Studies on the transmembrane disposition of the neural cell adhesion molecule N‐CAM

Abstract

The N‐CAMs are a group of surface glycoproteins involved in adhesive interactions of neurones. Related molecules of the mouse nervous system, identified in our laboratory, have been called BSP‐2 and shown to act as ligands in adhesion of neuroblastoma cells. Results presented in this report show that they are immunochemically identical with N‐CAM. A monoclonal anti‐(N‐CAM) antibody, that recognized a determinant accessible only after permeabilization of intact cells, was used to define the mode of association of the N‐CAMs with the plasma membrane. This antibody bound a 35000‐Mr fragment in lysates of trypsin‐treated neuroblastoma cells. It is concluded that the antibody reacts with a transmembrane or cytoplasmic domain of the molecules. The same antibody recognized the Mr‐180000 and Mr‐140000 proteins but not the Mr‐120000 chain, which copurify from adult mouse brain. The latter polypeptide was detected in the cytosol and could be partially released from brain membranes by osmotic shock. Part or all of the Mr‐120000 protein may thus lack a transmembrane segment. Our conclusion that the N‐CAM forms of higher Mr are transmembrane proteins was further corroborated by our finding that they contain phosphoserine residues, which can be labeled with (32P)phosphate in intact neuroblastoma cells.

Endocrine cells share expression of N‐CAM with neurones

“…It is not surprising that the 120 kDa glycoprotein was not detectable in PC12 cells since chromaffin cells in situ from which they derive do not appear to express it. Other tumour cell lines also show a limited expression of the N-CAM determinants [21]. Although it is accepted that the relative proportions of the three glycoproteins vary both with age and with brain region [7], it is of particular interest that the 180 and 120 kDa polypeptides were absent from the two endocrine tissues examined here while at the same time an additional N-CAM positive band of 49 kDa was prominent.…”

Progenitor cells of the olfactory receptor neuron lineage

“…After 6 days in vitro, cultures were fixed (15 min with 3.7% formaldehyde/5% sucrose in phosphate-buffered saline) and processed for NCAM immunoreactivity to reveal neuronal colonies. Cultures were incubated with undiluted H28 rat anti-NCAM hybridoma supernatant (Gennarini et al, 1984) and primary antibody detected with horseradish peroxidaseconjugated goat anti-rat IgG (Jackson, 1:500 dilution). The neuronal colony shown here contains many (Ͼ30) ORNs, bearing extensive neurites tipped with growth cones.…”

Homologous liver parenchymal cell-cell adhesion mediated by an endogenous lectin and its receptor

“…The washed cells were then counted in a haemocytometer using a phase contrast microscope. The yield of cells per gram of liver was approximately 100x10 6 .…”

“…Some of the major classes of CAM that have been identified in eukaryotes are fibronectins, cadherins, integrins, and Ig superfamily members. CAMs are large intrinsic membrane glycoproteins localized on the external cell surface [5,6]. Carbohydrate-mediated cell-cell recognition plays a vital role in the orderly development and functioning of multicellular organisms [7,8].…”