SUBCELLULAR-LOCALIZATION AND SUPRAMOLECULAR ORGANIZATION OF NEUROENDOCRINE-SPECIFIC PROTEIN-B (NSP-B) IN SMALL-CELL LUNG-CANCER

We have recently isolated and characterized a novel gene that is expre ssed in a neuroendocrine-specific fashion and was therefore designated neuroendocrine-specific protein (NSP) gene. The NSP-gene encodes thre e transcripts of different size, with unique 5'-sequences and complete ly overlapping 3'-sequences. The result ing proteins have an apparent molecular mass of 135 kDa as determined for NSP-A and 23 kDa as found for NSP-C. In the present study we focused on the biochemical characte rization and subcellular localization of NSP-B, so far only found to b e expressed in the neuroendocrine lung cancer cell line NCI-H82, and i ts relation to NSP-A. Transfection studies with the NSP-B transcript i n COS-1 cells, followed by immunoprecipitation, resulted in a set of p roteins ranging in molecular mass from 35 to 45 kDa, identical to NSP- Bs detected by immunoblotting in NCI-H82. In this cell line a major NS P-B triplet in the 43 to 45 kDa range and a 35 kDa NSP-B were consiste ntly detected. Only the 45 kDa NSP-B was found to be phosphorylated. T he observed pI values of the 43 to 45 kDa triplet ranged from 4.8 to 5 .0, while the 35 kDa NSP-B has a more basic pI value of 5.7. Gel filtr ation studies show that NSP-A and NSP-B form supramolecular aggregates with a molecular mass of over 500 kDa, present to a minor extent in t he phosphate buf fered saline soluble cell fraction, but mainly occurr ing in the membranous pellet fraction from which they can be solubiliz ed by Triton X-100. Complexes can either contain NSP-A or NSP-B separa tely, or comprise both these NSPs as concluded from immunoprecipitatio n and immunoaffinity chromatography studies. A subcellular distributio n of NSP-A, similar to that of NSP-B in NCI-H82, has been seen in the neuroendocrine lung cancer cell line SCLC-21H, in which NSP-A also occ urs in oligomeric complexes. Immunofluorescence studies show an associ ation of these NSP-aggregates with the endoplasmic reticulum, as concl uded from double-labeling studies using an antibody to a Ca2+-ATPase S ERCA-2b. Immunofluorescence studies in COS 1 cells transfected with th e NSP-B transcript showed that NSP-B also can interact with the endopl asmic reticulum as aggregates (reticulons) independently of NSP-A and NSP C.