Drugs that influence tubulin polymerization modulate thyrotropin-releasing hormone receptor number in AtT-20 cells
R Ravindra1, P J McIlroy, S A Patel
1Veterans Affairs Medical Center, North Chicago, IL 60064, USA.
Abstract:
In order to examine the role of cytoskeleton in modulating the cell surface receptors, AtT-20 cells (stably expressing thyrotropin-releasing hormone receptors) were incubated with drugs that are known to modify the tubulin-microtubule system. The binding of [3H]methyl thyrotropin-releasing hormone ([3H]mTRH) to intact cells increased as a function of time, and was linear from 1.25 x 10(6) to 6.25 x 10(6) cells/ml. Cells incubated with colchicine, vinblastine, and taxol for 16 hr were harvested and the cell concentration was determined using a haemocytometer. Because the drugs inhibited the cell proliferation at 100 nM, it was decided to examine the effect of 100 nM of each of the three drugs on the ability of [3H]mTRH to bind cell surface receptors. Cells were incubated with the drugs for 16 hr at 37 degrees. After the incubation, cells (5 x 10(6) cells/ml) from each group were assayed for [3H]mTRH binding. Colchicine, vinblastine, and taxol stimulated [3H]mTRH binding by up to 27, 27, and 21%, respectively, without altering the Ka of the ligand to the receptor. These results suggest that perturbation of cytosolic microtubules leads to a reorganization of the spatial location of hormone receptors.
Insights
Cytoskeleton modulation affects cell surface receptors. Perturbing microtubules with colchicine, vinblastine, and taxol increased [3H]methyl thyrotropin-releasing hormone binding to AtT-20 cells, suggesting receptor reorganization.
Area of Science:
- Cell Biology
- Biochemistry
- Pharmacology
Background:
- The cytoskeleton plays a crucial role in cellular processes, including the regulation of cell surface receptors.
- Thyrotropin-releasing hormone (TRH) receptors are important targets in various physiological pathways.
- Understanding how cellular structures influence receptor function is key to deciphering cell signaling.
Purpose of the Study:
- To investigate the role of the cytoskeleton, specifically microtubules, in modulating the function of cell surface receptors.
- To determine if drugs affecting the microtubule system alter the binding of [3H]methyl thyrotropin-releasing hormone ([3H]mTRH) to its receptors on AtT-20 cells.
Main Methods:
- AtT-20 cells, stably expressing TRH receptors, were treated with microtubule-disrupting agents (colchicine, vinblastine) and a microtubule-stabilizing agent (taxol) at 100 nM for 16 hours.
- Cell proliferation was assessed to determine drug efficacy.
- The binding affinity of [3H]mTRH to cell surface receptors was measured using radioligand binding assays.
Main Results:
- Colchicine, vinblastine, and taxol treatment significantly increased [3H]mTRH binding to AtT-20 cells by up to 27%, 27%, and 21%, respectively.
- These increases in binding occurred without altering the dissociation constant (Ka), indicating no change in ligand-receptor affinity.
- Drug treatments were optimized at 100 nM, a concentration that inhibited cell proliferation.
Conclusions:
- Perturbation of cytosolic microtubules influences the spatial organization and accessibility of hormone receptors on the cell surface.
- The cytoskeleton plays a dynamic role in regulating the cell surface expression and availability of TRH receptors.
- These findings highlight a link between microtubule dynamics and G protein-coupled receptor regulation.
Related Concept Videos
01:15Drugs that Stabilize Microtubules
01:27Transducer Mechanism: Enzyme-Linked Receptors
Major types that are helpful drug targets include:
01:26Neurochemical Transmission: Sites of Drug Action
01:21Drugs Affecting Neurotransmitter Release or Uptake
01:29Drugs Affecting Neurotransmitter Synthesis
01:11Pharmacogenetics of Drug Targets: β₂-Adrenergic Receptors, Apo E, Thymidylate Synthase