Cell culture, plasmids and transfections
Wild-type Neuro 2a (N2a wt) cells and Neuro 2a cells stably transfected with mouse Cpe (N2a-CPE, clone 17) were obtained from Dr Y P Loh (Bethesda, MD, USA). Cells were maintained in DMEM containing 10% FBS and stable transfectants were maintained in media containing 400 μg/ml G418. N2a wt cells were also stably transfected with the enzymatically inactive form of CPE, E300Q (Qian et al. 1999) using Lipofectamine 2000 (Invitrogen). Stable transfectants were selected in 800 μg/ml G418, pooled and maintained in 400 μg/ml G418. To examine the role of CPE in sorting proglucagon, N2a wt, N2a-CPE and N2a-E300Q cells were transfected with hamster pre-proglucagon (in pcDNA 3.1; a kind gift from Dr D F Steiner, Chicago, IL, USA). To examine the roles of other prohormone convertases, both N2a wt and N2a-CPE cells were transiently transfected with plasmids encoding PC1/3 and PC2 (kind gifts from Dr N G Seidah, Montreal, QC, Canada). To determine possible sorting signals, the sequence of proglucagon was mutated independently at three sites: at the processing site, R70K71; at the dibasic site within glucagon, R17R18; and at two leucines that were postulated to flank the helix structure within glucagon, L14 and L26. The processing site mutant, K71Q, was a kind gift from Dr D F Steiner (Chicago, IL, USA). We generated the mutation at the dibasic site, R18Q (forward primer sequence 5′-AAATACCTGGACTCCCGCCAAGCCCAAGAT-3′), and the double leucine-to-proline mutation was done in two steps; L14P was made using forward primer 1, 5′-TACAGCAAATACCCGGACTCCCGCCGAGCC-3′; and L26P was subsequently generated using forward primer 2, 5′-CAAGATTTTGTGCAGTGGCCGATGAACACC-3′. Bold sequences in primers indicate site of mutation. All site-directed mutagenesis reactions were carried out using the QuikChange II Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA) and results were confirmed by sequencing at the London Regional Genomics Facility, University of Western Ontario.
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