Complementary DNA cloning of a receptor for tumor necrosis factor and demonstration of a shed form of the receptor.
Tumor necrosis factor (TNF) receptor (TNFR) was isolated as a 68-kDa glycoprotein from UC/HeLa 2-5 cells developed from a parental B-cell line (UC cells) to overexpress the receptor. Tryptic digests of two separate TNFR preparations provided amino acid sequences of four different peptides. Amino-terminal analysis indicated the presence of the amino-acid sequence Val-Ala-Phe-Thr-Pro, reported to be the amino-terminal sequence of a 30-kDa urinary TNF-binding protein II. Examination of the cultured medium of UC/HeLa 2-5 cells showed an abundance of a 40-kDa TNF-binding protein, indicating that the previously cited 30-kDa TNF-binding protein II is likely to be a shed form of the TNFR. Based on the peptide sequences, oligonucleotides were synthesized, and two of these were used as primers in the polymerase chain reaction to amplify cDNA sequences from poly(A)+ RNA of UC/HeLa 2-5 cells. These PCR fragments were radiolabeled and used to screen a cDNA library made from UC/HeLa 2-5 mRNA. Further analysis identified cDNA sequences that encoded the amino acid sequences of all four TNFR peptides. RNA blot-hybridization analysis of UC/HeLa 2-5 mRNA revealed a 3.8-kilobase transcript of the same size as the mRNA in the parental UC cells. Genomic Southern blots indicated the presence of a single gene in parental cells and a second, amplified gene in TNFR-overexpressing cells, suggesting amplification of the transfected gene as a possible mechanism for the increase in TNFR numbers in UC/HeLa 2-5 cells.
Heller
R A
RA
Institute of Cancer and Developmental Biology, Syntex Research, Palo Alto, CA 94304.
Song
K
K
Onasch
M A
MA
Fischer
W H
WH
Chang
D
D
Ringold
G M
GM
D000595:Amino Acid Sequence; D001402:B-Lymphocytes; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics / Q000302:isolation & purification; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D015723:Gene Library; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D011956:Receptors, Cell Surface / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha / Q000378:metabolism
D004273:DNA, Neoplasm; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D018124:Receptors, Tumor Necrosis Factor; D014409:Tumor Necrosis Factor-alpha; D004247:DNA