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Secretory Trafficking Signal Encoded in the Carboxyl-Terminal Region of the CGβ-Subunit

Department of Developmental Biology, Washington University, School of Medicine, St. Louis, Missouri 63110

Address all correspondence and requests for reprints to: Irving Boime, Department of Developmental Biology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8103, St. Louis, Missouri 63110. E-mail: iboime{at}wustl.edu.

Although the LHβ- and chorionic gonadotropin-β- (CGβ) subunits share a high degree of sequence identity (>85%) in the first 114 amino acids, there is considerable sequence divergence at their carboxy ends. The CGβ-subunit terminates with a unique carboxyl-terminal extension (115–145; carboxyl-terminal peptide), which contains four O-linked oligosaccharides, whereas the LHβ-subunit bears a hydrophobic heptapeptide (115–121) at its carboxy terminus. LH is released through the regulated pathway in the pituitary, whereas CG is secreted constitutively from the placenta. We previously demonstrated in rat somatotroph-derived GH₃ cells that the LH is associated primarily with a regulated routing, and although the majority of CG was released constitutively from the cells, there was a fraction that was segregated through the regulated pathway. Moreover, we showed that the LHβ heptapeptide is a determinant for the regulated secretion of LH. Given that the primary evolutionary change between LHβ and CGβ occurred at the carboxy terminus, these data suggested that the presence of the CGβ carboxyl-terminal peptide region is responsible for the constitutive secretion of CG. A CG114 mutant (CGT) was constructed and expressed in GH₃ cells. Steady-state labeling and pulse-chase experiments demonstrated that the CGT entered the regulated pathway resulting in over 4-fold increase in the intracellular pool. The secretagogue, forskolin, stimulated CGT release over 3-fold, which was accompanied by a parallel intracellular decrease, and only marginal stimulation of CG was seen. Immunofluorescence demonstrated a unique membrane pattern of staining for CGT compared with dispersed cytoplasmic puncta for CG. Stimulation with forskolin caused a significant reduction in the relative fluorescence of CGT cells compared with a minor reduction for CG. These data show that the CGT analog resembles LH in its intracellular trafficking, further supporting the hypothesis that determinants at the carboxyl-terminal end of the CGβ-subunit evolved from the LHβ-subunit primarily to overcome the slow release and intracellular storage of LH resulting in rapid secretion of CG from the placenta.