Michael D. Sadick

Reevaluation of the roles of protein S and gas6 as ligands for the receptor tyrosine kinase Rse/Tyro 3

Recently, Stitt et al. (1995) reported that protein S (PS), but not Gas6, is a potent ligand for the receptor tyrosine kinase known as Rse, Tyro 3, Brt, Sky, and Tif (hereafter referred to as Rse/Tyro 3). PS is an abundant serum protein previously characterized as an essential anticoagu-lant. Gas6, which was identified as a gene whose expression is increased by growth arrest, shares 43% amino acid identity and overall domain organization with PS (Manfio-letti et al., 1993). Stitt et al. (1995) based their conclusions on experiments describing interspecies interactions of bovine Gas6 with murine Rse/Tyro 3 (mRse/Tyro 3) and on interactions of bovine and human PS (hPS) with mRse/ Tyro 3. Consistent with the results of Stitt et al. (1995), we identified bovine PS as a ligand for human Rse/Tyro 3 (hRse/Tyro 3), and we also found that hPS can act as a ligand for mRse/Tyro 3. However, when we analyzed the more relevant intraspecies interactions, we obtained different results. We found that human Gas6 (hGas6), but not hPS, acted as a potent ligand for hRse/Tyro 3 (Figure 1). The hRse/Tyro 3 we have studied is very likely the true homolog of mRse/Tyro 3 since they share 90% amino acid identity and a similar expression pattern (Mark et al., 1994; Lai et al., 1994). To characterize the Rse/Tyro 3 ligand, we constructed soluble receptor proteins containing the extraceltular domain of either hRse/Tyro 3 or mRseFryro 3 fused to the Fc portion of human immunoglobulin G1 (hRse-lgG and mRse-lgG). A similar fusion protein (termed Tyro 3-Fc) was utilized by Stitt et al. (1995) to characterize the binding of bovine Gas6 and hPS to mRse/Tyro 3. We first determined whether hRse-lgG or mRse-lgG differed in its ability to bind to hPS or hGas6 containing an epitope tag that allows for side-by-side comparison of the binding properties of the two proteins. Either h Rse-lgG or mRse-lgG was incubated with conditioned medium containing putative ligands. Complexes were captured with protein A (specific for the IgG fusion protein) and visualized with an antibody specific for the epitope-tagged putative ligand. While hGas6 was bound by hRse-lgG, it was not efficiently bound by mRse-lgG (Figure 2). The reciprocal result was obtained in analysis of binding of hPS to hRse-lgG and mRse-lgG; hPS was bound by mRse-lgG but not by hRse-lgG. These results are consistent with an apparent difference in affinity of hPS for human as opposed …

Antibody Binding Regions on Human Nerve Growth Factor Identified by Homolog- and Alanine-Scanning Mutagenesis

The binding specificities of a panel of mouse monoclonal antibodies (MAbs) to human nerve growth factor (hNGF) were determined by epitope mapping using chimeric and point mutants of NGF. Subsequently, the MAbs were used to probe NGF structure-function relationships. Six MAbs, which recognize distinct or partially overlapping regions of hNGF, were evaluated for their ability to block the binding of hNGF to the TrkA and p75 NGF receptors in various in vitro assays, which included blocking of TrkA autophosphorylation and blocking of NGF-dependent survival of dorsal root ganglion sensory neurons. Three MAbs (911,912,938) were potent blockers of all activities. Potent blocking of p75 binding occurs only with MAb 909, which recognizes an NGF region identified by mutagenesis as important for NGF-p75 binding. These results are consistent with recently proposed models of binding regions involved in NGF-TrkA and NGF-p75 interactions generated through mutagenic analysis and structure determination of the NGF-TrkA complex. These studies provide insight to the epitope specificities and potency of MAbs that would be useful for physiological NGF blocking studies.

Binding Protein-3-Selective Insulin-Like Growth Factor I Variants: Engineering, Biodistributions, and Clearance

Insulin-like growth factor I (IGF-I) is a potent anabolic peptide that mediates most of its pleiotropic effects through association with the IGF type I receptor. Biological availability and plasma half-life of IGF-I are modulated by soluble binding proteins (IGFBPs), which sequester free IGF-I into high affinity complexes. Elevated levels of specific IGFBPs have been observed in several pathological conditions, resulting in inhibition of IGF-I activity. Administration of IGF-I variants that are unable to bind to the up-regulated IGFBP species could potentially counteract this effect. We engineered two IGFBP-selective variants that demonstrated 700- and 80,000-fold apparent reductions in affinity for IGFBP-1 while preserving low nanomolar affinity for IGFBP-3, the major carrier of IGF-I in plasma. Both variants displayed wild-type-like potency in cellular receptor kinase assays, stimulated human cartilage matrix synthesis, and retained their ability to associate with the acid-labile subunit in complex with IGFBP-3. Furthermore, pharmacokinetic parameters and tissue distribution of the IGF-I variants in rats differed from those of wild-type IGF-I as a function of their IGFBP affinities. These IGF-I variants may potentially be useful for treating disease conditions associated with up-regulated IGFBP-1 levels, such as chronic or acute renal and hepatic failure or uncontrolled diabetes. More generally, these results suggest that the complex biology of IGF-I may be clarified through in vivo studies of IGFBP-selective variants.