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

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, K141R142 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 R142/A142pomc construct or the K141/A141pomc construct completely blocked cleavage at this site and yielded a 6K immunoreactive product that had the ACTH(1–13)NH2 sequence at the C-terminal end of the fusion protein. However, substitution of an alanine residue at R137, Q138, E139, and N140 had no effect on cleavage at the K141R142 cleavage site. Collectively, these results indicate that secondary structure N-terminal to the K141R142 does not appear to influence cleavage at this site. However, both K141 and R142 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.

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► Mouse α-TC1.9 cells express proprotein convertase 2 (PC2) but do not express proprotein convertase 1/3 (PC1/3).
► α-TC1.9 cells are capable of performing the reaction required for the posttranslational processing of POMC.
► Both K141 and R142 must be present at the endoproteolytic cleavage site N-terminal to the ACTH in POMC.