General Linker Diversification Approach to Bivalent Ligand Assembly: Generation of an Array of Ligands for the Cation-Independent Mannose 6-Phosphate Receptor.

A generalized strategy is presented for the rapid assembly of a set of bivalent ligands with a variety of linking functionalities from a common monomer. Herein, an array of phosphatase-inert mannose-6-phosphonate-presenting ligands for the cation-independent-mannose 6-phosphate receptor (CI-MPR) is constructed. Receptor binding affinity varies with linking functionality-the simple amide and 1,5-triazole(tetrazole) being preferred over the 1,4-triazole. This approach is expected to find application across chemical biology, particularly in glycoscience, wherein multivalency often governs molecular recognition.

Inhibition of insulin-like growth factor II (IGF-II)-dependent cell growth by multidentate pentamannosyl 6-phosphate-based ligands targeting the mannose 6-phosphate/IGF-II receptor.

The mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) binds M6P-capped ligands and IGF-II at different binding sites within the ectodomain and mediates ligand internalization and trafficking to the lysosome. Multivalent M6P-based ligands can cross-bridge the M6P/IGF2R, which increases the rate of receptor internalization, permitting IGF-II binding as a passenger ligand and subsequent trafficking to the lysosome, where the IGF-II is degraded. This unique feature of the receptor may be exploited to design novel therapeutic agents against IGF-II-dependent cancers that will lead to decreased bioavailable IGF-II within the tumor microenvironment. We have designed a panel of M6P-based ligands that bind to the M6P/IGF2R with high affinity in a bivalent manner and cause decreased cell viability. We present evidence that our ligands bind through the M6P-binding sites of the receptor and facilitate internalization and degradation of IGF-II from conditioned medium to mediate this cellular response. To our knowledge, this is the first panel of synthetic bivalent ligands for the M6P/IGF2R that can take advantage of the ligand-receptor interactions of the M6P/IGF2R to provide proof-of-principle evidence for the feasibility of novel chemotherapeutic agents that decrease IGF-II-dependent growth of cancer cells.

Cell non-autonomous regulation of hepatic IGF-1 and neonatal growth by Kinase Suppressor of Ras 2 (KSR2).

Individuals with poor postnatal growth are at risk for cardiovascular and metabolic problems as adults. Here we show that disruption of the molecular scaffold Kinase Suppressor of Ras 2 (KSR2) causes selective inhibition of hepatic GH signaling in neonatal mice with impaired expression of IGF-1 and IGFBP3. ksr2(-/-) mice are normal size at birth but show a marked increase in FGF21 accompanied by reduced body mass, shortened body length, and reduced bone mineral density (BMD) and content (BMC) first evident during postnatal development. However, disrupting FGF21 in ksr2(-/-) mice does not normalize mass, length, or bone density and content in fgf21(-/-)ksr2(-/-) mice. Body length, BMC and BMD, but not body mass, are rescued by infection of two-day-old ksr2(-/-) mice with a recombinant adenovirus encoding human IGF-1. Relative to wild-type mice, GH injections reveal a significant reduction in JAK2 and STAT5 phosphorylation in liver, but not in skeletal muscle, of ksr2(-/-) mice. However, primary hepatocytes isolated from ksr2(-/-) mice show no reduction in GH-stimulated STAT5 phosphorylation. These data indicate that KSR2 functions in a cell non-autonomous fashion to regulate GH-stimulated IGF-1 expression in the liver of neonatal mice, which plays a key role in the development of body length.

Dominant-negative effect of truncated mannose 6-phosphate/insulin-like growth factor II receptor species in cancer.

Oligomerization of the mannose 6-phosphate/insulin-like growth factor II receptor (M6P/IGF2R) is important for optimal ligand binding and internalization. M6P/IGF2R is a tumor suppressor gene that exhibits loss of heterozygosity and is mutated in several cancers. We tested the potential dominant-negative effects of two cancer-associated mutations that truncate M6P/IGF2R in ectodomain repeats 9 and 14. Our hypothesis was that co-expression of the truncated receptors with the wild-type/endogenous full-length M6P/IGF2R would interfere with M6P/IGF2R function by heterodimer interference. Immunoprecipitation confirmed formation of heterodimeric complexes between full-length M6P/IGF2Rs and the truncated receptors, termed Rep9F and Rep14F. Remarkably, increasing expression of either Rep9F or Rep14F provoked decreased levels of full-length M6P/IGF2Rs in both cell lysates and plasma membranes, indicating a dominant-negative effect on receptor availability. Loss of full-length M6P/IGF2R was not due to increased proteasomal or lysosomal degradation, but instead arose from increased proteolytic cleavage of cell-surface M6P/IGF2Rs, resulting in ectodomain release, by a mechanism that was inhibited by metal ion chelators. These data suggest that M6P/IGF2R truncation mutants may contribute to the cancer phenotype by decreasing the availability of full-length M6P/IGF2Rs to perform tumor-suppressive functions such as binding/internalization of receptor ligands such as insulin-like growth factor II.