It has become accepted that a fundamental activity of a steroid receptor with its bound ligand hormone working in a target cell is to either stimulate or repress the expression of genes. The steroid hormone 1α,25(OH)2D3, bound to its receptor, VDR, can activate as many 3,000 genes (micro array data) from the ~total 22,000 human genes. It is likely that these numbers are similar for the other steroid hormones as well as thyroid hormone.
It is also now understood that the hormones that bind nuclear receptors and initiate changes in gene expression also have receptor-mediated effects on processes independent of these nuclear actions. Table 1 compares the nuclear and nonnuclear (i.e., membrane initiated) actions of these hormones. Frequently these rapid nongenomic responses are generated within 1–2 minutes to 15–45 minutes. This contrasts with genomic responses, which generally take several hours to days to be fully functional and apparent and which can be blocked by inhibitors of transcription and translation.
Table1. Nuclear Receptors and Involvement in Genomic and/or Rapid Biological Responses
As one example, the steroid hormone 1α,25(OH)2D3, has been shown to initiate many biological responses via rapid response pathways that are independent of parallel genomic responses. Like the other steroid receptors, the VDR has a ligand binding domain (LBD) and a DNA binding domain that are essential to create a functional nuclear heterodimer with the retinoid X receptor. However, 1α,25(OH)2D3 is distinguished from the other steroid hormones in its conformational flexibility, as illustrated in Figure 1.
Fig1. The steroid hormone 1α,25(OH)2D3 is conformationally flexible and presents different 3-D shapes for binding to its receptor, the vitamin D receptor (VDR). Panel A presents the structure of two shapes of 1α,25(OH)2D3. The 6-s-cis presentation shape on the left side is in rapid exchange with the 6-s-trans or extended shape. The change from cis to trans occurs by a 360-degree rotation around the 6-7 single bond (see the two arrows). This interchange process occurs at the rate of several million times per second. This extreme rotation allows the molecule to assume a very large family of three-dimensional shapes. Panel B has two extreme presentations of 1α,25(OH)2D3. The top row is a space-filling three-dimensional representation of 6-s-cis shaped 1α,25(OH)2D3. The two dark images on the left end of the bowl shape each represent the space-filling of the 1α -OH group (bottom surface) and the 3β-OH group (upper surface). At the right-hand end of the bowl shape is the space filling view of carbon-25 OH group. On the right side is the three-dimensional planar shape of 1α,25(OH)2D3. The right-hand three-dimensional ligand shows a 6-s-trans presentation; overall it has a planar shape. The left dark image is the 1α-OH group, while the 3β-OH group is hidden on the “back” surface. At the right end of the planar shape is the dark image of the 25-OH group. The bottom labels for panel B are the VDR-GP (for the VDR genomic pocket engaged in gene transcription; left side) and the VDR-AP (for the VDR alternative pocket engaged in nongenomic responses; right side). In order for the VDR to generate genomic responses, it has been experimentally established that the VDR must have a bound 1α,25(OH)2D3 ligand that is bowl shaped. This can function to turn selected genes either on or off. For the VDR that is localized to the membrane caveolae where it produces rapid responses, it must have a bound 1α,25(OH)2D3 that is in the planar conformational shape. The different shapes of the VDR-GP and the VDR-AP ligands were evaluated by separate determination of the X-ray structure of the VDR-GP with a bound bowl-shape ligand and computer modeling of the VDR-AP with an AP ligand with a planar shape. Panel B has two extreme presentations of 1α,25(OH)2D3. The top row is a space-filling three-dimensional representation of 6-s-cis shaped 1α,25(OH)2D3; it has a bowl-like shape. The right-hand three-dimensional ligand shows a 6-s-trans presentation; it has a planar shape. The second row is a ball-and stick presentation of 1α,25(OH)2D3 in the bowl (left) or planar (right) shape. In order for the VDR to generate genomic responses, i.e., affect the rate of gene transcription, it has been experimentally established that the VDR must have a bound 1α,25(OH)2D3 ligand in the bowl shape. This is referred to as the VDR-GP (VDR genomic pocket). The VDR that is localized to the membrane caveolae and produces “rapid” responses must have a bound 1α,25(OH)2D3 that is in the planar conformational shape. This binding site is referred to as the VDR-AP (VDR alternative pocket).The different shapes of the VDR-GP and VDR-AP ligand were evaluated by separate determination of the X-ray structure of each and computer modeling of the AP ligand.
The subcellular location of VDR capable of producing both genomic and rapid responses is shown in Figure 2. The majority of the VDR is in the nucleus (80–85%) with the rest divided between the cytoplasm/ ER (10–15%) and caveoli (5%). It is the ~5% of caveolae-bound VDR that has been shown to be involved in voltage gated opening of chloride channels and exocytosis.
Fig2. Distribution of VDR in a target cell. Target cells for the steroid hormone 1α,25(OH)2D3 have VDR distributed between the nucleus (~80%), the cytosolic compartment (~15%), and the interior surface of the plasma membrane caveolae (~5%). The VDR can move by diffusion to these three compartments. The VDR monomer has hydrophobic bonds with the intracellular surface of the caveolae membrane. The X, Y, and Z red letters are outside the cell. The VDR, arriving from the nucleus and cytosol, docks with the surface of the caveolae facing the cytosol of the cell. When the caveolae becomes associated with a monomeric VDR with a bound 1α,25(OH)2D3 (red triangle), it moves (in this example) to the nearest chloride channel to activate rapid responses. However, the caveolae-associated VDR must have the 1α,25(OH)2D3 bound to the VDR-AP (alternate pocket), and not the VDR-GP (genomic pocket) for this to happen. This VDR/planar ligand is competent to initiate a variety of rapid responses (see Table 1). Illustrated in this figure is the VDR/1α,25(OH)2D3 initiated opening of chloride channels in the plasma membrane. In the nucleus of the cell, VDR with a bound 1α,25(OH)2D3 forms a heterodimer with the retinoid X receptor to activate gene transcription. Gene knock-out (KO) of the VDR in mice results in the loss of both 1α,25(OH)2D3 stimulated gene activation as well as the activation of rapid responses, including the opening of chloride channels, and even exocytosis from the cell.
The rapid membrane responses are not unique to the VDR receptors. Table 1 compares for the following receptors (thyroid β, estrogen α and β, glucocorticoid, cortisol, mineralocorticoid aldosterone, progesterone, androgen receptors, and the VDR). All the listed steroids have been shown to repeatedly participate in both activation and repression of genomic responses.
It has also been established that both the ERα and androgen receptors, in addition to the cell membrane caveolae, specifically bind to the cell’s mitochondria. In summary, steroid hormones clearly can stand alone with their receptors and initiate rapid nongenomic responses, genomic effects in the cell nucleus that are mediated by second messenger pathway, and also genomic effects where the steroid receptors function as transcription factors.
