Molecular cloning of the transmembrane component of the 13762 mammary adenocarcinoma sialomucin complex. A new member of the epidermal growth factor superfamily

• Published in: The Journal of biological chemistry 1992-08, Vol.267 (23), p.16341-16346
• Main Authors: SHENG, Z, WU, K, CARRAWAY, K. L, FREGIEN, N
• Format: Article
• Language: English
• Subjects: Adenocarcinoma; Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Animals; ascites sialoglycoprotein 2; ASGP-2 protein; Base Sequence; Biological and medical sciences; cDNA; Cloning, Molecular; DNA, Neoplasm - genetics; DNA, Neoplasm - isolation & purification; Epidermal Growth Factor - genetics; Fundamental and applied biological sciences. Psychology; Gene Library; genes; Glycoproteins; Humans; Mammary Neoplasms, Experimental; Membrane Proteins - genetics; Molecular Sequence Data; Molecular Weight; Mucins - analysis; Mucins - genetics; Multigene Family; nucleotide sequence; Poly A - genetics; Poly A - isolation & purification; prediction; Protein Conformation; Proteins; Rats; Recombinant Fusion Proteins - analysis; RNA - genetics; RNA - isolation & purification; RNA, Messenger; RNA, Neoplasm - genetics; RNA, Neoplasm - isolation & purification; Sequence Homology, Nucleic Acid; sialomucin; Sialomucins; Tumor Cells, Cultured
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(18)42007-8

Ascites sublines of the 13762 rat mammary adenocarcinoma have a cell surface sialomucin complex composed of the sialomucin ascites sialoglycoprotein-1 (ASGP-1) and the membrane-associated glycoprotein ASGP-2. The sialomucin complex is synthesized as a high M(r) precursor, pre-sialomucin complex (pSMC-1). To characterize the structure of the membrane-associated component of this complex, a lambda gt11 cDNA expression library was constructed using mRNA from 13762 rat mammary adenocarcinoma cells and screened with polyclonal antibody against ASGP-2. The strongest antibody-binding clone, designated lambda ASGP2.9-1, had a 1.3-kilobase (kb) insert, and hybridized to a 9-kb transcript in 13762 cell mRNA. The large size of this transcript was expected, since the estimated molecular mass of pSMC-1 is greater than 250 kDa. To obtain the full sequence of ASGP-2, a longer cDNA (5.4 kb), designated pASGP1/2.1, was subsequently cloned by screening a plasmid library with an oligonucleotide complementary to the 5' end of the phage insert. The amino acid sequence derived from nucleotide sequence of pASGP1/2.1 showed a 12-amino acid identity with amino acid sequence obtained from the NH2 terminus of ASGP-2, indicating the entire ASGP-2 coding region was included in the cDNA. Furthermore, an 18-amino acid identity with the NH2 terminus of a 6-kDa CNBr fragment of ASGP-2 was also observed in the cDNA sequence. The polypeptide contains several distinct domains, including a hydrophobic transmembrane domain, a short (20 residue) COOH-terminal cytoplasmic tail, and a large extracellular domain with 24 potential N-glycosylation sites. These properties correspond to features of ASGP-2 and pSMC-1 predicted by previous biochemical studies. Most interestingly, the extracellular domain contains two cysteine-rich sequences, each of which has a segment with strong similarities to proteins with epidermal growth factor activity. Since our recent studies show that ASGP-2 can modulate epidermal growth factor receptor phosphorylation activity, these results provide structural evidence to support the role of the heterodimeric sialomucin complex as a bifunctional modulator of cellular interactions and cell proliferation.