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Characteristics of somatostatin desensitization in the pituitary tumor cell line AtT-20
N Mahy1, M Woolkalis, D Manning
1Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia.
Abstract:
The molecular mechanisms of somatostatin (SRIF) desensitization were investigated in the anterior pituitary tumor cell line AtT-20. Previous studies have shown that pretreatment of AtT-20 cells with SRIF analogs desensitizes the cells to SRIF inhibition of hormone release, cyclic AMP formation and calcium influx. This desensitization may involve a change in the properties of the SRIF receptors. Pretreatment of AtT-20 cells with Trp8-SRIF reduced the binding of the SRIF analog [125I]CGP 23996 (des-Alal, Gly2-[desamino-Cys3, Tyr11]-3, 14-dicarbasomatostatin) to AtT-20 cell membranes. The loss of [125I]CGP 23996 binding was dependent on the time of Trp8-SRIF treatment and was reversible. The ability of GTP analogs to inhibit [125I]CGP 23996 binding was reduced after Trp8-SRIF treatment, suggesting that the SRIF receptor and the inhibitory G proteins become uncoupled during desensitization. This is indicated further by the decrease in SRIF stimulation of GTPase activity and SRIF inhibition of forskolin-stimulated adenylyl cyclase activity in desensitized membranes. The reduction and recovery of SRIF inhibition of adenylyl cyclase activity after Trp8-SRIF pretreatment has a similar time course as the changes in [125I]CGP 23996 binding. GTP inhibition of forskolin-stimulated adenylyl cyclase activity is also reduced in SRIF-desensitized membranes. The loss of the GTP effect occurs rapidly and does not fully recover after Trp8-SRIF pretreatment. The levels of ADP-ribosylation of inhibitory GTP binding protein, the relative quantity of the alpha subunits of the inhibitory G proteins and their electrophoretic mobility after 2-dimensional gel electrophoretic analysis, are not altered in SRIF-desensitized membranes.(ABSTRACT TRUNCATED AT 250 WORDS)
Insights
Somatostatin (SRIF) desensitization in pituitary cells involves SRIF receptor uncoupling from inhibitory G proteins. This process affects hormone release and cyclic AMP formation, with molecular changes observed in receptor binding and GTPase activity.
Area of Science:
- Endocrinology
- Molecular Cell Biology
- G protein-coupled receptor signaling
Background:
- Somatostatin (SRIF) analogs desensitize anterior pituitary cells to SRIF's inhibitory effects on hormone release, cyclic AMP (cAMP) formation, and calcium influx.
- Desensitization may involve alterations in SRIF receptor properties and downstream signaling pathways.
Purpose of the Study:
- To investigate the molecular mechanisms underlying somatostatin (SRIF) desensitization in the AtT-20 anterior pituitary tumor cell line.
- To elucidate the role of SRIF receptors and inhibitory G proteins in the desensitization process.
Main Methods:
- Radioligand binding assays using [125I]CGP 23996 to measure SRIF receptor occupancy.
- Assessment of GTP analog inhibition of radioligand binding.
- Measurement of SRIF-stimulated GTPase activity.
- Evaluation of SRIF and GTP inhibition of forskolin-stimulated adenylyl cyclase activity.
- Analysis of inhibitory G protein subunit levels and modifications via ADP-ribosylation and 2D gel electrophoresis.
Main Results:
- Trp8-SRIF pretreatment reduced [125I]CGP 23996 binding to AtT-20 cell membranes in a time-dependent and reversible manner.
- GTP analog inhibition of radioligand binding, SRIF stimulation of GTPase activity, and SRIF inhibition of adenylyl cyclase activity were diminished in desensitized membranes, suggesting receptor-G protein uncoupling.
- GTP inhibition of adenylyl cyclase was also reduced, indicating a broader impact on G protein signaling.
- Levels and modifications of inhibitory G protein subunits remained unaltered.
Conclusions:
- SRIF desensitization in AtT-20 cells is associated with the uncoupling of SRIF receptors from inhibitory G proteins.
- This uncoupling affects multiple downstream signaling events, including adenylyl cyclase activity.
- The molecular basis of desensitization does not appear to involve changes in the quantity or modification of inhibitory G protein subunits.