α-TC1.9 cells – A model system for analyzing the endoproteolytic processing of POMC

Abstract

The mouse α-TC1.9 endocrine cell line was used to analyze the amino acid requirements for endoproteolytic processing at the paired basic amino acid cleavage site, K 141R 142 that is N-terminal to the ACTH sequence in the POMC proprotein of the anuran amphibian, Silurana tropicalis. Real-Time PCR analysis of non-transfected α-TC1.9 cells indicated that these cells endogenously express the pc2 (proprotein convertase 2) gene, but do not express the pc1/ 3 (proprotein convertase 1/3) gene or the pomc gene. In addition, immunocytochemical analysis and RIA analysis of non-transfected α-TC1.9 cells did not detect the presence of POMC-related products in these cells. For this study the open reading frame of a S. tropicalis POMC cDNA (wild-type) was placed into an expression vector and transiently transfected into α-TC1.9 cells. Two days after transfection the steady-state levels of α-MSH-related and β-endorphin-related end-products were nearly the same as the steady-state levels of these POMC-related end-products in extracts of the S. tropicalis intermediate pituitary. Transient transfection of either the R 142/A 142 pomc construct or the K 141/A 141 pomc construct completely blocked cleavage at this site and yielded a 6K immunoreactive product that had the ACTH(1–13)NH 2 sequence at the C-terminal end of the fusion protein. However, substitution of an alanine residue at R 137, Q 138, E 139, and N 140 had no effect on cleavage at the K 141R 142 cleavage site. Collectively, these results indicate that secondary structure N-terminal to the K 141R 142 does not appear to influence cleavage at this site. However, both K 141 and R 142 are required for the integrity of this cleavage site. Finally, this study indicates that α-TC1.9 cells should be useful for studying the amino acid requirements for the other endoproteolytic cleavage sites in the S. tropicalis POMC proprotein.