Rat CNS myelin at 0.5C2 g of total protein/well was dried overnight onto the coated wells and used β3-AR agonist 1 as a substrate (Shen et al., 1998). almost as effectively as full-length MAG. To determine whether the inhibition site mapped to Ig-3, Ig-4, or Ig-5, we made chimeric molecules of various combinations of these three MAG Ig domains fused to Ig domains from another Ig family member, sialoadhesin (Sn), which also binds to sialic acid in the same linkage as MAG. The MAG-Sn molecules were expressed in CHO cells and all contained five Ig domains and were able to bind sialic acid. However, only the chimeric molecules made up of MAG Ig-5 inhibited neurite outgrowth. Furthermore, peptides corresponding to sequences in MAG Ig-5, but not Ig-4 or Sn Ig-5, are able to block inhibition of neurite outgrowth by both wild-type MAG and CNS myelin. We conclude that this inhibition site on MAG is usually carried by Ig domain name 5 and that this site is unique from your sialic-acid binding site. at 37C and β3-AR agonist 1 washed twice. The medium was changed to DMEM and 1 107 human erythrocytes in HEPES-DMEM were added and incubated for 1 h at 37C. Unbound erythrocytes were washed off and cells were examined and photographed under a phase-contrast microscope and photographed. Neurite outgrowth on transfected CHO cells. Confluent monolayers of CHO cells expressing wild-type MAG, truncated MAG, MAG-Sn chimeras, or control CHO cells were established over a 24 h period in individual Cryab chambers of an eight-well tissue culture slide (Lab-Tek). Cocultures were established as explained previously (Doherty et al.,1990; Mukhopadhyay et al., 1994) by adding 5 104 P5 cerebellar neurons in Sato media to the CHO cell monolayers. MAG or Sn peptides were included at 50 g/ml as indicated. After 16C18 h, the cocultures were fixed for 30 min with 4% paraformaldehyde, and permeabilized with ice-cold methanol for 2 min. The cells were then blocked for 20 min with DMEM made up of 10% β3-AR agonist 1 FCS and incubated for 2 h with a rabbit polyclonal antibody against growth-associated protein 43 (Space43) (1:4,000; a gift from R. Curtis and G. Wilkins, Imperial College, London, UK). Cells were washed three times with 2% PBS-BSA (2%) and then incubated for 30 min at room temperature with a biotinylated donkey anti-rabbit Ig (1:300; Amersham Biosciences, Arlington Heights, IL), washed three times, and incubated with streptavidin-conjugated Texas reddish (1:300; Amersham Biosciences) for 45 min. After three more washes, the slides were mounted in Permafluor (Baxter Healthcare Miami, β3-AR agonist 1 FL) and viewed with a Zeiss fluorescent microscope. The length of the longest neurite for each Space43-positive neuron was decided for the first 180C200 neurons encountered when scanning the slide in a systematic manner using the Uncor image-analysis program. Myelin preparation. Myelin was purified from rat CNS white matter following the Norton protocol (Norton and Poduslo, 1973). After the final hypotonic shock, the membranes were centrifuged and resuspended in 10 mm HEPES. The protein concentration of the preparation was decided and used immediately as a substrate in the neurite outgrowth assay. Neurite outgrowth on immobilized myelin. The wells of an eight-chamber tissue culture slides (Lab-Tek) were first coated with 16.6 g/ml poly-l-lysine at room temperature for 1 h. Rat CNS myelin at 0.5C2 g of total protein/well was dried overnight onto the coated wells and used as a substrate (Shen et al., 1998). A total of 2 104 P5 cerebellar neurons suspended in Sato media with 2% FBS were plated into each well and incubated at 37C for 24 h. As a control, poly-l-lysine was used as a substrate. Fixation, immunostaining, and measurement of neurite outgrowth were as explained above for neurite outgrowth on CHO cells. Results The β3-AR agonist 1 inhibition site on MAG is within Ig domains 3, 4, and 5 Previously, we established that sialic acid binding by MAG is not necessary for it to inhibit neurite outgrowth (Tang et al., 1997). Furthermore, we showed that a soluble, truncated chimeric form of MAG consisting of MAG Ig domains 1C3 fused to the Fc region of human IgG [MAG(d1C3)-Fc] and bound to neurons in a sialic acid-dependent manner but, did not inhibit neurite outgrowth (Tang et al., 1997)..