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Expression In Heterologous Cells Of The Unusual Cytoplasmic Domain Of Rat Brain 5B4/NCAM-ld.

P. Ramos, L. Ellis
Published 1989 · Biology, Medicine

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Monoclonal antibody 5B4 recognizes a carboxy-terminal epitope common to large (approximately 180 kDa) and short (approximately 140 kDa) forms of neural cell adhesion molecules (NCAM-ld and NCAM-sd, respectively). The deduced primary sequence of rat brain 5B4/NCAM-ld predicts a large cytoplasmic domain (390 amino acids, Mr 39,284) of striking amino acid composition (52% proline, alanine, serine and threonine) and little predicted alpha or beta secondary structure: its function is unknown. To directly test the deduced topology of the protein, and especially the solubility and stability of its unusual cytoplasmic domain, we have constructed a cDNA expression vector designed to express this domain independently as a soluble protein (designated 5B4cyt) in a heterologous cell system (simian Cos cells). 5B4cyt is indeed soluble, but migrates anomalously on SDS-PAGE under denaturing and reducing conditions as two species of approximately 77 and approximately 80 kDa. In pulse chase experiments, the approximately 77 kDa band chases into the approximately 80 kDa band with a t1/2 of approximately 1 h. The difference in mobility is apparently a consequence of the rapid phosphorylation of the approximately 77 kDa species. The approximately 88 kDa phospho-form is reasonably stable with a t1/2 of approximately 6 h. These results are consistent with the deduced topology of 5B4/NCAM-ld, and demonstrate the feasibility of this experimental approach for exploring the biochemistry and structure of its unusual cytoplasmic domain.
This paper references
Immunolocalization of a neuronal growth-dependent membrane glycoprotein
I. Wallis (1985)
Efficient expression in insect cells of a soluble, active human insulin receptor protein-tyrosine kinase domain by use of a baculovirus vector.
L. Ellis (1988)
Biosynthesis of the D2-cell adhesion molecule: post-translational modifications, intracellular transport, and developmental changes
J. Lyles (1984)
Sequence of a cDNA clone encoding the polysialic acid-rich and cytoplasmic domains of the neural cell adhesion molecule N-CAM.
J. Hemperly (1986)
NCAM: the molecule and its genetics
U. Rutishauser (1986)
Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose
L. Ellis (1986)
The 5B4 antigen expressed on sprouting neurons contains α-2,8-linked polysialic acid
J. Rosenberg (1986)
Human muscle neural cell adhesion molecule (N-CAM): Identification of a muscle-specific sequence in the extracellular domain
G. Dickson (1987)
Identification of a cDNA clone that contains the complete coding sequence for a 140-kD rat NCAM polypeptide
S. Small (1987)
Sulfation and phosphorylation of the neural cell adhesion molecule, N-CAM.
B. Sorkin (1984)
Studies on transformation of Escherichia coli with plasmids.
D. Hanahan (1983)
SV40-transformed simian cells support the replication of early SV40 mutants
Y. Gluzman (1981)
Neural cell adhesion molecule: structure, immunoglobulin-like domains, cell surface modulation, and alternative RNA splicing.
B. Cunningham (1987)
Studies on the transmembrane disposition of the neural cell adhesion molecule N-CAM. A monoclonal antibody recognizing a cytoplasmic domain and evidence for the presence of phosphoserine residues.
G. Gennarini (1984)
Truncation of the ectodomain of the human insulin receptor results in secretion of a soluble insulin binding protein from transfected CHO cells
L. Ellis (1988)
Promoter-regulatory region of the major immediate early gene of human cytomegalovirus.
D. Thomsen (1984)
Cell surface modulation of the neural cell adhesion molecule resulting from alternative mRNA splicing in a tissue-specific developmental sequence
B. Murray (1986)
Isolation and sequence of λgt11 cDNA clones encoding the 5B4 antigen expressed on sprouting neurons
P. Ramos (1989)
Metallothionein-human GH fusion genes stimulate growth of mice.
R. Palmiter (1983)
An analysis of 5'-noncoding sequences from 699 vertebrate messenger RNAs.
M. Kozak (1987)
Production of human c-myc protein in insect cells infected with a baculovirus expression vector.
C. Miyamoto (1985)
A cascade of tyrosine autophosphorylation in the beta-subunit activates the phosphotransferase of the insulin receptor.
M. White (1988)
Nerve growth cones isolated from fetal rat brain. IV. Preparation of a membrane subfraction and identification of a membrane glycoprotein expressed on sprouting neurons
L. Ellis (1985)
Cell adhesion and the molecular processes of morphogenesis.
G. Edelman (1985)

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