| Dipeptidyl peptidase IV (DPPIV) is a serine exopeptidase that cleaves N-terminal dipeptides from polypeptides if the second residue in the polypeptide is a proline or an alanine. As a membrane glycoprotein, DPPIV is expressed on the cell surface of many cell types as homodimers. DPPIV has been under extensive studies for more than two decades from different aspects since its original identification in rat liver and kidney. Recent studies have shown DPPIV may be involved in many biological processes, including interaction with extracellular matrix, metabolism and uptake of proline-containing peptides in the small intestine and kidney proximal tubules, regulation of lymphocyte activation and proliferation, and maturation and degradation of peptide hormone and neuropeptides (for example, substance P). In a process of identification and characterization of membrane glycoproteins that are expressed on distinct membrane domains in polarized cells (for example, liver hepatocytes and epithelial cells of intestine and kidney tubules), we successfully produced antibodies to a membrane glycoprotein with Mr. of 110,000 (originally denoted gp110) that is expressed in the bile canaliculus of hepatocytes and in the apical membrane domain of epithelial cells of small intestine and kidney proximal tubules. We have identified that this gp110 is DPPIV. Using antibodies to DPPIV, we isolated the full-length cDNA for DPPIV for the first time. After subcloning the cDNA into an in vitro expression vector pGEM-4Z, RNA was produced in vitro with Sp6 polymerase. RNA produced was used to study the membrane topology of DPPIV using rabbit reticulocyte lysate translation and translocation systems. These studies revealed that DPPIV has a type II membrane orientation (a single transmembrane domain located in the N-terminal region with most of the polypeptide in the extracellular side and only N-terminal 6 amino acids in the cytoplasmic side). Further studies have demonstrated that N-terminal 34 amino acid sequence (defined as N-terminal signal/anchor sequence) is sufficient for the signal/anchor function in translocating the polypeptide across microsomal membranes and anchoring the polypeptide in the membrane. Site-directed mutagenesis studies of this N-terminal signal/anchor sequence have revealed some important structural features of this type of targeting sequence for the rough endoplasmic reticulum (RER) (RER-targeting sequence) as compared to other types of RER-targeting sequences. After subcloning the DPPIV cDNA into the inducible eukaryotic expression vector pMSG, we have obtained stable transfected Chinese hamster ovary (CHO) cells that produce enzymatically active DPPIV. By Western and Northern blotting analysis, we were able to conclude that expression of DPPIV in rat tissues is mainly regulated at the mRNA levels. |
