The binding of a monoclonal antibody reactive with pp60v-src to the rat CNS both in vitro and in vivo: evidence that the epitope is present intracellularly as well as being associated with a number of antigenically related polypeptides located externally in the plasma membrane only in the synaptic region.
A monoclonal antibody, F4, has been produced which reacts with an epitope possessing an unusual subcellular distribution. It binds to the external surface of the neuronal plasma membrane only in the region of the synapse. This is evidenced by binding of F4 to presynaptic terminals in unfixed cultures of rat cerebellum and to preparations of unfixed synaptosomes. In addition, much larger amounts of the epitope are present intracellularly. In fixed nervous tissue, the epitope is found in many neurons, and is associated mainly with presynaptic plasma membranes, synaptic vesicles, postsynaptic densities (cerebral cortex and hippocampus, but not cerebellum), rough endoplasmic reticulum, and the Golgi apparatus. The epitope is especially abundant in large neurons (e.g. pyramidal cells). Similar amounts of epitope are present in the chromaffin cells of the adrenal medulla. It is also expressed in ependymal cells in the brain, and in epithelial cells present in ducts of the medulla, but not cortex, of the kidney. However, the epitope is not found in glial cells in the brain, or in either liver, spleen, skeletal muscle, or testes. F4 is not species specific, as it binds to postmortem adult human cerebral cortex and neonatal cerebellum in a manner as described for the rat. It also binds to homogenates of brains of fish, chicken and mouse. The appearance of the epitope during development of the cerebellum in vivo and in vitro occurs in parallel with the differentiation of neurons and formation of synapses, though small amounts are also present in neuronal precursor cells. The F4 antibody can detect nanogram amounts of pp60v-src on immunodots. The strength of this reaction is high enough that F4 can be used to demonstrate pp60v-src-like immunoreactivity in Rous Sarcoma virus-transformed chick embryo fibroblasts. However, present evidence suggests that it may be premature to assign the immunocytochemical reactivity of F4 in the brain exclusively to pp60c-src. This conclusion is based on the fact that F4 reacts with several polypeptides from synaptic plasma membranes on Western blots of renaturing, two-dimensional gels that are dissimilar in size to pp60c-src, and from the fact that it can cross-react, albeit weakly, with several other serine protein kinases in an immunodot assay. Appreciation of this cross-reactivity, and of the evolutionary conservation of the epitope, as well as its sensitivity to denaturation, has led to our working hypothesis that F4 binds to a conformational epitope present on several polypeptides that may be most perfectly represented by some aspect of the catalytic domain of tyrosine protein kinases.
Lasher
R S
RS
Department of Cellular and Structural Biology, University of Colorado Medical School, Denver 80262.
Erickson
P F
PF
Mena
E E
EE
Cotman
C W
CW
D000818:Animals; D000911:Antibodies, Monoclonal / Q000378:metabolism*; D001921:Brain / Q000276:immunology* / Q000378:metabolism / Q000648:ultrastructure; D002478:Cells, Cultured; D000939:Epitopes; D007150:Immunohistochemistry; D008297:Male; D008565:Membrane Proteins / Q000032:analysis / Q000276:immunology*; D051379:Mice; D008810:Mice, Inbred C57BL; D008854:Microscopy, Electron; D015688:Oncogene Protein pp60(v-src); D012191:Retroviridae Proteins / Q000276:immunology*; D013569:Synapses / Q000276:immunology* / Q000648:ultrastructure
D000911:Antibodies, Monoclonal; D000939:Epitopes; D008565:Membrane Proteins; D012191:Retroviridae Proteins; D015688:Oncogene Protein pp60(v-src)