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Agonist-induced desensitization of adenylyl cyclase in Y1 adrenocortical tumor cells
1Banting and Best Department of Medical Research, University of Toronto, Ontario, Canada.
Abstract:
Y1 adrenocortical tumor cells (Y1DS) and Y1 mutants resistant to ACTH-induced desensitization of adenylyl cyclase (Y1DR) were transfected with a gene encoding the mouse beta 2-adrenergic receptor (beta 2-AR). Transfectants expressed beta 2-ARs that were able to stimulate adenylyl cyclase activity and steroid biosynthesis. These transfectants were used to explore the basis for the DR mutation in Y1 cells. We demonstrate that beta-adrenergic agonists desensitize the adenylyl cyclase system in transfected Y1DS cells whereas transfected Y1DR cells are resistant to desensitization by beta-adrenergic agonists. The fate of the beta 2-ARs during desensitization was evaluated by photoaffinity labelling with [125I]iodocyanopindolol diazerine. Desensitization of Y1DS transfectants was accompanied by a modest loss in receptor density that was insufficient to account for the complete loss of responsiveness to beta-adrenergic agonists. The extent of receptor loss induced by beta-adrenergic agonists in Y1DR transfectants exceeded that in the Y1DS transfectants indicating that the mutation which protects Y1DR cells from agonist-induced desensitization is prior to receptor down-regulation in the desensitization pathway. From these results we infer that ACTH and isoproterenol desensitize adenylyl cyclase by a common pathway and that receptor loss is not a major component of the desensitization process in these cells.
Insights
Y1DR cells, resistant to beta-adrenergic agonist desensitization, show that receptor loss is not the primary cause of adenylyl cyclase desensitization. The protective mutation occurs before receptor down-regulation.
Area of Science:
- Endocrinology
- Molecular Biology
- Cell Signaling
Background:
- Adrenocortical tumor cells (Y1DS) and mutants resistant to ACTH desensitization (Y1DR) are used to study adenylyl cyclase regulation.
- The beta 2-adrenergic receptor (beta 2-AR) mediates responses to beta-adrenergic agonists.
Purpose of the Study:
- To investigate the molecular basis of resistance to beta-adrenergic agonist-induced desensitization in Y1DR cells.
- To determine the role of receptor down-regulation in the desensitization pathway of adenylyl cyclase.
Main Methods:
- Transfection of Y1DS and Y1DR cells with the mouse beta 2-AR gene.
- Stimulation of adenylyl cyclase activity and steroid biosynthesis.
- Photoaffinity labeling with [125I]iodocyanopindolol diazerine to assess receptor density during desensitization.
Main Results:
- Transfected Y1DS cells showed desensitization to beta-adrenergic agonists, while Y1DR cells were resistant.
- Desensitization in Y1DS cells involved a modest, insufficient loss of beta 2-AR density.
- Y1DR cells exhibited greater beta 2-AR loss than Y1DS cells, indicating the protective mutation precedes receptor down-regulation.
Conclusions:
- The mutation conferring resistance in Y1DR cells acts upstream of receptor down-regulation in the desensitization pathway.
- Adrenocorticotropic hormone (ACTH) and beta-adrenergic agonists likely utilize a common pathway to desensitize adenylyl cyclase.
- Receptor loss is not the principal mechanism for beta-adrenergic agonist-induced adenylyl cyclase desensitization in these cells.