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 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 COS-1 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.(ABSTRACT TRUNCATED AT 250 WORDS)
Senden
N H
NH
Department of Molecular Cell Biology & Genetics, University of Limburg, Maastricht/The Netherlands.
van de Velde
H J
HJ
Broers
J L
JL
Timmer
E D
ED
Kuijpers
H J
HJ
Roebroek
A J
AJ
Van de Ven
W J
WJ
Ramaekers
F C
FC
D000818:Animals; D000911:Antibodies, Monoclonal; D018288:Carcinoma, Small Cell / Q000737:chemistry*; D004721:Endoplasmic Reticulum / Q000737:chemistry; D000882:Haplorhini; D015151:Immunoblotting; D008175:Lung Neoplasms / Q000737:chemistry*; D046911:Macromolecular Substances; D008970:Molecular Weight; D009363:Neoplasm Proteins / Q000032:analysis*; D009419:Nerve Tissue Proteins / Q000032:analysis*; D011233:Precipitin Tests; D013347:Subcellular Fractions / Q000737:chemistry*; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D046911:Macromolecular Substances; D009363:Neoplasm Proteins; D009419:Nerve Tissue Proteins