Related Experiment Videos
Activation and partial proteolysis of variant glucocorticoid receptors, studied by two-phase partitioning
Abstract:
In cultured lines of mouse lymphoma cells, resistant to glucocorticoids is frequently associated with the occurrence of glucocorticoid receptors with an abnormally low affinity (nt-) or an abnormally high affinity (nti) for nuclei and DNA. We have investigated whether the abnormal affinities for DNA are correlated with alterations in charge and surface properties of the receptors, that would be revealed through the partition coefficient in aqueous dextran/poly(ethylene glycol) two-phase systems. We have found that none of the receptor variants is defective in the activation step per se, and that only the nti receptors are abnormal in partition properties. Partial proteolysis of wild-type and nt- receptors with alpha-chymotrypsin produces forms which are indistinguishable from the nti receptors with respect to partition coefficients. Upon alpha-chymotrypsin treatment the wild-type receptors attain DNA-binding properties identical to those of the nti receptors, while the nt- receptors, in spite of some increase in DNA affinity, still bind less firmly to DNA than the alpha-chymotrypsin-treated wild-type receptors. alpha-Chymotrypsin treatment of the various receptor types also produces an increase in the binding to dextran sulphate, but the dextran sulphate affinity is higher and varies less between different receptor types than the DNA affinity. Trypsin-treated receptors were found to be devoid of affinity for DNA and dextran sulphate.
Insights
Glucocorticoid receptor variants with altered DNA affinity were studied. Only high-affinity variants showed changes in surface properties, suggesting altered receptor charge affects DNA binding in resistant lymphoma cells.
Area of Science:
- Molecular Biology
- Cell Biology
- Biochemistry
Background:
- Glucocorticoid resistance in mouse lymphoma cells is often linked to altered glucocorticoid receptor (GR) affinity for nuclei and DNA.
- Receptor variants include those with abnormally low (nt-) or high (nti) affinity for DNA.
Purpose of the Study:
- To investigate if abnormal DNA affinities of GR variants correlate with changes in receptor charge and surface properties.
- To assess these properties using partition coefficients in aqueous two-phase systems.
Main Methods:
- Utilized cultured mouse lymphoma cell lines with glucocorticoid resistance.
- Employed aqueous dextran/poly(ethylene glycol) two-phase systems to determine receptor partition coefficients.
- Performed partial proteolysis using alpha-chymotrypsin and trypsin on wild-type and variant receptors.
- Assessed DNA and dextran sulfate binding affinities of treated and untreated receptors.
Main Results:
- No GR variant showed defects in the activation step.
- Only nti (high-affinity) receptors exhibited abnormal partition properties, indicating altered surface characteristics.
- Partial proteolysis of wild-type and nt- receptors with alpha-chymotrypsin yielded forms with partition properties similar to nti receptors.
- Alpha-chymotrypsin treatment normalized DNA-binding properties of wild-type receptors to match nti receptors, while nt- receptors remained less bound.
- Proteolysis increased dextran sulfate binding, but this varied less between receptor types than DNA affinity.
- Trypsin treatment abolished DNA and dextran sulfate binding.
Conclusions:
- Abnormal DNA binding affinities in glucocorticoid-resistant lymphoma cells are associated with altered receptor surface properties, particularly in high-affinity variants.
- Proteolytic modification can mimic the altered surface and DNA-binding characteristics of nti receptors.
- Receptor charge and surface properties, as revealed by partition coefficients, play a role in DNA interaction and may contribute to glucocorticoid resistance mechanisms.