Cytoplasm

Dimerization strengthened

Biological response

Major groove Transcriptional complex

Nucleus

Major groove Transcriptional complex

Translation VWiRNAs j< A

FIGURE 10-14 Action of the glucocorticoid receptor on a target cell.

is unavailable at the surface of the unactivated form of the larger receptor complex (Figure 10-14). The properties listed here pertain to the activation or DNA-binding (or nuclear binding) form of the steroid-receptor complex.

A large number of in vitro experiments describe the conditions under which the activation-transformation process occurs (see Figure 10-14). The steroid-receptor complex undergoes a further conformational change that enables it to translocate to the cell nucleus, where it brings about an increase in the rate of synthesis of certain mRNAs coding for the translation of specific enzymes in the cytoplasm. The activated-transformed state of the steroid-receptor complex can be determined by its ability to bind to DNA. Another view of activation, which is not incompatible with the conformational change idea, is that a low-molecular-weight inhibitor is bound to the receptor in the unactivated form, thereby holding receptor subunits together in a way that obscures its DNA-binding sites (Figure 1014). Upon activation, this inhibitor dissociates from the surface of the receptor, exposing the Zn2+ fingers of the DNA-binding domain after other proteins have dissociated. Considerable work has now been accomplished on the mode of entry of activated-transformed cytoplasmic receptors into the nucleus. The receptors apparently are phosphorylated and associate with specific proteins (nucleoporins) that ferry the receptors through the nucleopore. In addition, hsp 70 may bind to this transportation complex and provide the energy required for transport via its ATPase activity.

The receptor for nuclear localization signals is a sequence of seven or eight amino acids that are presumed to be the binding site for nucleoporins or other specific proteins that affect attachment to the nucleo-pore and subsequent transport. Nucleoporin proteins are found in both the cytoplasmic and nucleoplasmic compartments, suggesting their carrier role in the process of translocation. A nuclear translocation signal was first demonstrated for the SV40 T antigen and has the sequence 126-PKKKRKV, indicating the importance of basic amino acids. Nuclear localization sequences for various steroid receptors are as follows: human glucocorticoid receptor, 491-RKTKKKIK; human mineralocorticoid receptor, 673-RKSKKLGK; human androgen receptor, 628-RKLKKLGN; human progesterone receptor, 637-RKFKKFNK; human estrogen receptor, 256-RKDRRGGR; human vitamin D3 receptor, 102-RKREMILL; and human thyroid hormone receptor, 179-KRLAKRKL. The second amino acid, lysine, is required and is present in this position in all receptors cited. It is likely that molecules recognizing the nuclear localization sequence have an acidic amino acid sequence. In support of this idea, it has been shown that antibodies to the sequence DDDED, which could be considered the acidic counterpart of the SV40 T antigen nuclear localization sequence KKKRK, blocked all of the nuclear-targeted protein receptors. It is likely that a cytoplasmic receptor binds to this sequence and assists in transport through the nucleo-pore. It is also possible that the "receptor" is dissociated from the protein being transported to the nucleoplasmic side, generating concentrations of molecules on both sides of the nucleopore. In addition, as mentioned before, the transport process may involve the energy of ATP, and possibly hsp 70, a known ATPase, may be involved in the transport process. Once in the nucleoplasm, the receptor is released from its carriers and is able to interact with chromatin. The nuclear localization sequence (NLS) receptors may involve at least two cytoplasmic proteins of about 60,000 and 76,000 molecular weight. It has even been hypothesized that the hsp 56, which has been shown to be an immunophilin, could be the NLS-binding protein for the cytoplasmic glucocorticoid receptor. Also, this molecular weight is close to the 60,000 mentioned earlier. This protein has an acidic sequence (rabbit) 140-EDLTDDED-147 that conceivably could interact with the rat glucocorticoid receptor NLS, 510-RKTKKKIK-517. These proteins bind directly to the NLS sequence of the steroid receptor and probably act as transporting proteins to and through the nucleopore.

In order to appreciate the structure of the nucleopore, a schematic representation is shown in Chapter 1. Its dimensions are about 80 nm in length and 100 nm in diameter. The protein molecular weight of the complex is estimated at 125 million by scanning electron microscopy. The aperture in the center can change from 10 nm in the closed state to 40 nm in the open state. There is evidence for fibrillar connections between a pore complex and the nucleoskeleton, the cytoskeleton, and other pore complexes. Presumably, solid-state transport could occur through this structure, which is involved in the nuclear import of proteins and export of RNAs with the assistance of the nucleoporins. The locations of nucleoporin residence are not clear, but they could be attached to the spokes. The nucleoporins are a family of proteins that contain carbohydrate structures and are sensitive to the action of wheat germ agglutinin. The nucleoporins could be different from the nuclear localization signal receptors in the cytoplasm, but they could play a structural role in the nucleopore-transporting process. Figure 10-15 shows models of the structural organization of the nucleoporin family. Once in the nucleus, the exposed Zn2+ fingers of the activated receptor complex permit interaction with the major groove of DNA. A second site on the steroid-receptor complex in the steroid-binding domain assists in the specific binding to DNA.

With the use of cell culture, several types of cells have been isolated: cells with normal function of the receptor that respond to glucocorticoid and cells that apparently have a defective receptor. Other types are

Variable Central Repetitive Domain Variable Carboxy-Terminal Domain

Ammo-Terminal Domain Typical for e.g. Heptad-Repeat

Nucleoporin Protein Family in NSP1 and p62 i i

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Cure Tennis Elbow Without Surgery

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