Molecular characterization of an apical early endosomal glycoprotein from developing rat intestinal epithelial cells.

The apical endosomal compartment is thought to be involved in the sorting and selective transport of receptors and ligands across polarized epithelia. To learn about the protein components of this compartment, we have isolated and sequenced a cDNA that encodes a glycoprotein that is located in the apical endosomal tubules of developing rat intestinal epithelial cells. The deduced amino acid sequence predicts a protein of 1216 amino acids with a molecular mass of 133,769 Da. The deduced amino acid sequence together with amino-terminal amino acid sequencing indicate that there is a cleaved 21-amino acid signal sequence at the NH2-terminal portion of the molecule. There is a single hydrophobic region near the carboxyl terminus that has the characteristics of a membrane-spanning domain and a 36-amino acid cytoplasmic tail. We have found that the major form of this protein in intestinal epithelial cells has a molecular mass of 55-60 kDa, which is significantly smaller than the size predicted from the cDNA sequence, suggesting that the protein is synthesized as a large precursor and processed to the smaller form. The smaller form remains associated with the membrane, however, possibly through noncovalent association with the transmembrane portion of the molecule or with another membrane protein. The extracytoplasmic domain is cysteine-rich, with three cysteine-rich repeats that are similar to cysteine repeats present in several receptor proteins. However, there is no other significant similarity to other proteins in the GenBank. The cytoplasmic tail contains a possible internalization motif and several consensus motifs for serine/threonine kinases. Northern blot analysis suggests a single abundant message, and Southern blot analysis is consistent with a single gene and the absence of pseudogenes for this unique endosomal protein.