Proteoglycan Introduction

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Heparan sulfate proteoglycans (HSPGs) bind via their heparan sulfate (HS) gly-cosaminoglycan chains to a large variety of extracellular ligands. These ligands include components of the extracellular matrix, other cell surface receptors, viruses, proteases and their inhibitors, and growth factors. The interaction of growth factors with HS has been proposed to affect growth factor function, if by no means other than limiting growth factor diffusion. For certain growth factors, however, work over the past decade has shown that HS binding has a more direct role in signaling. It is proposed that the HS chain participates directly in the assembly of these growth factors with their signaling receptor and may even act as a regulator of the signaling (1,2). This role for HSPGs has been shown for epidermal growth factor family members, most notably heparin-binding EGF and the heregulins (3), hepatocyte growth factor/scatter factor (4), and for the members of the fibroblast growth factor (FGF) family (5,6).

The role of HSPGs as regulators of growth factor action is best characterized for the FGF family, and this family will be the focus of this chapter. HSPGs regulate FGF signaling by participating in the formation of a ternary signaling complex comprised of the FGF ligand, FGF receptor tyrosine kinase (RTK), and HS (7,8). HS-binding sites on both the FGF ligand (9) and the RTK (10) facilitate assembly of this signaling complex. Because the HS structure is known to have considerable variation, particularly in its sulfation pattern, it becomes an important question as to whether this variation is specific and serves to regulate the formation of these signaling complexes. The 19 FGF family members currently known signal through RTKs that are encoded by four genes (FGFR1-4). In addition, all FGFRs with the exception of FGFR4 are subject to RNA splice variation with profound effects on ligand specificity [reviewed in (11)]. HSPGs have been shown to be both promoters and inhibitors of FGF signaling

From: Methods in Molecular Biology, Vol. 171: Proteoglycan Protocols Edited by: R. V. Iozzo © Humana Press Inc., Totowa, NJ

(12,13). This dual role can be explained by differential binding of domains within the HS chain to FGF ligand and its RTK. For example, a HS that contains a specific sulfation sequence for both the FGF and the RTK is predicted to behave as a stimulator of FGF signal transduction, whereas a sulfation sequence that binds the FGF ligand but fails to recognize the RTK is postulated to be a competitive inhibitor. Specificity in the binding interactions between HS and the other signaling partners is made possible by the remarkable diversity of HS polysaccharide chains. In fact, the information density present in HS chains exceeds that of nucleic acids or polypeptides (14).

Experimental evidence is also accumulating in support of the notion that specific HSPG core proteins bear specific FGF-modulating HS chains (15,16). It is unclear whether this is a uniform behavior of a specific core protein, since literature reports have attributed FGF stimulatory and inhibitory activities to basically all existing HSPG classes, or whether this depends on the cell type that expresses the HSPG. Divergent reports on the activity of specific HSPGs are likely to reflect differences in the cell type of origin and metabolic state of the experimental model. In vivo, HSPGs are widely distributed, but show remarkable tissue- and cell type-specific patterns of expression. It is also becoming increasingly clear that HSPGs are not passive FGF co-receptors, but are themselves dynamically regulated with dramatic effects on FGF signal transduction (17).

An important question regarding HSPG regulation of FGF signaling, therefore, is whether the HS chains are expressed with specific regulatory properties and whether this specific expression occurs within a specific tissue or cell type. Much of the information on HS regulation of FGF signaling has been acquired using heparan sulfate isolated in bulk from tissues. These extracts would of course represent a mixture of HSPGs stemming from multiple cellular and extracellular sources, and any location-specific information would inescapably be lost. Although the question can also be addressed by extracting HSPGs from cells in culture and examining the effects of the isolated HSPG preparations on FGF signaling, the clear disadvantage of this approach is that there is no reason to believe that cell lines that have escaped normal growth control would still be equipped with HSPGs equivalent to their counterparts in vivo. Therefore, we have developed different assays that allow us to localize HS chains in situ and to explore their abilities to assemble a ternary complex with FGF and RTK, thereby providing direct information of their potential role in FGF signaling (18,19).

The strategy that will be described is to reconstitute the signaling complex in situ in a stepwise fashion by adding each of its components as a binding probe (illustrated schematically in Fig. 1). As a first step, the HS chains in the tissue need to be localized. This can be achieved by using an antibody directed against the "stubs" remaining on HSPGs after heparitinase digestion (anti-delta-HS antibody 3G10) (20). This localization is important for establishing whether all of the HS participates in formation of a signaling complex, or whether this is limited to only a specific HS population. Since monoclonal antibody 3G10 reacts with HS sugar moieties rather than the protein, it detects all HSPGs regardless of the core protein identity. Next, HS capable of binding the FGF under investigation needs to be identified. This is done by incubating the tissue sections with the FGF and detecting bound growth factor (see Fig. 1A). Due to

Fig. 1. Schematic representation of the in situ binding assays. (A) Assay to detect growth factor binding to tissue HSPGs. Tissue sections are incubated with growth factor and bound growth factor is detected with antibody directed against the growth factor protein or a biotin tag. This assay distinguishes the following classes of HSPGs: (1) HSPGs that do not bind FGF and are expected not to play a role in signaling; (2) HSPGs that bind FGF and may be positive or negative regulators of signaling. (B) Assay to distinguish positive and negative regulators of growth factor signaling. This assay allows the differentiation of the following classes of HSPGs: (1) HSPGs that do not bind FGF; (2a) HSPGs that bind FGF, but the HSPG/growth factor complex does not bind soluble receptor (These HSPGs are expected to sequester FGF and therefore not to promote signaling.); (2b) HSPGs that not only bind FGF, but assemble the complete signaling complex (These HSPGs are expected to promote signaling.). AP, alkaline phosphatase; FR1cECD, FGFR-1c extracellular domain.

Fig. 1. Schematic representation of the in situ binding assays. (A) Assay to detect growth factor binding to tissue HSPGs. Tissue sections are incubated with growth factor and bound growth factor is detected with antibody directed against the growth factor protein or a biotin tag. This assay distinguishes the following classes of HSPGs: (1) HSPGs that do not bind FGF and are expected not to play a role in signaling; (2) HSPGs that bind FGF and may be positive or negative regulators of signaling. (B) Assay to distinguish positive and negative regulators of growth factor signaling. This assay allows the differentiation of the following classes of HSPGs: (1) HSPGs that do not bind FGF; (2a) HSPGs that bind FGF, but the HSPG/growth factor complex does not bind soluble receptor (These HSPGs are expected to sequester FGF and therefore not to promote signaling.); (2b) HSPGs that not only bind FGF, but assemble the complete signaling complex (These HSPGs are expected to promote signaling.). AP, alkaline phosphatase; FR1cECD, FGFR-1c extracellular domain.

the abundant expression of HS is tissues, this can be performed relatively easily using either frozen and paraffin-embedded tissue sections. Finally, the ability of the FGF/ HS complexes to bind FGF RTK is tested by adding a soluble tagged receptor construct (see Fig. 1B). The binding pattern of this construct to the FGF provides a detailed picture of which HS populations are able to bind the FGF and promote its binding to RTK.

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