Many neuroendocrine peptides and proteins undergo post- translational proteolytic processing to generate the bioactive forms. For example, both insulin and the insulin receptor require proteolytic processing. Until recently, only carboxypeptidase E (CPE) was associated with peptide/protein processing in neuroendocrine cells. Our finding that fat/fat mice lack CPE activity due to a point mutation but are still capable of limited peptide/protein processing suggests that another carboxypeptidase contributes to this processing step in vivo. A search for additional CPE-like enzymes in the secretory pathway of neuroendocrine cells led to our discovery of carboxypeptidase (CPD), an integral membrane protein that contains a 58 residue cytoplasmic C-terminal tail. A key issue in considering the physiological role of an enzyme is the intracellular location. We have found that CPD is primarily found in the trans Golgi network (TGN), cycles to the cell surface and returns to the TGN. The role of phosphorylation of CPD on trafficking will be the focus of the first specific aim. The cytoplasmic tail of CPD contains several consensus sites for phosphorylation. The function of these and other elements within the cytoplasmic tail of CPD will be studied in Aim 2 by analysis of deletion and point mutations. Since few TGN proteins have been studied in detail, the studies on CPD will be important for understanding the specific elements involved in trafficking of TGN proteins.
In Aim 3, we will investigate proteins that interact with the C- terminal region of CPD. Recently, several proteins that interact with the cytosolic region of CPD have been identified, an he overall goal is to determine the physiological significance of these interactions. In addition, we will also screen for other CPD-binding proteins using affinity chromatography and the yeast two-hybrid method. This will provide a better understanding of the proteins, either known or novel, that interact with CPD. These studies on CPD will advance our knowledge of protein trafficking in neuroendocrine cells.
| Kuliawat, Regina; Kalinina, Elena; Bock, Jason et al. (2004) Syntaxin-6 SNARE involvement in secretory and endocytic pathways of cultured pancreatic beta-cells. Mol Biol Cell 15:1690-701 |
| Kalinina, Elena V; Fricker, Lloyd D (2003) Palmitoylation of carboxypeptidase D. Implications for intracellular trafficking. J Biol Chem 278:9244-9 |
| Kalinina, Elena; Varlamov, Oleg; Fricker, Lloyd D (2002) Analysis of the carboxypeptidase D cytoplasmic domain: Implications in intracellular trafficking. J Cell Biochem 85:101-11 |
