NEUROENDOCRINE-SPECIFIC PROTEIN-C (NSP-C) - SUBCELLULAR-LOCALIZATION AND DIFFERENTIAL EXPRESSION IN RELATION TO NSP-A

A mouse monoclonal antibody RNL-4, as well as rabbit polyclonal antise rum POL-8 were raised against a synthetic peptide, encompass ing the f irst twenty unique amino-terminal amino acid residues of NSP-C. The sp ecificity of both immunoreagents was established in an ELISA assay usi ng the synthetic peptide and by their immunoreactivity to NSP-C fusion proteins. Immunofluorescence analysis of COS-1 cells, transfected wit h NSP-C cDNA, showed staining of the endoplasmic reticulum with RNL-4 and POL-8. No cross-reactivity of these reagents with NSP-A or NSP-B w as seen. Immunohistochemical studies in normal human tissues showed ex pression of NSP-C in tissues of neural and neuroendocrine origin, i.e. neurons of the central and peripheral nervous system, the neurohypoph ysis, adrenal medulla, adenohypophysis, pars intermedia, and in sporad ic neuroendocrine cells of the lung. Expression of NSP-C was found in several small cell lung cancer (SCLC) cell lines, in non-SCLC cell lin es with neuroendocrine features, but not in typical non-SCLC cell line s. Also, in a neuroblastoma cell line NSP-C expression was observed. I mmunoblotting and immunoprecipitation studies with RNL-4 and POL-8 ide ntified the 23 kDa NSP-C polypeptide in these cell lines. Immunofluore scence microscopy showed that also in these cell lines N8P-C is locate d at the endoplasmic reticulum, as shown before for NSP-A and NSP-B. I n some of the cell lines coexpression of NSP-A and NSP-C was observed, while in others only one of the two could be detected. The differenti al expression of NSP-A and NSP-C in these cell lines is confirmed by i mmunoblotting and was also evident at the mRNA level. When NSP-A and N SP-C were coexpressed, the number of NSP-C-positive cells was always l ess than the number of NSP-A-positive cells. A partial colocalization of NSPs was observed in the endoplasmic reticulum. Cell fractionation studies revealed that both proteins are retained in the membranous fra ction of the cell, from which they can be solubilized by Triton X-100. Immunoprecipitation analyses under native conditions indicate that NS P-C does not need to associate with NSP-A to form high molecular weigh t NSP-reticulons.