This Data Analysis Problem does not appear in the textbook.

Source:  Morello, J.-P., A. Salahpour, A. Laperriére, V. Bernier, M.-F. Arthus, M. Lonergan, U. Petäjä-Repo, S. Angers, D. Morin, D. G. Bichet, M. Bouvier. 2000. Pharmacological chaperones rescue cell-surface expression and function of misfolded V2 vasopressin receptor mutants. J. Clin. Investigation 105: 887–895. 

Corresponding chapter(s) in the textbook: Chapter 12 (and 15 and 17)

Review the following terms before working on the problem: vasopressin, peptide hormones, mutation, vasopressin receptor, nephrogenic diabetes insipidus, protein folding, deletion, chaperones, myc protein, [35S]methionine labeling, pulse-chase labeling, membranes, centrifugation, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, autoradiography

Experiment

Vasopressin (antidiuretic hormone) is a peptide hormone secreted by the posterior pituitary that promotes water resorption in the kidney. Mutations in the vasopressin receptor (V2R) gene may lead to congenital nephrogenic diabetes insipidus, a disease characterized by inability of the kidneys to concentrate urine. Some mutations lead to misfolded V2R protein, which is functional but cannot reach the surface of the kidney cell. One such mutation leads to the formation of V2R with a 3-amino acid deletion (del 62-64 V2R). Small molecules called pharmacological chaperones, which help with the proper folding of such mutant proteins, are being tested as a treatment for this genetic disease.

The effect of a chemical chaperone, a small, membrane-permeable molecule (SR121463A) on the metabolism of wild-type (wt) and del 62-64 V2R proteins was tested in this experiment. Wt and mutant proteins tagged with a myc-tag were expressed in human embryonic kidney cells. (The myc-tag is a short peptide from the myc transcription factor that can be efficiently recognized by a specific antibody.) Cultures were grown in the absence or presence of the chemical chaperone labeled with [35S]methionine for 30 minutes, and followed by a chase in nonradioactive medium for the time periods indicated in the figure. Crude membrane fractions were isolated by centrifugation of cell lysates, solubilized, and immunoprecipitated with an anti-myc antibody. The immunoprecipitated proteins were fractionated by SDS-polyacrylamide gel electrophoresis, and autoradiography was performed.

Figure

Source: Morello, J.-P., A. Salahpour, A. Laperriére, V. Bernier, M.-F. Arthus, M. Lonergan, U. Petäjä-Repo, S. Angers, D. Morin, D. G. Bichet, M. Bouvier. 2000. Pharmacological chaperones rescue cell-surface expression and function of misfolded V2 vasopressin receptor mutants. J. Clin. Investigation 105: 887–895.

Questions

1. Why was pulse-chase labeling used in this experiment?

2. What is the relationship between proteins p and m? Suggest an explanation for their different electrophoretic mobilities.

3. Interpret the difference between the behavior of wt and mutant V2R proteins.

4. How does SR121463A affect the metabolism of wt V2R?

5. How does SR121463A affect the metabolism of the mutant V2R?