The H1 and H2 polypeptides associate to form the asialoglycoprotein receptor in human hepatoma cells.

Antibody-induced degradation and chemical cross-linking experiments have been carried out to assess the nature of the interaction between the two asialoglycoprotein-receptor polypeptides, H1 and H2, synthesized in HepG2 cells. Incubation of HepG2 cell monolayers with anti-H1 antibody caused a specific and equal loss of both H1 and H2 polypeptides. The same result was obtained with anti-H2 antibody. Control serum did not affect the level of H1 or H2 not did anti-H1 or anti-H2 antibodies affect the level of the transferrin receptor. The chemical cross-linking reagent, difluorodinitrobenzene, has been used to demonstrate that H1 can be cross-linked to H2 in HepG2 cell microsomal membranes. Dimer and trimer species with apparent molecular masses of 93 and 148 kD, respectively, were readily observed upon chemical cross-linking and some dimers and trimers were immunoreactive with both anti-H1 and anti-H2 antibodies. The putative trimer, possibly two H1 and one H2 molecules, is a minimum estimate of the true size of the asialoglycoprotein receptor in intact HepG2 cell, and it is possible that larger hetero-oligomeric forms of the receptor exist. The results of both types of experiments indicate that H1 and H2 form an oligomeric complex in HepG2 cells and thus, both polypeptides constitute the human asialoglycoprotein receptor.

Cloning and characterization of a murine band 3-related cDNA from kidney and from a lymphoid cell line.

Anion exchange is a nearly ubiquitous cellular transport function which contributes to the regulation of cell pH and of cell volume. However, the only plasma membrane anion exchanger of known identity and sequence is erythroid band 3. Both hybridization and immunologic data support the presence of band 3-related mRNAs and proteins in nonerythroid tissues. We have used low stringency hybridization with the murine band 3 cDNA to clone a band 3-related cDNA from murine kidney and from 70Z/3 pre-B cells. The cDNA encodes a band 3-related protein (B3RP) of 1237 amino acids, with a predicted mass of 137 kDa. The carboxyl-terminal hydrophobic domain of B3RP has an amino acid sequence 67% identical to that of band 3, with a very similar predicted secondary structure. The amino-terminal hydrophilic domain of B3RP has two sections. The section adjacent to the putative membrane-associated segment is 33% identical in amino acid sequence to the amino-terminal, cytoplasmic domain of band 3. The other, far amino-terminal section of B3RP has no correspondent in the band 3 sequence. B3RP mRNA is present in a variety of epithelial and other tissues and probably encodes an anion exchange protein of wide distribution.

The 5' region of the p53 gene: evolutionary conservation and evidence for a negative regulatory element.

The 5' regions of the mouse, rat and human functional p53 genes were isolated and analysed. All three genes possess a non-coding exon, comprising exclusively 5' untranslated sequences. This exon contains extensive diad symmetry near the 5' end of p53 mRNA, possibly allowing for the formation of a stable hairpin structure in this mRNA. The nucleotide sequence within this hairpin element is highly conserved among the species. A DNA stretch of 225 bp preceding the p53 mRNA cap site possesses distinct promoter activity when assayed in the CAT system. However, this activity is practically abolished when further upstream p53 sequences (approximately 120 bp) are included in front of the CAT gene. This suggests that the control of p53 gene expression is complex and involves a negative regulatory element.