Proteins that import into the nucleus pass through the nuclear pore complex (NPC), which is a large structure of approximately 10/6 megadaltons composed of at least 100 different proteins. Typically, short stretches of basic amino acids serve as nuclear localization signal sequences (NLSs) to target karyophilic proteins to the NPC. The NLS of the glucocorticoid receptor (GR), NL1, is comprised of multiple, interacting segments of basic amino acids and is interdigitated within the receptor's DNA-binding domain. A number of soluble factors have been identified whose function in distinct steps in nuclear import have been established from in vitro assays. Included in this group is the cytoplasmic 70 kDa heat shock protein, hsp70, which has been shown to be required for the in vivo and in vitro nuclear import of various karyophilic proteins, such as the simian virus 40 large tumor antigen (SV40 TAg). However, GRs, which utilize either a hormone-dependent or hormone-independent pathway, do not require hsp70 for nuclear import in vitro. Thus, one of the specific aims of this proposal is to determine the basis for differential hsp70 requirements of GR versus SV40 TAg nuclear import. The questions that will be addressed by these studies include: 1. Does GR NL1 removed from its natural context maintain its hsp70-independent activity? 2. Does denaturation of the NLS alter its requirement for hsp70? 3. Is hsp70 required during translation for appropriate folding of the GR NL1? 4. Are GR NL1 and TAg NLS distinguished by their relative affinities for hsp70 or their activation of hsp70 ATPase activity? 5. Are GR and TAg distinguished by interactions made with NPC components during hsp70-independent versus hsp70-dependent nuclear import? While there is little disagreement concerning the role of ATP in nuclear import, the role of ATP in nuclear export remains an unresolved issue, not only for steroid receptors, but for other shuttling proteins as well. In ATP-depleted cells, GRs remain localized within the nucleus and, in fact, are associated with the nuclear matrix. The restoration of cellular ATP levels leads to the release of matrix-bound GR in vivo. We hypothesize that molecular chaperones, which utilize the energy of nucleotide binding, release or hydrolysis, may facilitate the delivery of steroid receptors to, or their release from, the nuclear matrix. Thus, in Specific Aim 2 we will use a combination of biochemical, molecular, and genetic approaches to uncover the mechanisms responsible for dynamic interactions of GR with the nuclear matrix, and identify the proteins that participate in this subnuclear trafficking of the receptor. The specific questions which will be addressed in this aim include: 1. What factors are required to release GRs from the nuclear matrix in vitro? 2. Which domain within the GR is responsible for reversible nuclear matrix binding? 3. Which proteins interact with domains of the rat GR that are required for binding to, or release from, the nuclear matrix?
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