Design of first and second-generation DHFR DD and GDNF fusion proteins. Diagram of wild-type GDNF protein showing the signal peptide, precursor protein, furin cleavage site (F), and mature peptide. First-generation DD designs: N-terminal fusion of Y100I DD mutant to GDNF after signal peptide (DD-G) and C-terminal fusion of N18T DD mutant to GDNF (G-DD). Second-generation DD designs: N-terminal fusion of Y100I DD to GDNF 9 residues after signal peptide (SP9-DD-G) and C-terminal fusion of N18T DD mutant to GDNF after an additional furin cleavage site (G-F-DD). DD, destabilizing domains; DHFR, dihydrofolate reductase; GDNF, glial cell line–derived neurotrophic factor.