Chapter 2:  Cells and within cells

Cells

Translation

Receptor Mechanisms

    Neurons

   Proteins, Protein Structure and Protein Analysis

    Glia

   Epigenetics

   Protein folding in the cell

Within cells

   Transcription

   Post-translational modifications of proteins

   Noncoding RNAs

   Protein degradation in the cell - Autophagy

   Membranes and Membrane Proteins

   miRNAs and the brain

   Protein secretion / Secretory pathway

   The Exctracellular Matrix

The receptors for steroid hormones are transcription factors which regulate gene expression. Steroids are very hydrophobic hormones which can cross the blood-brain barrier and have important actions on the brain. Thus, an understanding of the mechanism of action of steroid hormones is important in considering brain function. As an introduction to the next module on transcription factors as receptors (which will concern primarily receptors for steroids), first a short overview of general principles of gene transcription (see also "Transcription") and intracellular signal transduction pathways will be given ,

A gene has two regions, a regulatory region and a coding region. The coding region is in the 3' end of the gene (so-called downstream). The coding region is transcribed into RNAby the enzyme RNA polymerase II. The RNA produced is called the primary transcript (heteronuclear RNA). It is further enzymatically processed to give rise to the mature messenger RNA (mRNA) which is subsequently translated into protein.

In the processing of the primary transcript the introns are cut out and the  exons (ex = expression) are spliced together to form the mRNA.In the regulatory region one finds the so-called TATA box and promoter elements where transcription factors (with such names as Transcription Factor IIB etc) bind. These transcription factors, collectively called basal transcription factors,  form a binding site for the RNA polymerase. They and the polymerase are sufficient to give a very low level of basal transcription of the gene into RNA.

Further upstream on the gene are enhancer elements which are the sites for binding of yet more transcription factors (also referred to as activating transcription factors). The transcription factors which bind here are often dimers. The binding of these transcription factors causes the DNA to loop such that the transcription factors contact the RNA polymerase and  the basal transcription factors. This complex formation strongly activates the polymerase to produce the primary RNA transcript.

First, consider G-protein-coupled receptors....

G-protein-coupled receptor mechanisms generating cyclic AMP can lead to phosphorylation and activation of a transcription factor called CREB (Cyclic-AMP Responsive Element Binding protein). As its name implies, CREB binds to an enhancer element on the gene called CRE (Cyclic-AMP Responsive Element). CREB is a homodimer with phosphorylation sites for protein kinase A (PKA). Upon activation the free catalytic subunit of PKA enters the nucleus (nucleus indicated in red in figure to right) where it phosphorylates CREB, which in its phosphorylated form can bind to CRE and enhance gene expression. CREB received its name for the action of cyclic AMP on its activity.

Further research has shown, however, that the activity of this transcription factor is regulated by a large number of kinases and thus that CREB is truely an integrator of converging signaling pathways. Also, CREB has been shown to be a large familiy of related proteins.

Use the link to the left  to learn more about the CREB family of transcription factors.

Besides activating kinase (thus potentially effecting CREB activity) Ca²⁺ can also signal directly to the nucleus via Ca²⁺ waves. These waves are generated at the membrane of the cell and are propagated to the nucleus via Ca²⁺-induced Ca²⁺ release (CICR). Propagation of the signal involves the mobilization of Ca²⁺ from intracellular stores (see figure to left). Once in the nucleus it is thought that Ca²⁺ would bind to calmodulin or some other Ca²⁺ binding proteins found in the nucleus to ultimately act on gene expression. Recently a transcription factor directly regulated by Ca²⁺ has been discovered. This factor has been given the name DREAM.

Use the link to the right to obtain more information about DREAM.

In considering the CREB family, we have already discussed tyrosine receptor kinase signaling via the MAP Kinase cascade and CREB. This signaling begins at the membrane with the activation of phospholipase C gamma. In this case activated PLC gamma generates diacyl glycerol (DAG) at the membrane. The DAG then activates PKC. The activated PKC phosphorylates another kinase to start a cascade of kinase activations known as the MAP kinase cascade. This ultimately leads to the phosphorylation of transcription factors in the nucleus, including CREB and another one with the name c-Jun (c for cellular because there is also an ocogene form, v-Jun). c-Jun is not phosphorylated by PKA nor CaM Kinases, thus making this pathway somewhat unique for the growth factors (although it can be activated by receptor mechanisms working through Gq and PLC-beta).

So, in conclusion......

All the membrane-bound receptors, i.e. ion channel receptors (e.g. NMDA receptor via Ca²⁺), tyrosine kinase receptors, and G-protein-coupled receptors, have links to the nucleus for the regulation of gene expression.

For steroids the link to the nucleus is more direct, namely the receptors themselves are transcription factors. The steroids, being hydrophobic, has no trouble in crossing the cell membrane to find their intracellular receptors. The receptors can be in the cytosol or in the nucleus (depends on which steroid is under consideration). Binding of the steroid induces dimerization and, if the receptor was cytosolic, it now moves to the nucleus. In the nucleus the activated receptor now binds enhancer elements of genes. This leads to enhanced gene expression.

The first gene activated can be a transcription factor. An example is the transcription factor c-fos (c because of "cellular", there is also an oncogene form, "viral", v-fos). c-fos dimerizes with another transcription factor, c-jun, to form a complex which can bind to an enhancer called AP-1. The gene coding for c-fos contains a CRE, the binding site for CREB. Thus, the expression of c-fos can be enhanced by PKA and Ca²⁺ calmodulin dependent protein kinase (CaCal/PK). Because, in this case, it is the first gene to be activated the c-fos gene is termed an "immediate early gene". The c-fos protein then activates, via the AP-1 site on the late gene, the expression of the late gene. Other signals can act directly on the late gene.

In the example illustrated above, the MAP kinase cascade is acting on the late gene by phosphorylating and thus activating transcription factors that can bind to enhancer elements on the late gene (such as c-jun or other transcription factors). Similarly,  the  receptor for glucocorticoid (a steroid hormone) is a transcription factor which, in the steroid-bound form,  binds to the glucocorticoid-responsive element (GRE) to induce gene expression.

The expression of an immediate early gene (such as c-fos) relative to the late gene is shown to the left. The immediate early gene is normally only expressed for a very short time (+/- 2h) whereas expression of the late gene, while slow to be initiated, remains active for many hours or even days. This property of c-fos has been exploited in a method to locate brain regions that have just been activated. The premise is that if the brain region has activated within the last two hours then the neurons in this region will show high expression of c-fos (thus the name "c-fos method"). In this method either antibodies against c-fos, or cDNA probes for c-fos mRNA are used to examine for c-fos expression in  tissue sections of the brain. The method has been instrumental in gaining insight into the function of specific brain areas.

For example, have you ever wondered what brain region is activated when a bird begins to sing? If so, now you can find out. The link to the right will give you an illustration of an application of the c-fos method to locate brain regions responsible for singing in song-birds. Not all late genes are regulated by c-fos so one must view a negative result with the method very carefully (i.e. the negative brain area may in fact be activated by another immediate early gene).