Characterisation of insulin-like growth factor-binding protein-3 binding to a novel receptor on human platelet membranes

Abstract

Insulin-like growth factor-binding protein-3 (IGFBP-3) is an important regulator of insulin-like growth factor (IGF) bioavailability and IGF-independent growth responses. IGFBP-3 is stored within the alpha granules of platelets, permitting its rapid and concentrated delivery at sites of platelet lysis. Previous studies have demonstrated a lack of mRNA for IGFBP-3 in platelets and in the megakaryocytes from which platelets are formed, indicating that IGFBP-3 is endocytosed from the extracellular milieu. In this study, the binding of IGFBP-3 to platelet membranes was characterised to determine whether specific cell-surface IGFBP-3 receptors exist that might account for IGFBP-3 uptake into the alpha granules by megakaryocytes. IGFBP-3 binding to platelets was saturable, requiring at least 4.6 nM 125I-labelled IGFBP-3 to occupy all binding sites present on 100 μg of platelet membranes. Non-linear regression analysis revealed the presence of a single class of high-affinity binding sites for IGFBP-3 on platelets, with a Kd between 2.6 × 10-10 and 8.0 × 10-10 M and 1.51-4.89 × 1011 binding sites/mg of platelet membrane. Kinetic analysis of 125I-IGFBP-3 binding to platelet membranes demonstrated a forward rate (kon) of 8.1 × 108 per M per min. The reverse rate constant (koff) was calculated to be 1.6 × 10-1 per min (t1/2=4.2 min) and confirmed experimentally to be 3.3 × 10-1 per min (t1/2=2.1 min). Binding of 125I-IGFBP-3 to platelet membranes was inhibited in a dose-dependent manner by recombinant Escherichia coli IGFBP-3. In contrast, rat IGFBP-4 was not able to compete with 125I-IGFBP-3 for platelet binding sites. Additionally, concentrations of IGF-I ranging from a 15-fold to a 40 000-fold molar excess caused a consistent 20% reduction in 125I-IGFBP-3 binding. The mechanism of this slight reduction is unknown, but suggests that IGF-I does not compete directly with IGFBP-3 for receptor binding sites. However, it does not preclude the possibility that IGF-I may be endocytosed into the alpha granules as part of an IGF-I-IGFBP-3 complex. These results demonstrate the presence of high-affinity binding sites for IGFBP-3 on human platelet membranes. The nature and kinetics of the binding reaction are characteristic of a receptor-ligand interaction. This receptor may be involved in the endocytosis of circulating IGFBP-3 by megakaryocytes for packaging within the alpha granules of platelets. It is unknown if it is present in other tissues.