Antipeptide antibodies recognize c-erbA and a related protein in human A431 carcinoma cells.
Peptides corresponding to the deduced amino acid residues 15-29 of the amino-terminal region and 445-456 of the carboxyl-terminal region of the human placental c-erbA protein (hc-erbA-beta) were synthesized and used to produce site-specific rabbit polyclonal antipeptide sera. Antibodies to the carboxyl-terminal peptide (C-91) and the amino-terminal peptide (N-98) specifically immunoprecipitated the hc-erbA-beta proteins synthesized in vitro. Furthermore, 68% and 48% of the T3-binding activity of the hc-erbA-beta protein were immunoabsorbed by antibodies C-91 and N-98, respectively. These results indicate that C-91 and N-98 recognized hc-erbA-beta proteins. These antibodies were used to study the subcellular distribution of hc-erbA-beta protein in human cultured cells. Nuclear extracts were prepared from human A431 carcinoma cells; C-91 immunoabsorbed 50% of the specific T3-binding activity in these extracts. These results provide structural evidence to confirming that hc-erbA-beta is the T3 nuclear receptor. Cells were metabolically labeled with [35S]methionine, and the cytosolic extracts were immunoprecipitated by C-91 or N-98. A protein with a mol wt of 58,000 (Cp58) was specifically immunoprecipitated by N-98 or C-91. Peptide mapping by V8 digestion and cyanogen bromide cleavage showed that the Cp58 molecules immunoprecipitated by N-98 or C-91 was identical. Indirect immunofluorescence using N-98 or C-91 indicated that Cp58 is present exclusively in the cytoplasm. Other human cultured cells, HepG2, MCF-7, IM-9, and KB, were also evaluated, and similar results were found. These results raised the possibility that a precursor of hc-erbA-beta may be present in the cytosol. The functional significance of the hc-erbA-beta-related cytosolic Cp58 remains to be established.
Fukuda
T
T
Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.
Willingham
M C
MC
Cheng
S Y
SY
D000906:Antibodies / Q000276:immunology; D000918:Antibody Specificity; D002467:Cell Nucleus / Q000378:metabolism; D003488:Cyanogen Bromide; D003600:Cytosol / Q000032:analysis / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D006801:Humans; D007163:Immunosorbent Techniques; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis* / Q000276:immunology; D010920:Placenta / Q000032:analysis; D011498:Protein Precursors / Q000032:analysis*; D011518:Proto-Oncogene Proteins / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D011988:Receptors, Thyroid Hormone / Q000378:metabolism; D012697:Serine Endopeptidases / Q000378:metabolism; D014284:Triiodothyronine / Q000378:metabolism; D014407:Tumor Cells, Cultured
D000906:Antibodies; D010446:Peptide Fragments; D011498:Protein Precursors; D011518:Proto-Oncogene Proteins; D011988:Receptors, Thyroid Hormone; D014284:Triiodothyronine; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D003488:Cyanogen Bromide