Abstract. We analyzed the binding of fibronectin to integrin a5/31 in various cells; in some cells fibronectin bound with low affinity (e.g., K562 cells) whereas in others (e.g., CHO), it bound with high affinity (Kd ,~ 100 nM) in an energy-dependent manner. We constructed chimeras of the extracellular and transmembrane domains of c~3 joined to the cytoplasmic domains of otsBl. The affinity state of these chimeras was assessed by binding of fibrinogen or the monoclonal antibody, PACl. The cytoplasmic domains of 0~5/~ conferred an energy-dependent high affinity state on otnbB3 in CliO but not K562 cells. Three additional tx cytoplasmic domains (ix2, otv~, ot~B) conferred PAC1 binding in CHO cells, while three others (otM, OiL, av) did not. In the high affinity ot chimeras, cotransfection with a truncated (~3A724) or mutated (~3(S752"-~P)) 83 subunit abolished high affinity binding. Thus, both cytoplasmic domains are required for energy-dependent, cell type-specific affinity modulation. In addition, mutations that disrupted a highly conserved o~ subunit GFFICR motif, resulted in high affinity binding of ligands to Otnb/~3. In contrast to the chimeras, the high affinity state of these mutants was independent of cellular metabolism, cell type, and the bulk of the ~ subunit cytoplasmic domain. Thus, integrin cytoplasmic domains mediate inside-out signaling. Furthermore, ' the highly conserved GFFKR motif of the c~ Subunit cytoplasmic domain maintains the default low affinity state.

In the adult mammalian CNS, chondroitin sulfate proteoglycans (CSPGs) and myelin–associated inhibitors (MAIs) stabilize neuronal structure and restrict compensatory sprouting following injury. The Nogo receptor family members NgR1 and NgR2 bind to MAIs and have been implicated in neuronal inhibition. Here we show that NgR1 and NgR3 bind with high–affinity to the glycosaminoglycan moiety of proteoglycans and participate in CSPG inhibition in cultured neurons. Nogo receptor triple mutants (NgR123−/−), but not single mutants, show enhanced axonal regeneration following retro–orbital optic nerve crush injury. The combined loss of NgR1 and NgR3 (NgR13−/−), but not NgR1 and NgR2 (NgR12−/−), is sufficient to mimic the NgR123−/− regeneration phenotype. Regeneration in NgR13−/− mice is further enhanced by simultaneous ablation of RPTPσ, a known CSPG receptor. Collectively, these results identify NgR1 and NgR3 as novel CSPG receptors, demonstrate functional redundancy among CSPG receptors, and provide unexpected evidence for shared mechanisms of MAI and CSPG inhibition.

Glycosaminoglycan (GAG) side chains endow extracellular matrix proteoglycans with diversity and complexity based upon the length, composition and charge distribution of the polysaccharide chain. Using cultured primary neurons, we show that specific sulfation in the GAG chains of chondroitin sulfate mediates neuronal guidance cues and axonal growth inhibition. Chondroitin-4-sulfate (CS-A), but not chondroitin-6-sulfate (CS-C), exhibits a strong negative guidance cue to mouse cerebellar granule neurons. Enzymatic and gene-based manipulations of 4-sulfation in the GAG side chains alter their ability to direct growing axons. Furthermore, 4-sulfated chondroitin sulfate GAG chains are rapidly and significantly increased in regions that do not support axonal regeneration proximal to spinal cord lesions in mice. Thus, our findings show that specific sulfation along the carbohydrate backbone carries instructions to regulate neuronal function. Supplementary material available online at

The retinoid-X receptor (RXR) regulates multiple hormonal pathways through heterodimerization with nuclear receptors such as the all-trans retinoic acid receptor (RAR). The orphan nuclear receptor NGFI-B (also called Nur77) can heterodimerize with RXR. Here we show that nerve growth factor (NGF) induces the phosphorylation of Ser 105 of NGFI-B in PC12 phaeochromocytoma cells, resulting in translocation of the NGFI-B-RXR heterodimer complex out of the nucleus using nuclear export signals within NGFI-B. As a consequence of the redistribution of RXR, the transcriptional activity of RXR-RAR is reduced. NGFI-B-mediated nuclear export of receptors may serve as a mechanism for crosstalk between NGF and retinoid pathways.