CALEB Binds via Its Acidic Stretch to the Fibrinogen-like Domain of Tenascin-C or Tenascin-R and Its Expression Is Dynamically Regulated after Optic Nerve Lesion

Abstract

Recently, we described a novel chick neural transmembrane glycoprotein, which interacts with the extracellular matrix proteins tenascin-C and tenascin-R. This protein, termed CALEB, contains an epidermal growth factor-like domain and appears to be a novel member of the epidermal growth factor family of growth and differentiation factors. Here we analyze the interaction between CALEB and tenascin-C as well as tenascin-R in more detail, and we demonstrate that the central acidic peptide segment of CALEB is necessary to mediate this binding. The fibrinogen-like globe within tenascin-C or -R enables both proteins to bind to CALEB. We show that two isoforms of CALEB in chick and rodents exist that differed in their cytoplasmic segments. To begin to understand the in vivo function of CALEB and since in vitro antibody perturbation experiments indicated that CALEB might be important for neurite formation, we analyzed the expression pattern of the rat homolog of CALEB during development of retinal ganglion cells, after optic nerve lesion and during graft-assisted retinal ganglion cell axon regeneration by in situ hybridization. These investigations demonstrate that CALEB mRNA is dynamically regulated after optic nerve lesion and that this mRNA is expressed in most developing and in onethird of the few regenerating (GAP-43 expressing) retinal ganglion cells.

Phosphorylation of Neuroglycan C, a Brain-specific Transmembrane Chondroitin Sulfate Proteoglycan, and Its Localization in the Lipid Rafts

“…The NGC kinase activity coprecipitated with NGC was partially inhibited by DRB and heparin (inhibitors for CKII) but not by GF109203X (an inhibitor of PKC), at concentrations around their IC 50 in the in vitro kinase reaction (Fig. 6C).…”

“…The phosphorylation of the ectodomains of these proteins is considered to be a mechanism to modify molecular interactions at the cell surface. NGC has been shown to interact with tenascin through its acidic cluster (50) and with ErbB3 tyrosine kinase through the epidermal growth factor motif 2 of the ectodomain. Although no molecules have been identified to interact directly with the chondroitin sulfate attachment region of the NGC ectodomain which is supposed to be phosphorylated, the ectodomain phosphorylation of NGC could change the cellular physiology of the NGC-expressing cells 2 S. Higashiyama, unpublished observation.…”

“…6C). Other reagents, KN-62, which is a specific inhibitor of Ca 2ϩ /calmodulin-dependent kinase II, and CKI-7, which is a specific inhibitor of CKI, had no effect on the phosphorylation of NGC even at concentrations 10 times higher than their IC 50 or K i (data not shown).…”

“…6C). DRB, a CKII-specific inhibitor with an IC 50 of 4 -10 M, partially inhibited the phosphorylation of NGC at concentrations around IC 50 . Heparin, an inhibitor of CKI (IC 50 , 24 g/ml) and CKII (IC 50 , 0.15 g/ml), also partially inhibited the phosphorylation of NGC.…”

“…DRB, a CKII-specific inhibitor with an IC 50 of 4 -10 M, partially inhibited the phosphorylation of NGC at concentrations around IC 50 . Heparin, an inhibitor of CKI (IC 50 , 24 g/ml) and CKII (IC 50 , 0.15 g/ml), also partially inhibited the phosphorylation of NGC. GF109203X, a specific inhibitor of PKC with an IC 50 of 10 nM, did not inhibit the NGC kinase activity at 10 nM but partially inhibited it at extremely high concentrations (Fig.…”