Maturation of Receptor Proteins in Eukaryotic Expression Systems

Abstract

Transient and stable expression in eukaryotic cells is commonly used to examine receptor function. Characterization of the V2 vasopressin receptor synthesized in transiently transfected cells revealed the presence of large quantities of immature protein and a small fraction of fully mature protein. The immature protein was characterized by its sensitivity to endoglycosidase H treatment, abnormal migration in SDS PAGE, and a tendency to form aggregates. Prevention of protein glycosylation by mutagenesis increased the fraction of mature protein produced, but did not eliminate the need for the maturation step. On the other hand, stably transfected cells produce almost exclusively mature receptor protein with a t_1/2 of 6 h, while the immature form has a t_1/2 of 20 min. In the absence of N-linked glycosylation the t_1/2 of the mature V2 receptor in stably transfected cells was reduced to 4.5 h. In transient expression experiments the immature receptor proteins exhibited a prolonged t_1/2 of about 8 h. Comparison of the half life of the immature form of the wild type and the R137H mutant V2 receptor did not reveal differences despite the lower amounts of mutant mature receptor detected by binding.