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Regulation of adenylyl cyclase activity by beta-adrenergic agonists in a desensitization-resistant mutant cell line
1Banting and Best Department of Medical Research Department of Pharmacology, University of Toronto, Ontario, Canada.
Abstract:
Mutant clones resistant to ACTH-induced desensitization of adenylyl cyclase (Y1DR) were previously isolated from the Y1 mouse adrenocortical tumor cell line. In this study, both parental Y1 cells (Y1DS) and a Y1DR mutant were transfected with a gene encoding the mouse beta 2-adrenergic receptor, and transfectants isolated from both Y1DS and Y1DR cells were shown to express beta 2-adrenergic receptors. These transfectants responded to the beta-adrenergic agonist isoproterenol with increases in adenylyl cyclase activity and steroidogenesis and changes in cell shape. The transfectants were analyzed to determine whether the Y1DR mutation was specific for ACTH-induced desensitization of adenylyl cyclase or also affected desensitization of adenylyl cyclase via the beta 2-adrenergic receptor. Treatment of intact Y1DS transfectants with isoproterenol caused a rapid desensitization of the adenylyl cyclase system to further stimulation by the beta-adrenergic agonist. Treatment of intact cells with isoproterenol did not affect ACTH-stimulated adenylyl cyclase activity, indicating that desensitization was agonist specific or homologous. Y1DR transfectants were resistant to the desensitizing effects of isoproterenol in intact cells as well as in cell homogenates. These results indicate that the mutation in Y1DR transfectants affects a component that is common to the pathways of isoproterenol-induced desensitization and ACTH-induced desensitization of adenylyl cyclase. As determined using the hydrophilic beta-receptor antagonist CGP-12177, isoproterenol caused a rapid sequestration of cell surface receptors in both Y1DS and Y1DR transfectants. From these results we infer that the DR phenotype does not arise from mutations affecting receptor sequestration and that receptor number does not limit the response to isoproterenol in these transfectants.
Insights
Mutant mouse cells resistant to ACTH desensitization also resist beta-adrenergic receptor desensitization. This suggests a common mechanism underlies desensitization for both ACTH and beta-adrenergic receptors in adenylyl cyclase regulation.
Area of Science:
- Cellular Biology
- Endocrinology
- Molecular Pharmacology
Background:
- Mutant clones (Y1DR) resistant to adrenocorticotropic hormone (ACTH)-induced desensitization of adenylyl cyclase were previously identified in Y1 mouse adrenocortical tumor cells.
- Understanding the molecular basis of receptor desensitization is crucial for comprehending cellular signaling regulation.
Purpose of the Study:
- To investigate whether the Y1DR mutation affects desensitization of adenylyl cyclase induced by beta 2-adrenergic receptors.
- To determine if the mutation impacts a common pathway for ACTH and beta-adrenergic receptor desensitization.
Main Methods:
- Transfection of parental Y1 cells (Y1DS) and Y1DR mutants with a gene encoding the mouse beta 2-adrenergic receptor.
- Stimulation of adenylyl cyclase activity and steroidogenesis using the beta-adrenergic agonist isoproterenol.
- Analysis of receptor sequestration using a hydrophilic beta-receptor antagonist (CGP-12177).
Main Results:
- Y1DS and Y1DR transfectants expressed functional beta 2-adrenergic receptors, responding to isoproterenol with increased adenylyl cyclase activity and steroidogenesis.
- Isoproterenol induced homologous desensitization of adenylyl cyclase in Y1DS transfectants, which was specific and did not affect ACTH signaling.
- Y1DR transfectants exhibited resistance to isoproterenol-induced desensitization in both intact cells and cell homogenates, indicating a common desensitization component.
Conclusions:
- The Y1DR mutation affects a component common to both isoproterenol- and ACTH-induced adenylyl cyclase desensitization pathways.
- Receptor sequestration is not the cause of the Y1DR phenotype, and receptor number does not limit the response to isoproterenol.