Methods for Preparing Extracellular Matrix and Quantifying Insulin-like Growth Factor-Binding Protein Binding to the ECM - Methods in Molecular Biology: Extracellular Matrix Protocols - page 210

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Methods for Preparing Extracellular Matrix
and Quantifying Insulin-like Growth
Factor-Binding Protein Binding to the ECM
Bo Zheng and David R. Clemmons
1. Introduction
The insulin-like growth factor-binding proteins (IGFBPs) are carrier pro-
teins that are present in all extracellular fluids (1) . Similarly, insulin-like growth
factor-I (IGF-I) and -II are synthesized by connective tissue cells that are
present within the stroma of all tissues, and therefore their presence is ubiqui-
tous in all organs (2) . IGF receptors are present in all tissues, which accounts
for balanced growth of organs, and deletion of the IGF-I gene results in a bal-
anced decrease in organ growth, whereas overexpression of IGF-I results in a
symmetric increase in growth (3) .
The principle role of the IGFBPs is to determine the distribution of IGF-I
and -II among cells and tissues and to provide controlled access to receptors
(1) . Because there is excess binding capacity in all interstitial fluids, all the
IGF that circulates is in a bound form. Because protein-bound IGF-I and -II
cannot bind to their receptors, mechanisms need to exist to release IGF-I and -II
under controlled conditions. Three events have been described that result in
controlled release. Because in all cases, the affinity of unmodified forms of
IGFBPs in solution is higher than the receptor, these modifications must be
ones that result in a reduction in IGFBP affinity in order to result in a more
favorable equilibrium. These include proteolysis, binding to cell surfaces or
extracellular matrix (ECM), and phosphorylation or dephosphorylation. This
chapter concerns association of IGF binding proteins with ECM. ECM binding
results in an 8-15-fold reduction in affinity of IGFBP-5 for IGF-I or -II, and
therefore it is physiologically significant (4) .