Biomedical subjects

J I Jones

Publications and source records attributed to J I Jones.

27 records · Page 2Linked to original sources

PubMed1993

Human IGFBP-1 is phosphorylated on 3 serine residues: effects of site-directed mutagenesis of the major phosphoserine.

Human IGFBP-1 is phosphorylated by cells in culture and is present in both phosphorylated and nonphosphorylated forms in human fetal serum and amniotic fluid. We have found immunoprecipitable [32P]IGFBP-1 in the conditioned media of both Chinese hamster ovary (CHO) cells (stabley transfected and secreting human IGFBP-1) and human hepatoma (HepG2) cells metabolically labelled with [32P]orthophosphate. Phosphoamino acid analysis of this [32P]IGFBP-1 demonstrates that only serine residues are phosphorylated. Four phosphorylated isoforms of IGFBP-1 can be separated from one nonphosphorylated form by nondenaturing gel electrophoresis. Since we have shown that the nonphosphorylated form of IGFBP-1 has a lower affinity for IGF-I compared to phosphorylated forms and a greater potentiating effect of IGF-I actions, we determined which serine residues in human IGFBP-1 are phosphorylated. After metabolically labelling IGFBP-1 with 32P, the purified phosphoprotein was digested first with trypsin and then with endoproteinase Glu-C. By radiosequencing the resulting 32P-labelled phosphopeptides, we found 3 serine residues to be phosphorylated. Approximately 70% of incorporated 32P was attributed to Ser101, while Ser169 accounted for approximately 25% and Ser119 for 5%. To investigate the physiologic importance of Ser101, this residue (and the nonphosphorylated Ser98) were changed to alanine by site directed mutagenesis of a human IGFBP-1 expression vector, followed by transfection into CHO cells. The [Ala98,101]IGFBP-1 purified from the conditioned media of these cells had the following characteristics: 1) when labelled with [32P]orthophosphate, it contained 63% less radioactivity than wild type IGFBP-1; 2) when analyzed by nondenaturing gel electrophoresis, it contained none of the most rapidly migrating and most rapidly migrating and most highly phosphorylated isoform, more of the nonphosphorylated isoform, and more of the most slowly migrating phosphorylated isoform; and 3) its affinity for IGF-I was reduced 2.5-fold and was midway between wild type IGFBP-1 from transfected CHO cells and dephosphorylated IGFBP-1. We conclude that Ser101 represents the major site of phosphorylation of IGFBP-1 and that while phosphorylation of Ser101 increases affinity of IGFBP-1 for IGF-I, phosphorylation of Ser169 and/or Ser119 also contributes to the high affinity of fully phosphorylated IGFBP-1.

Alanine↗

PubMed1991

Phosphorylation of insulin-like growth factor (IGF)-binding protein 1 in cell culture and in vivo: effects on affinity for IGF-I.

The insulin-like growth factors (IGF-I and IGF-II) are present in extracellular fluids bound to specific IGF-binding proteins (IGFBPs). We and others have reported varying biologic activity of different preparations of IGFBP-1 that appeared to have identical amino acid sequences and molecular sizes. This observation prompted us to determine whether IGFBP-1 undergoes posttranslational modifications. Immunoprecipitation was used to show that Chinese hamster ovary cells (transfected with a human IGFBP-1 cDNA construct) and human hepatoma (HepG2) cells secrete 32P-labeled IGFBP-1 following incubation with [32P]orthophosphate. Phospho amino acid analysis of 32P-labeled IGFBP-1 revealed only phosphoserine residues. A method was developed that could separate nonphosphorylated IGFBP-1 from four or five phosphorylated isoforms. Using this technique we demonstrated that human amniotic fluid and human fetal serum contain a large proportion of nonphosphorylated IGFBP-1, as well as phosphorylated forms. In contrast, HepG2 cells and human decidual cells secrete predominantly the phosphorylated isoforms. These observations suggest that IGFBP-1 is secreted as a phosphoprotein and is subsequently dephosphorylated in vivo. Binding studies showed that the phosphorylated IGFBP-1 secreted by HepG2 cells has a 6-fold higher affinity for IGF-I than it does after dephosphorylation. We conclude that IGFBP-1 is phosphorylated and that this phosphorylation is a physiologically important posttranslational modification.

Animals↗

PubMed1990

Binding of [125I]iodipine to parathyroid cell membranes: evidence of a dihydropyridine-sensitive calcium channel.

The parathyroid cell is unusual, in that an increase in extracellular calcium concentrations inhibits PTH release. Calcium channels are glycoproteins that span cell membranes and allow entry of extracellular calcium into cells. We have demonstrated that the calcium channel agonist (+)202-791, which opens calcium channels, inhibits PTH release and that the antagonist (-)202-791, which closes calcium channels, stimulates PTH release. To identify the calcium channels responsible for these effects, we used a radioligand that specifically binds to calcium channels. Bovine parathyroid cell membranes were prepared and incubated under reduced lighting with [125I] iodipine (SA, 2000 Ci/mmol), which recognizes 1,4-dihydropyridine-sensitive calcium channels. Bound ligand was separated from free ligand by rapid filtration through Whatman GF/B filters. Nonspecific binding was measured by the inclusion of nifedipine at 10 microM. Specific binding represented approximately 40% of the total binding. The optimal temperature for [125I] iodipine binding was 4 C, and binding reached equilibrium by 30 min. The equilibrium dissociation constant (Kd) was approximately 550 pM, and the maximum number of binding sites was 780 fmol/mg protein. Both the calcium channel agonist (+)202-791 and antagonist (-)202-791 competitively inhibited [125I] iodipine binding, with 50% inhibition concentrations of 20 and 300 nM, respectively. These data indicate the presence of dihydropyridine-sensitive calcium channels on parathyroid cell membranes.

Animals↗

PubMed1976