Subcellular localization and supramolecular organization of neuroendocrine-specific protein B (NSP-B) in small cell lung cancer
Senden, N.H.; van de Velde, H.J.; Broers, J.L.; Timmer, E.D.; Kuijpers, H.J.; Roebroek, A.J.; Van de Ven, W.J.; Ramaekers, F.C.
European Journal of Cell Biology 65(2): 341-353
1994
ISSN/ISBN: 0171-9335 PMID: 7720728 Document Number: 438732
We have recently isolated and characterized a novel gene that is expressed in a neuroendocrine-specific fashion and was therefore designated neuroendocrine-specific protein (NSP)-gene. The NSP-gene encodes three transcripts of different size, with unique 5'-sequences and completely overlapping 3'-sequences. The resulting 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 characterization and subcellular localization of NSP-B, so far only found to be expressed in the neuroendocrine lung cancer cell line NCI-H82, and its relation to NSP-A. Transfection studies with the NSP-B transcript in COS1 cells, followed by immunoprecipitation, resulted in a set of proteins 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 NSP-B triplet in the 43 to 45 kDa range and a 35 kDa NSP-B were consistently detected. Only the 45 kDa NSP-B was found to be phosphorylated. The 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 filtration 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 the phosphate buffered saline soluble cell fraction, but mainly occurring in the membranous pellet fraction from which they can be solubilized by Triton X-100. Complexes can either contain NSP-A or NSP-B separately, or comprise both these NSPs as concluded from immunoprecipitation and immunoaffinity chromatography studies. A subcellular distribution 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 occurs in oligomeric complexes. Immunofluorescence studies show an association of these NSP-aggregates with the endoplasmic reticulum, as concluded from double-labeling studies using an antibody to a Ca-2+ -ATPase SERCA-2b. Immunofluorescence studies in COS-1 cells transfected with the NSP-B transcript showed that NSP-B also can interact with the endoplasmic reticulum as aggregates (reticulons) independently of NSP-A and NSP-C.