Abnormal intracellular localization and transport of a PTH-R mutant protein

Objectives: A replacement of proline with leucine at position 132 of the receptor for parathyroid hormone (PTH)/parathyroid hormone-related peptide (PTHrP), i.e., PTH-R, has been discovered in human Blomstrand's lethal chondrodysplasia. As skeletal deformities in this type of chondrodysplasia appear to relate to compromised receptor binding to its ligand, we examined the possibility that rat PTH-R carrying P132L mutation (PTH-R^P132L) would result in abnormal intracellular localization and transport. Methods: Osteoblastic MC3T3-E1 cells were transfected with pAlter-Max expression vectors containing cDNAs encoding either wild-type PTH-R or mutant PTH-R^P132L. Using these cells, the expression levels of genes related to skeletal development were examined by semi-quantitative RT-PCR and the cell homogenates from these cells were used for cyclic AMP accumulation and Western blot analyses. The cellular localization of these receptor-proteins was examined by immunofluorecence and immunoelectron microscopy. Results: The cells expressing the wild-type PTH-R produced a receptor protein with a molecular mass of 66.3 kDa, which localized its immunoreactivity mainly on the cell surfaces. In contrast, the PTH-R^P132L was hardly detected on the cell surfaces, but accumulated within the rough-surfaced endoplasmic reticulum. Consistent with this localization, the cells expressing the mutant receptor failed to generate cyclic AMP in response to PTH. Furthermore, a remarkably weaker intensity of the 66.3 kDa band compared with the wild-type counterpart suggests that PTH-R^P132L is prone to degradation in the transfected cells. Conclusion: These findings indicate that defective transport of PTH-R^P132L to the cell surface would be the molecular basis for Blomstrand's chondrodysplasia.