In addition to the well-characterized genomic mechanism of steroid action that uses
intracellular receptors, steroid hormones also signal through nongenomic processes
that use membrane receptors. A membrane receptor for corticosterone (CORT) has
been described in brains of the roughskin newt (Taricha granulosa). This receptor is
believed to be a G-protein coupled receptor because corticosterone binding is inhibited
by guanyl nucleotides and enhanced by Mg�����. The studies described in this thesis use
biochemical, pharmacological and molecular techniques to characterize the newt
neuronal membrane glucocorticoid receptor (mGR) in pursuit of its molecular
identification. The mGR was successfully solubilized from newt neuronal membranes
and conditions were defined that maintained corticosterone binding activity for further
study. The solubilized receptor was partially purified using standard chromatographic
techniques and an immobilized ligand affinity resin (CORT-Sepharose). These
chromatographic studies were combined with the use of a novel photoaffinity ligand
(azido-CORT) to biochemically characterize the mGR protein, finding that it is an
acidic glycoprotein with an apparent molecular weight of 63 kDa and an isoelectric
point of approximately 5.0. Pharmacological studies with mGR showed that a subset
of kappa opioid ligands displaced corticosterone from the receptor binding site with K[subscript i]
values in the nanomolar to low-micromolar range. The interaction of mGR with kappa
opioid ligands was specific because no mu-, delta-, or orphanin-specific opioid ligands
were effective at displacing corticosterone from the receptor. These data suggest that
the newt neuronal mGR may be a kappa-opioid like receptor. Finally, molecular
studies were used to clone a novel newt brain protein, neuronal axonal protein 22
(NAP-22), that was identified in a protein differential display strategy designed to
identify mGR. Studies with the cloned and expressed NAP-22 protein suggest that it is
not the mGR but, instead, may be a mGR-associated protein. These studies provided
new information about the biochemical and pharmacological properties of mGR, and
may have discovered a protein that is associated with the newt neuronal mGR. / Graduation date: 1999
| Identifer | oai:union.ndltd.org:ORGSU/oai:ir.library.oregonstate.edu:1957/33492 |
| Date | 06 May 1999 |
| Creators | Evans, Simon J. |
| Contributors | Moore, Frank L. |
| Source Sets | Oregon State University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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