Independent signals determine the subcellular localization of NEP in prostate cancer cells.
NEP (Neutral endopeptidase 24.11) is a cell surface enzyme that hydrolyzes bioactive neuropeptides implicated in the transition from androgen-dependent prostate cancer (PC) to androgen-independent PC. We report the cloning and sequence analyses of NEP cDNAs from human androgen-responsive LNCaP PC cells and prostatic stromal cells. To investigate the functional role of a nuclear localization sequence (NLS) detected within the N-terminus and of an endoplasmic reticulum retention signal within the C-terminus, NEP-GFP expression vectors were constructed containing the whole NEP gene, fragments encoding the N-terminus/C-terminus of the protein (5(')NEP-GFP/3(')NEP-GFP), and 5(')NEP-GFP constructs lacking the NLS. 3(')NEP-GFP transfected cells showed plasma membrane/cytoplasmic fluorescence whereas the 5(')NEP-GFP fusion protein was also detected in the nucleus. The omission of the NLS resulted in no reduction in nuclear and an increase in cytoplasmic staining. The results suggest that the analyzed structural motifs determine the subcellular distribution of NEP in epithelial LNCaP PC cells and stromal prostatic cells and therefore could be responsible for the altered cellular localization of NEP observed in PC.
Gomes
Iara
I
Department of Anatomy and Cell Biology, Philipps-University, Marburg, Germany. iaragomes@web.de
Aumüller
Gerhard
G
Wennemuth
Gunther
G
Bette
Michael
M
Albrecht
Martin
M
D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000378:metabolism; D018014:Gene Transfer Techniques; D006031:Glycosylation; D049452:Green Fluorescent Proteins; D006801:Humans; D006868:Hydrolysis; D008164:Luminescent Proteins / Q000378:metabolism; D008297:Male; D008856:Microscopy, Fluorescence; D008957:Models, Genetic; D008969:Molecular Sequence Data; D015260:Neprilysin / Q000096:biosynthesis* / Q000235:genetics; D010957:Plasmids / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000201:enzymology*; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D012984:Software; D014162:Transfection
D018076:DNA, Complementary; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D015260:Neprilysin