Molecular cloning of the transmembrane component of the 13762 mammary adenocarcinoma sialomucin complex. A new member of the epidermal growth factor superfamily.
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.
Sheng
Z
Z
Department of Molecular Biology and Biochemistry, University of Miami School of Medicine, Florida 33101.
Wu
K
K
Carraway
K L
KL
Fregien
N
N
D000230:Adenocarcinoma; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004273:DNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D004815:Epidermal Growth Factor / Q000235:genetics*; D015723:Gene Library; D006801:Humans; D008325:Mammary Neoplasms, Experimental; D008565:Membrane Proteins / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009077:Mucins / Q000032:analysis / Q000235:genetics*; D005810:Multigene Family; D011061:Poly A / Q000235:genetics / Q000302:isolation & purification; D011487:Protein Conformation; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger; D012334:RNA, Neoplasm / Q000235:genetics / Q000302:isolation & purification; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000032:analysis; D012689:Sequence Homology, Nucleic Acid; D052116:Sialomucins; D014407:Tumor Cells, Cultured
D004273:DNA, Neoplasm; D008565:Membrane Proteins; D009077:Mucins; D012333:RNA, Messenger; D012334:RNA, Neoplasm; D011993:Recombinant Fusion Proteins; D052116:Sialomucins; D011061:Poly A; D004815:Epidermal Growth Factor; D012313:RNA