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Down-regulation of gonadotropin receptors in a murine Leydig tumor cell line
Abstract:
The murine Leydig tumor cell line, MLTC-1, has specific cell surface receptors for human chorionic gonadotropin (hCG), which are coupled to adenylate cyclase. When the cells were exposed to hCG, there was a loss of these receptors (down-regulation), which was both dose- and time-dependent. Down-regulation was inhibited by lowering the temperature, removing bound hormone or blocking protein synthesis with cycloheximide. Down-regulation was found to be a biphasic event. The initial phase was dependent upon the binding of hormone and had a half-time of approximately 3 h. The second phase occurred around 8 h after exposing the cells to the hormone and was apparently independent of bound hCG. It was related to increases in cyclic AMP since it could be mimicked by incubating the cells with choleragen or dibutyryl cyclic AMP and isobutylmethylxanthine. The reappearance of hormone receptors began approximately 24-32 h after the initial exposure to hCG and was complete within 48 h. Adenylate cyclase activity in membranes from control and down-regulated cells responded equally well to Mn2+ and NaF indicating that neither the catalytic or regulatory component (G/F) of the adenylate cyclase system had been lost during downregulation. Cholate extracts of control and down-regulated cells also were equally effective at reconstituting isoproterenol-stimulated adenylate cyclase activity in S49 cyc-membranes (which lack a functional G/F). Thus, down-regulation did not impair the ability of G/F to couple receptors to the catalytic component of adenylate cyclase.
Insights
Human chorionic gonadotropin (hCG) down-regulates its own receptors on MLTC-1 cells in a biphasic manner. Receptor reappearance occurs within 48 hours, with no impairment to adenylate cyclase function.
Area of Science:
- Endocrinology
- Cell Biology
- Molecular Pharmacology
Background:
- MLTC-1 cells possess cell surface receptors for human chorionic gonadotropin (hCG) linked to adenylate cyclase.
- Exposure to hCG triggers a dose- and time-dependent loss of these receptors, a process termed down-regulation.
Purpose of the Study:
- To investigate the mechanisms and kinetics of hCG receptor down-regulation in MLTC-1 cells.
- To determine the impact of down-regulation on the adenylate cyclase system's components and function.
Main Methods:
- MLTC-1 cells were treated with varying doses and times of hCG exposure.
- Experiments involved temperature manipulation, hormone removal, protein synthesis inhibition (cycloheximide), and measurement of cyclic AMP.
- Adenylate cyclase activity was assessed in cell membranes and through reconstitution assays with S49 cyc-membranes.
Main Results:
- Down-regulation occurred in two phases: an initial hormone-binding dependent phase (t½ ≈ 3 h) and a later phase (≈ 8 h) related to cyclic AMP increases.
- Receptor levels recovered between 24-32 hours and were complete by 48 hours post-hCG exposure.
- Adenylate cyclase catalytic and regulatory components (G/F) remained functional, as evidenced by Mn2+/NaF stimulation and reconstitution assays.
Conclusions:
- hCG receptor down-regulation in MLTC-1 cells is a complex, biphasic process.
- The down-regulation mechanism does not compromise the integrity or coupling ability of the adenylate cyclase system.
- Receptor resensitization occurs within 48 hours, suggesting a dynamic regulation of the hCG receptor system.