genetics , Binding Sites , Binding, Competitive , Humans , Kinetics , Ligands , Lipoproteins, HDL/chemistry , Lipoproteins, HDL/genetics , Lipoproteins, HDL/metabolism , Liver Neoplasms, Experimental/metabolism , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/genetics , Peptide Fragments/metabolism , Protein Structure, Tertiary , Tumor Cells, CulturedABSTRACT
The role of the centrosomes in microtubule nucleation remains largely unknown at the molecular level. gamma-Tubulin and the two associated proteins h103p (hGCP2) and h104p (hGCP3) are essential. These proteins are also present in soluble complexes containing additional polypeptides. Partial sequencing of a 76- kD polypeptide band from these complexes allowed the isolation of a cDNA encoding for a new protein (h76p = hGCP4) expressed ubiquitously in mammalian tissues. Orthologues of h76p have been characterized in Drosophila and in the higher plant Medicago. Several pieces of evidence indicate that h76p is involved in microtubule nucleation. (1) h76p is localized at the centrosome as demonstrated by immunofluorescence. (2) h76p and gamma-tubulin are associated in the gamma-tubulin complexes. (3) gamma-tubulin complexes containing h76p bind to microtubules. (4) h76p is recruited to the spindle poles and to Xenopus sperm basal bodies. (5) h76p is necessary for aster nucleation by sperm basal bodies and recombinant h76p partially replaces endogenous 76p in oocyte extracts. Surprisingly, h76p shares partial sequence identity with human centrosomal proteins h103p and h104p, suggesting a common protein core. Hence, human gamma-tubulin appears associated with at least three evolutionary related centrosomal proteins, raising new questions about their functions at the molecular level.
