Endocrinology, Vol 123, 2646-2652, Copyright © 1988 by Endocrine Society

ARTICLES

Antipeptide antibodies recognize c-erbA and a related protein in human A431 carcinoma cells

T Fukuda, MC Willingham and SY Cheng
Laboratory of Molecular Biology, National Cancer Institute, Bethesda, Maryland 20892.

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.

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