Glucocorticoid receptor (GR) proteins that enter the nucleus after binding hormone ligand in v-mos-transformed cells, are rapidly depleted from that compartment and subsequently redistributed to the cytoplasm. GRs that have acquired this property (i.e. inefficient nuclear re-entry) in hormone treated v-mos-transformed cells can be classified as desensitized, since they cannot re-enter the after secondary hormone treatments. Nuclear entry of ligand-bound GRs is not by itself sufficient to desensitize GRs in v- mos-transformed cells, as antagonist-bound receptors are efficiently retained within the nucleus and not redistributed to the cytoplasm. Thus, the action of v-mos oncoproteins appear to be restricted to GR proteins that are recycling between the nuclear and cytoplasmic compartment. The long term goals of this proposal are to uncover the mechanisms responsible for this oncoprotein-mediated alteration in the subcellular compartmentalization of this signal transducing protein. In particular, we hope to reveal the molecular and biochemical basis for the apparent block in GR recycling that occurs in v-mos-transformed cells. Since GR dephosphorylation is a critical component of proposed receptor recycling pathways, we will examine the phosphorylation state of desensitized receptors by 2-dimensional mapping of phosphotryptic peptides generated from 32P-labeled immunopurified receptors. Qualitative and/or quantitative differences in the phosphorylation profile of GRs that are unable to recycle may be revealed. The effects of okadaic acid, a specific protein phosphatase type 1 and 2A inhibitor, on recycling and phosphorylation of Grs in vivo will be examined by indirect immunofluorescence and 2- dimensional tryptic mapping, respectively. Purified protein phosphatases types 1 and 2A will be used in vitro to dephosphorylate in vivo 32P-labeled immunopurified GRs and specific sites of PP1 and PP2A dephosphorylation identified by 2-dimensional tryptic mapping. In this way dephosphorylation of specific phosphoserine residues within the GR that are associated with a recycling pathway may be identified. Immunoprecipitations and glycerol gradient sedimentation analyses will be used to in order to determine whether other cellular factors are associated with desensitized receptors. We will also ascertain whether, nuclear- and DNA-binding activity of desensitized receptors can be recovered in vitro through dissociation of putative desensitized receptor-multiprotein complexes by high salt, elevated temperatures, and/or dephosphorylation with purified PP1 and PP2A. Finally, we will use a novel genetic selection scheme to screen for potential GR, v-mos oncoprotein phosphatase, and cellular mutations that lead to the reversal of receptor desensitization. Through these studies we hope to gain a better understanding of how regulated modification of a signal transducing protein influences its subcellular compartmentalization and association with ancillary factors that modify its function.
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